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.     

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3×105CFU.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。     

ATP生物发光法检测E.coliO157:H7的研究

ATP生物发光法是近年来发展最快的定量微生物检测分析技术之一,具有快速、简便、灵敏度高等特点,已在食品、化工、环境等众多领域得到了广泛应用,主要是通过间接测定活菌总数来监测样品清洁度或卫生状况.文章利用ATP生物发光法建立了E coliO157;H7与光源计数值的定量线性模型,研究了pH 7.4下四种不同的缓冲液(KH2PO4-NaOH,NazHPO+-C6H8O7,PBS、Tris-HCl)、质量浓度为10 g·L-1下五种不同的化学物质(NaOH,NaCl,NaH2PO+4KCl,MgCl2)对E coliO157:H7光源计数值的影响.结果表明:Tris-HCI缓冲液对茵液的稀释效果最好,其对不同稀释浓度的区分能见度好,并且背景信号最小;MgCl2对发光有明显的增强作用,而NaOH,NaCl,NaHz PO4,KCl对发光有较大的抑制作用,其中NaOH对发光的抑制作用最大,达60.56%.同时,ATP生物发光法与传统的培养法相关性良好(r=0.9608),检测限可达103cells·mL-1.     

ATP生物发光法检测E. coliO157∶H7的研究

ATP生物发光法是近年来发展最快的定量微生物检测分析技术之一,具有快速、简便、灵敏度高等特点,已在食品、化工、环境等众多领域得到了广泛应用,主要是通过间接测定活菌总数来监测样品清洁度或卫生状况。文章利用ATP生物发光法建立了E. coliO157∶H7与光源计数值的定量线性模型,研究了pH 7.4下四种不同的缓冲液（KH2PO4-NaOH,Na2HPO4-C6H8O7,PBS、Tris-HCl）、质量浓度为10 g.L^-1下五种不同的化学物质（NaOH,NaCl,NaH2PO4,KCl,MgCl2）对E.coliO157∶H7光源计数值的影响。结果表明：Tris-HCl缓冲液对菌液的稀释效果最好,其对不同稀释浓度的区分能见度好,并且背景信号最小;MgCl2对发光有明显的增强作用,而NaOH,NaCl,NaH2PO4,KCl对发光有较大的抑制作用,其中NaOH对发光的抑制作用最大,达60.56%。同时,ATP生物发光法与传统的培养法相关性良好（r=0.9608）,检测限可达103cells.mL^-1。     

不同浓度FeCl3·6H2O,CuSO4·5H2O和MnCl2·4H2O溶液核磁共振横向弛豫时间研究

利用核磁共振技术,测量了不同浓度的FeCl3.6H2O,CuSO4.5H2O和MnCl2.4H2O水溶液的核磁共振信号及横向弛豫时间.实验结果表明横向弛豫时间T2与顺磁离子浓度成反比关系,并由实验结果获得了通过核磁共振横向弛豫时间测量离子浓度的方法.     

单分子动力学研究大肠杆菌单链结合蛋白与单链DNA的结合过程

大肠杆菌单链结合蛋白(E.coli SSB)具有与单链DNA(single-stranded DNA,ssDNA)结合的性质,起着保护ssDNA及引导相关蛋白质反应的重要作用,然而,它与ssDNA的结合过程及其细节尚未得到确定的研究结果.本文采用单分子磁镊对E.coli SSB/ssDNA复合体进行拉力测试,并采用单分子化学反应动力学方法对测试结果进行分析.研究发现:E.coli SSB在ssDNA上的结合过程分为两个不同的阶段,一个是在临界力下快速的结合缠绕阶段,一个是随着力的进一步减小逐步缠绕的阶段.从中得到了E.coli SSB与ssDNA的化学反应系数,并得到了相应的自由能参数.采用自由能校准的方法,得到了SSB与ssDNA结合的完整自由能曲线,从而揭示了E.coli SSB与ssDNA的结合特征.本文的分析方法也可以应用于相似的化学反应的研究中.     

基于免疫磁分离和酶促信号放大传感器的构建及应用研究

甲胎蛋白(AFP)作为肿瘤标志物,其检测在肝癌早期诊断过程中有着重要意义。磁分离技术能够实现快速无损分离,可以有效简化实验过程。本实验用免疫磁珠作为固相载体连接捕获抗体,并与酶标二抗通过AFP连接形成夹心结构,酶催化外加底物使其显色,最终用紫外分光光度法检测底物浓度间接实现对溶液中AFP的定量检测。体系对AFP的检测范围为10~80ng·mL-1,检出限为3.6ng·mL-1。该方法灵敏度高,重现性好,为AFP的检测提供了新的思路和方法。     

基于近红外量子点的荧光共振能量转移生物探针构建及应用

本论文构建了基于近红外量子点In P/Zn S和Cy7(C45H44K3N3O16S4)的荧光共振能量转移(FRET)体系,完成了不同p H值和不同浓度下的FRET体系转换效率的检测。检测结果显示:当量子点浓度保持不变时,随着染料浓度的增加,体系转换效率也随之增加,当In P/Zn S量子点与Cy7浓度比为1∶250时,转换效率高达68%。细胞测试结果表明,FRET体系对p H值有较高敏感度,对细胞微环境p H值的检测精度可达0.1,该体系可以作为敏感型FRET探针用于生物微环境检测。     

