基于氧化铝纳米通道的电化学生物传感器研究

将制备好的氧化铝纳米通道经与3-氨丙基三乙氧基硅烷反应使其表面修饰了氨基后,再在含生物素的缓冲溶液(pH 5.5)中反应12h,制成表面固定了生物素的氧化铝纳米通道,通道孔径约50nm。另取PVC管一段,在其顶端用PVC/THF混合液粘附制备好的聚合物膜,再将上述修饰好的氧化铝纳米通道用有机硅橡胶粘在敏感膜的底部,作为工作电极待用。以生物素-亲和素体系为模型,用经修饰的氧化铝纳米通道为识别载体进行电位法检测,实现了亲和素的检测。亲和素的质量浓度在0.10~0.60mg·L-1范围内与相应的电位变化值之间呈线性关系,检出限(3σ)为0.05mg·L-1。试验结果验证了氧化铝纳米通道电位生物传感器测定生物大分子的可行性。     

检测大肠杆菌的免疫生物传感器研究进展

大肠杆菌是人体内常见的食源性致病菌,对其检测极为重要。免疫生物传感器技术作为目前研究较热的一种技术,在大肠杆菌的检测方面有不错的进展。本文综述了电化学、光学、压电等类型免疫传感器近十年的研究进展,从检测原理、抗体固定方式、修饰材料、检测能力等方面进行了综述,并对各自的优劣势进行了讨论,对免疫传感器的发展趋势进行了总结与展望。     

大肠杆菌O157:H7微滴数字PCR定量方法的建立

以大肠杆菌O157：H7(E. coli O157：H7)rfbE基因为靶基因,建立了可对其准确定量的微滴数字PCR( ddPCR)方法。对ddPCR反应中的探针浓度进行了优化,考察了方法的线性范围、精密度、定量限和检出限。最终确定ddPCR 反应中的最佳探针浓度为300 nmol/L。 E. coli O157：H7基因组 DNA 浓度范围为4~1.25×105拷贝/20μL ddPCR反应液时,ddPCR方法线性相关系数( R2)为0.999。当DNA浓度为760~88400拷贝/20μL 时,方法的精密度最好( RSD<5%)。本方法的定量限为4拷贝/20μL,检出限为3拷贝/20μL。特异性验证结果表明,建立的ddPCR方法特异性良好,对13份猪肉、牛肉和鸡肉样品的检测结果与定量PCR方法检出结果一致。     

NaOH蚀刻玻碳电极的大肠杆菌DNA电化学生物传感器的构建及检测

以NaOH蚀刻后的玻碳电极为基底制备了高灵敏与高选择性的大肠杆菌电化学DNA传感器。经NaOH蚀刻后的玻碳电极被活化且在电极表面形成羧基层,为DNA探针的固定提供了更多位点,在偶联剂EDC/NHS的作用下,端氨修饰的探针DNA与羧基的羧氨反应使其以肽键的形式固定在电极表面,极大地提升了传感器的灵敏度。通过电化学循环伏安法(CV)和差分脉冲法(DPV)对所制备传感器的灵敏度和选择性进行表征,得到该传感器对大肠杆菌的线性检测范围为12.5~62.5 nmol/L,检出限可达1.20×10~(-9)mol/L。     

纳米金标记抗体增强SPR检测大肠杆菌O157∶H7

将金纳米粒子(AuNPs)标记的大肠杆菌O157∶H7(E.coli O157∶H7)的多克隆抗体(PAb)作为二抗,采用氨基偶联法将PAb固定在传感器表面作为一抗,通过三明治方法用双通道表面等离子体子共振(SPR)传感器对E.coli O157∶H7进行检测,并与SPR直接法检测进行了比较.结果表明,直接法的检出限为103cfu/mL,线性范围为103～109cfu/mL;AuNPs增强三明治法的检出限为10 cfu/mL,线性范围为10～1010cfu/mL,灵敏度比直接法提高了100倍,且具有更宽的检测范围.本方法不仅检测时间短,而且具有良好的选择性和重现性.     

基于生物素-亲和素系统的压电免疫传感器检测相思子毒素研究

利用生物素-亲和素系统的放大作用和纳米金质量扩增效应,建立了压电免疫传感器检测相思子毒素的新方法.首先在石英晶体的金电极上依次组装二巯基丙酸、EDC和NHS进行表面修饰,然后通过亲和素固定生物素标记相思子毒素多抗来制备敏感膜,利用纳米金的质量扩增效应设计了一种"毒素-纳米金标记单抗"复合物,成功实现了对相思子毒素的检测,提高了传感器灵敏度和重现性.本传感器对相思子毒素响应的线性范围为0.05～5 mg/L; 回归方程为Δf＝50.81CAbrin+67.11(r=0.9903,n＝10,P<0.0001); 检测灵敏度为50.81 Hz · L/mg.     

基于二氧化硅荧光纳米颗粒与核酸染料SYBRGreenⅠ的双色显微成像技术用于E.coliO157:H7的检测

将免疫荧光纳米标记技术与激光共聚焦显微成像方法相结合,发展了一种基于二氧化硅荧光纳米颗粒和核酸染料SYBR Green Ⅰ的双色显微成像技术用于大肠杆菌O157:H7的检测.采用联吡啶钌(RuBpy)二氧化硅荧光纳米颗粒对羊抗大肠杆菌O157:H7抗体进行修饰,基于抗体-抗原相互作用实现了其对目标大肠杆菌O157:H7...     

基于二氧化硅荧光纳米颗粒与核酸染料SYBRGreenⅠ的双色显微成像技术用于E.coliO157:H7的检测

将免疫荧光纳米标记技术与激光共聚焦显微成像方法相结合,发展了一种基于二氧化硅荧光纳米颗粒和核酸染料SYBR Green Ⅰ的双色显微成像技术用于大肠杆菌O157:H7的检测.采用联吡啶钌(RuBpy)二氧化硅荧光纳米颗粒对羊抗大肠杆菌O157:H7抗体进行修饰,基于抗体-抗原相互作用实现了其对目标大肠杆菌O157:H7的特异性标记;同时以核酸染料SYBR Green Ⅰ对细菌进行染色,将细菌和纳米颗粒团聚体区分开,实现了对大肠杆菌O157:H7的双色标记,并通过激光共聚焦显微镜进行免分离的荧光成像检测.结果表明,该方法可用于缓冲溶液体系和混合细菌样品中目标大肠杆菌O157:H7的特异性检测,在仅含5％目标菌的混合样品中仍能观察到具有明显黄色荧光的大肠杆菌O157:H7,且整个检测步骤包括样品预处理可在3h内完成.该方法则具有较好的灵敏度,可检出2.6×103 Cell/mL的目标细菌样品.若采用针对其它病原菌细胞壁抗原的特异性抗体,则有望发展成为一种通用技术用于多种病原菌的快速和灵敏检测.     

微悬臂生物传感器检测生物素-亲和素相互作用

将生物素共价键合到微悬臂的硅表面, 利用微悬臂传感技术在流动注样条件下研究了生物素-亲和素的动态作用过程, 同时利用原子力显微镜(AFM)力刻蚀技术验证了生物素化硅表面结合亲和素的有效性. 结果显示亲和素与生物素作用产生较大的表面应力驱动微悬臂偏转, 悬臂偏转的程度和速率与亲和素的浓度相关, 亲和素浓度越大, 微悬臂偏转程度也越大, 同时反应速率也更快. 这项工作为开发新型的无标记检测技术, 研究生物分子之间相互作用提供了有益的探索.     

菲涅耳反射型光纤生物传感器研究

构建了一种基于菲涅耳反射原理的光纤生物传感器,以生物素-链霉亲和素体系为模型验证了其进行生物传感的可行性.首先,利用硅烷偶联剂,在切平整的普通单模光纤端面修饰上生物素,将其作为吸附链霉亲和素的生物敏感膜.然后,将光纤端面浸入不同浓度的链霉亲和素溶液中,测量宽带光源经过光纤端面反射后的光谱.实验结果表明,反射光谱的相对功率与链霉亲和素溶液浓度之间呈线性关系,测量得到灵敏度为15.38 dB/(mg/mL).用牛血清白蛋白代替链霉亲和素,测量得到的反射光谱曲线与在之前浸入生物素后的反射光谱曲线基本重合,证明此生物传感器的选择性好.最后以人免疫球蛋白G与山羊抗人免疫球蛋白G体系验证了此结构进行生物传感的实用性.     