Atmospheric pressure plasma jet utilizing Ar and Ar/H_2O mixtures and its applications to bacteria inactivation

An atmospheric pressure plasma jet generated with Ar with H2O vapor is characterized and applied to inactivation of Bacillus subtilis spores. The emission spectra obtained from Ar/H2O plasma shows a higher intensity of OH radicals compared to pure argon at a specified H2O concentration. The gas temperature is estimated by comparing the simulated spectra of the OH band with experimental spectra. The excitation electron temperature is determined from the Boltzmann’s plots and Stark broadening of the hydrogen Balmer Hβline is applied to measure the electron density. The gas temperature, excitation electron temperature, and electron density of the plasma jet decrease with the increase of water vapor concentration at a fixed input voltage. The bacteria inactivation rate increases with the increase of OH generation reaching a maximum reduction at 2.6%(v/v) water vapor. Our results also show that the OH radicals generated by the Ar/H2O plasma jet only makes a limited contribution to spore inactivation and the shape change of the spores before and after plasma irradiation is discussed.     

荧光免疫及磁免疫层析法检测莱克多巴胺的研究

分别将量子点和超顺纳米磁珠作为荧光探针和磁信号探针应用于免疫反应中,构建了检测莱克多巴胺的荧光免疫和磁免疫层析的分析方法,并成功应用于尿液中莱克多巴胺的检测。两种方法均基于免疫竞争模式,在荧光免疫分析方法中,量子点偶联上识别莱克多巴胺的抗体,样品中莱克多巴胺和包被在ELISA板上莱克多巴胺的完全抗原竞争结合量子点,样品中莱克多巴胺的浓度越高,ELISA板上吸附的量子点越少,所测荧光强度值越低,该方法的检出限为1 ng·mL-1,检测时间为4 h。在磁免疫层析方法中,检测线上特异性捕获的纳米磁珠颜色的深浅和尿液中莱克多巴胺浓度成反比例关系,即莱克多巴胺的浓度越高,检测线的颜色越浅,该方法的定性检出限为10 ng·mL-1,检测时间为15 min。两种方法各有优缺点,基于量子点的荧光免疫分析法在痕量检测和定量分析方面具有优势,而磁免疫层析法更适合于现场快速检测。     

Recovery of Escherichia Coli O157:H7 and Salmonella in Milk and Cream of Chicken Soup from High Hydrostatic Pressure (HHP) and Bacteriocin Applications Upon Storage

By the application of HHP; more than 8 log cycle reduction was achieved for E. coli O157:H7 933 and Salmonella FDA in milk and cream of chicken soup in 5 min. Storage of HHP treated milk samples showed absence of both bacteria for 24 h at 4 °C and for 48 h at 37 °C. The population of the strains in cream of chicken soup exceeded their initial value after 5 days of storage at 25 °C. BP 1 was combined with HHP and used for inhibition of surviving bacterial species in cream of chicken soup. There was at least 7 log cycle reduction in E. coli O157:H7 933 and Salmonella FDA after 5 and 7 days of storage at 25 °C respectively, when compared to HHP alone. Combination of HHP with BP 1 extended the shelf life of cream of chicken soup for both bacterial species an additional 2 days at 25 °C when compared with HHP treatment alone.     

Synergistic antibacterial effects of ultrasound and thyme essential oils nanoemulsion against Escherichia coli O157:H7

Essential oil nanoemulsions have been proven to have stronger antimicrobial effects compared to the essential oil alone or coarse emulsion. Sonoporation could be the promising candidate to trigger a synergistic effect with thyme essential oil nanoemulsion (TEON) and produce a more effective antibacterial efficacy. Therefore, in this study, the bactericidal effects of ultrasound (US) in combination with TEON treatments against Escherichia coli (E. coli) O157:H7 were investigated. The remarkable synergistic effects of US (20 kHz, 255 W/cm², 9 min) and TEON (0.375 mg/mL) treatments at 22 °C reduced E. coli O157:H7 populations by 7.42 ± 0.27 log CFU/mL.The morphological changes of cells exposed to different treatments were observed by scanning electron microscopy and transmission electron microscopy. The results showed that the synergistic effects of the ultrasound and TEON treatments altered the morphology and interior microstructure of organism cells. Laser scanning confocal microscopy (LSCM) images revealed that the combination treatments of ultrasound and TEON altered the permeability of cell membranes, and this affected the integrity of E. coli O157:H7 cells. This was further indicated by the high amounts of nucleic acids and proteins released from these cells following treatment.The results from this study illustrated the mechanisms of the synergistic effects of sonoporation and TEON treatments and provided valuable information for their potential in food pasteurization.     

Using fluorescence measurement of zinc ions liberated from ZnS nanoparticle labels in bioassay for <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7

In this study, an alternative approach using ZnS nanoparticle biolabels as fluorescence signal transducers is reported for the immunoassay of E. coli O157:H7 in tap water samples. Instead of measuring the fluorescence of ZnS nanoparticles in the assay, the fluorescence signal is generated through the binding of zinc ions released from nanoparticle labels with zinc-ion sensitive fluorescence indicator Fluozin-3. In the assay, ZnS nanoparticles around 50 nm in diameter were synthesized, bioconjugated, and applied for the detection of E. coli O157:H7. The assay shows a detection range over two orders of magnitude and a detection limit around 1000 colony-forming units (cfu) of E. coli O157:H7.     

Vibrio parahaemolyticus, enterotoxigenic Escherichia coli, enterohemorrhagic Escherichia coli and Vibrio cholerae

This review highlighted the following: (i) pathogenic mechanism of the thermostable direct hemolysin produced by Vibrio parahaemolyticus, especially on its cardiotoxicity, (ii) heat-labile and heat-stable enterotoxins produced by enterotoxigenic Escherichia coli, especially structure-activity relationship of heat-stable enterotoxin, (iii) RNA N-glycosidase activity of Vero toxins (VT1 and VT2) produced by enterohemorrhagic Escherichia coli O157:H7, (iv) discovery of Vibrio cholerae O139, (v) isolation of new variant of Vibrio cholerae O1 El Tor that carries classical ctxB, and production of high concentration of cholera toxin by these strains, and (vi) conversion of viable but nonculturable (VBNC) Vibrio cholerae to culturable state by co-culture with eukaryotic cells.     

Characterization of self-assembled monolayers (SAMs) on silicon substrate comparative with polymer substrate for Escherichia coli O157:H7 detection

This article presents the characterization of two substrates, silicon and polymer coated with gold, that are functionalized by mixed self-assembled monolayers (SAMs) in order to efficiently immobilize the anti-Escherichia coli O157:H7 polyclonal purified antibody.A biosurface functionalized by SAMs (self-assembled monolayers) technique has been developed. Immobilization of goat anti-E. coli O157:H7 antibody was performed by covalently bonding of thiolate mixed self-assembled monolayers (SAMs) realized on two substrates: polymer coated with gold and silicon coated with gold. The F(ab′)₂ fragments of the antibodies have been used for eliminating nonspecific bindings between the Fc portions of antibodies and the Fc receptor on cells. The properties of the monolayers and the biofilm formatted with attached antibody molecules were analyzed at each step using infrared spectroscopy (FTIR-ATR), atomic force microscopy (AFM), scanning electron microscopy (SEM) and cyclic voltammetry (CV). In our study the gold-coated silicon substrates approach yielded the best results.These experimental results revealed the necessity to investigate each stage of the immobilization process taking into account in the same time the factors that influence the chemistry of the surface and the further interactions as well and also provide a solid basis for further studies aiming at elaborating sensitive and specific immunosensor or a microarray for the detection of E. coli O157:H7.     

DyxTbyY1-x-yP5O14晶体的荧光性质

研究了DyxTbgY1-x-yP5O14晶体的荧光特征,Dy3+离子对Tb3+离子5D3能级的猝灭作用和对5D4能级的敏化作用,讨论了Tb3+离子和Dy3+离子浓度间的关系及对荧光强度和荧光寿命的影响。     

超声波条件下降解染料废水的工艺条件研究

采用Fenton试剂对模拟染料废水的降解效果进行研究。结果表明,H2O2投加量、Fe2+投加量、pH值条件、超声处理时间的改变对染料废水的处理效果影响很大。对酸性染料:当pH为4.5,30%H2O2投加的体积分数为30mL/L,Fe2+投加的质量浓度为400mg/L,反应时间为40min时为降解反应的最佳操作条件。对碱性染料,正交试验表明当pH为4、30%H2O2投加的体积分数16mL/L、Fe2+投加的质量浓度为300mg/L、反应时间为60min时为降解反应的最佳操作条件,其降解率达98.46%,COD的去除率达到96.7%。     

UV照射大肠杆菌对其吸收光谱的影响

探讨了样品池溶液及分光光度计内置紫外光对大肠杆菌吸收光谱测定的影响。发现样品池溶液(2.8 mL双蒸水+0.1 mL生理盐水+0.1 mL LB培养基)较为合理,既给细菌提供了有利的生存环境,又对细胞的紫外吸收影响较小;研究同时发现在257 nm波段150 s内细胞吸收强度基本没有变化,说明内置的紫外光不会影响试验测定结果。在此基础上,研究了UV照射0,20,40,60,80,120 s大肠杆菌吸收光谱的变化情况。结果显示随着UV照射剂量的增加,大肠杆菌吸收光谱存在一个先降后升再降的现象。该现象反映了辐射中细胞损伤死亡和修复存活的生物学效应。该研究从光谱学角度探讨了UV对大肠杆菌细胞的损伤作用,对利用光谱技术研究细胞的辐射损伤、死亡和修复具有一定参考意义。