Binding Characteristics Study of DNA based Aptamers for E. coli O157:H7

E. coli O157:H7 is a pathogenic bacterium producing verotoxins that could lead to serious complications such as hemolytic uremia syndrome. Fast detection of such pathogens is important. For rapid detection, aptamers are quickly gaining traction as alternative biorecognition molecules besides conventional antibodies. Several DNA aptamers have been selected for E. coli O157:H7. Nonetheless, there has not been a comparative study of the binding characteristics of these aptamers. In this work, we present a comprehensive analysis of binding characteristics including binding affinity (K_d) and binding capacity (B_max) of DNA-based aptamers for E. coli O157:H7 using qPCR. Our results show that aptamer E18R has the highest binding capacity to E. coli 157:H7 and the highest specificity over non-pathogenic E. coli strains K12 and DH5α. Our study also finds that the common biotin-tag modification at 5′ end typically changes the binding capacity significantly. For most of the selected aptamers, the binding capacity after a biotin-tag modification decreases. There exists a discrepancy in the binding capability between the selected aptamer and the aptamer used for detection. Our study also shows that a lower concentration of Mg²⁺ ions in the binding buffer leads to a decrease in the binding capacity of E17F and E18R, while it does not affect the binding capacity of S1 and EcoR1.     

基于甘露糖功能化的水凝胶用于E.coli O157: H7的检测

利用伴刀豆球蛋白A(Con A)的多价结合能力, 结合水凝胶技术与核酸染色技术发展了一种基于甘露糖功能化的水凝胶检测大肠杆菌(E.coli)O157: H7的方法. 以过硫酸铵(APS)为催化剂, 四甲基乙二胺(TEMED)为加速剂, 用丙烯酰胺(AAm)、N,N-二甲基双丙烯酰胺和N-丙烯酰氧琥珀酰亚胺(NAS)合成水凝胶, 通过氨基化甘露糖与NAS发生交联反应, 制备了甘露糖功能化的水凝胶. 当甘露糖功能化的水凝胶加入与Con A共孵育后的菌悬液中时, 由于Con A既能与甘露糖特异性结合, 又能与E.coli O157: H7表面的O-抗原发生免疫反应而紧密连接, 使目标菌被捕获到水凝胶表面, 采用核酸染料SYBR Green Ⅰ对捕获细菌进行染色, 实现了对E.coli O157: H7的核酸标记, 最后通过活体荧光成像系统对水凝胶进行荧光成像, 从而实现对待测样品的检测. 研究结果表明, 该方法可应用于缓冲液体系和混合细菌样品中E.coli O157: H7的特异性检测, 且整个检测步骤包括样品预处理可在2 h内完成. 该方法成本低、易操作, 且具有较好的灵敏度, 可检出3.7×10¹ Cells/mL的目标细菌样品.     

A novel electrochemical biosensor based on dynamic polymerase-extending hybridization for E. coli O157:H7 DNA detection

A novel biosensor based on single-stranded DNA (ssDNA) probe functionalized aluminum anodized oxide (AAO) nanopore membranes was demonstrated for Escherichia coli O157:H7 DNA detection. An original and dynamic polymerase-extending (PE) DNA hybridization procedure is proposed, where hybridization happens in the existence of Taq DNA polymerase and dNTPs under controlled reaction temperature. The probe strand would be extended as long as the target DNA strand, then the capability to block the ionic flow in the pores has been prominently enhanced by the double strand complex. We have investigated the variation of ionic conductivity during the fabrication of the film and the hybridization using cyclic voltammetry and impedance spectroscopy. The present approach provides low detection limit for DNA (a few hundreds of pmol), rapid label-free and easy-to-use bacteria detection, which holds the potential for future use in various ss-DNA analyses by integrated into a self-contained biochip.     

一种实用的基于化学发光和磁性纳米颗粒的E.coli O157:H7免疫鉴定方法

化学发光磁酶免疫已经被应用于检测病原体,但是由于针对相应病原体的抗体筛选和修饰等的步骤耗时费力,不适于对多种病原体进行筛查.制备了兔抗大肠杆菌(E.coli)O157:H7的免疫磁性纳米颗粒,富集病原菌后与鼠抗E.coli O157:H7的单克隆抗体形成双抗夹心,采用碱性磷酸酶标记的马抗鼠IgG与单抗结合,加入碱性磷酸酶的化学发光底物试剂3-(2'-螺旋金刚烷)-4-甲氧基-4-(3'-羟基)苯-1,2-二氧杂环丁烷磷酸检测化学发光.实验研究了底物缓冲液、碱性磷酸酶浓度对化学发光强度的影响,比较了NaBH4和甘氨酸对免疫磁珠剩余活性醛基的封闭效果以及本方法检测E.coli O157:H7的特异性和敏感性.结果表明,碱性磷酸酶与底物在c缓冲液中反应的化学发光强度最高,碱性磷酸酶浓度决定了化学发光的强度和持续时间,NaBH4对活性醛基的封闭效果优于甘氨酸,以D群宋内氏志贺氏菌、B群福氏志贺氏菌、鼠伤寒沙门氏菌、金黄色葡萄球菌和霍乱弧菌及E.coli Top10f'为对照的比较实验显示,该检测方法具有良好的特异性,以1mL的菌液为检测体积时对E.coli O157:H7的检测灵敏度为103cell/mL,整个方法的检测时间约为3h.该方法适用于对多样本进行筛查.     

Chemiluminescent enzyme-linked immunosorbent assay on a strip to detect Escherichia coli O157:H7

A fast and sensitive chemiluminescent enzyme-linked immunosorbent assay method to measure pathogenic bacteria, Escherichia coli O157:H7, on immuno-chromatographic membrane was studied. Non-specific binding of proteins on membrane strip was controlled to attain the best performance of immunosensor by optimising the composition of a running buffer. The specificity of the proposed immunostrip was confirmed by conducting experiments for four different micro-organisms. A chemiluminescent signal could be successfully generated from a proposed immunostrip sensing system, and a significant change in the chemiluminescent light intensity with the concentration of target microbes was obtained. E. coli O157:H7 could be quantitatively measured in the range of 1.1?×?10^3?–1.1?×?10⁷ CFU (colony forming units) mL^?1 within 16?min by using the developed chemiluminescent immunostrip.     

Microfluidic based impedance biosensor for pathogens detection in food products

A MEMS‐based impedance biosensor was designed, fabricated, and tested to effectively detect the presence of bacterial cells including E. coli O157:H7 and Salmonella typhimurium in raw chicken products using detection region made of multiple interdigitated electrode arrays. A positive dielectrophoresis based focusing electrode was used in order to focus and concentrate the bacterial cells at the centerline of the fluidic microchannel and direct them toward the detection microchannel. The biosensor was fabricated using surface micromachining technology on a glass substrate. The results demonstrate that the device can detect Salmonella with concentrations as low as 10 cells/mL in less than 1 h. The device sensitivity was improved by the addition of the focusing electrodes, which increased the signal response by a factor between 6 and 18 times higher than without the use of the focusing electrodes. The biosensor is selective and can detect other types of pathogen by changing the type of the antibody immobilized on the detection electrodes. The device was able to differentiate live from dead bacteria.     

A sensitive lateral flow biosensor for Escherichia coli O157:H7 detection based on aptamer mediated strand displacement amplification

Foodborne diseases caused by pathogens are one of the major problems in food safety. Convenient and sensitive point-of-care rapid diagnostic tests for food-borne pathogens have been a long-felt need of clinicians. Commonly used methods for pathogen detection rely on conventional culture-based tests, antibody-based assays and polymerase chain reaction (PCR)-based techniques. These methods are costly, laborious and time-consuming. Herein, we present a simple and sensitive aptamer based biosensor for rapid detection of Escherichia coli O157:H7 (E. coli O157:H7). In this assay, two different aptamers specific for the outmembrane of E. coli O157:H7 were used. One of the aptamers was used for magnetic bead enrichment, and the other was used as a signal reporter for this pathogen, which was amplified by isothermal strand displacement amplification (SDA) and further detected by a lateral flow biosensor. Only the captured aptamers on cell membrane were amplified, limitations of conventional DNA amplification based method such as false-positive can be largely reduced. The generated signals (red bands on the test zone of a lateral flow strip) can be unambiguously read out by the naked eye. As low as 10 colony forming units (CFU) of E. coli O157:H7 were detected in this study. Without DNA extraction, the reduced handling and simpler equipment requirement render this assay a simple and rapid alternative to conventional methods.     

Rapid detection of Escherichia coli O157:H7 spiked into food matrices

Food poisoning causes untold discomfort to many people each year. One of the primary culprits in food poisoning is Escherichia coli O157:H7. While most cases cause intestinal discomfort, up to 7% of the incidences lead to a severe complication called hemolytic uremic syndrome which may be fatal. The traditional method for detection of E. coli O157:H7 in cases of food poisoning is to culture the food matrices and/or human stool. Additional performance-based antibody methods are also being used. The NRL array biosensor was developed to detect multiple antigens in multiple samples with little sample pretreatment in under 30 min. An assay for the specific detection of E. coli O157:H7 was developed, optimized and tested with a variety of spiked food matrices in this study. With no sample pre-enrichment, 5 × 10³ cells mL⁻¹ were detected in buffer in less than 30 min. Slight losses of sensitivity (1-5 × 10⁻⁴ cell mL⁻¹⁾ but not specificity occur in the presence of high levels of extraneous bacteria and in various food matrices (ground beef, turkey sausage, carcass wash, and apple juice). No significant difference was observed in the detection of E. coli O157:H7 in typical culture media (Luria Broth and Tryptic Soy Broth).