QCM immunosensor with nanoparticle amplification for detection of Escherichia coli O157:H7

A quartz crystal microbalance (QCM) immunosensor was developed and evaluated for detection of Escherichia coli O157:H7. The immunosensor was fabricated by self-assembling of protein A and affinity-purified anti-E. coli O157:H7 antibodies on the gold electrode of an AT-cut piezoelectric quartz crystal. To enhance the sensitivity of the QCM immunosensor, nanoparticle-antibody conjugates, which were prepared using streptavidin-conjugated nanoparticles (145 nm diameter) and biotinylated anti-E. coli antibodies, were used for signal amplification. After the binding of E. coli O157:H7 cells with the antibodies immobilized on the electrode, nanoparticle-antibody conjugates were introduced as mass amplifiers. Compared to the direct detection of E. coli O157:H7, the binding of the nanoparticle conjugates further resulted in a decrease in resonant frequency and an increase in resonant resistance, and the detection sensitivity was improved by five orders of magnitude by lowering the detection limit from 10⁷ to 10² CFU/mL. The sensor specificity and nonspecific adsorption of nanoparticle-antibody conjugates were also investigated.     

Preparation and characterization of phytosterol-loaded nanoparticles with sodium caseinate/dextran conjugates

Sodium caseinate (SC)/dextran conjugates were prepared via Maillard reaction under controlled dry-heating conditions. Moreover, the nanoparticles of phytosterols (PS) encapsulated by SC or SC/dextran were produced using the emulsion evaporation method. The encapsulation efficiency (78.81 ± 5.22%) of PS in SC/dextran nanoparticles was higher than that (73.5 ± 2.78%) in SC nanoparticles. Compared with the compact and dense structure of SC nanoparticles, SC/dextran nanoparticles existed as relatively loose aggregates. The result of differential scanning calorimetry demonstrated that the encapsulation of PS greatly decreased its crystallinity. The released rates of PS from SC and SC/dextran nanoparticles under acidic gastric conditions were 8.59% and 4.73%, respectively. After 7 h of intestinal digestion, the released rate (52.19%) of PS from SC/dextran nanoparticles was significantly higher than that from SC (32.67%) nanoparticles. Therefore, SC/dextran conjugates prepared by the Maillard reaction are more suitable to be used as wall material for the nano-encapsulation of PS.     

基于荧光微球的免疫层析法结合免疫磁珠分离技术快速定量检测鼠伤寒沙门氏菌

为了建立一种能快速、灵敏、特异检测鼠伤寒沙门氏菌(Salmonella typhimurium,ST)的方法,本研究以荧光微球(FM)为标记物建立了荧光微球免疫层析检测法(FM-ICA),将羧基化的磁性微球与抗体偶联制备了抗-ST免疫磁珠(IMB),并将FM-ICA与IMB联合用于富集和检测三种食品样本中的ST。在优化条件下,FM-ICA检测的线性范围为3.16×10~6~2×10~9 CFU/m L,回收率为80%~120%,变异系数均小于5%。当菌液浓度大于1×10~8 CFU/m L时,结果显示与同属沙门氏菌的交叉反应;而当菌液浓度小于或等于1×108CFU/m L时,常见的12株非目标菌检测结果为阴性,特异性较好。FM-ICA+IMB联合检测三种模拟食品时的回收率为38%~55%,其中,西红柿对回收率影响较小,鸡蛋和猪肉影响较大。然而,联合检测的灵敏度可达到1×10~5 CFU/m L,比单一FM-ICA方法提高了30倍,同时,整个检测过程可在2 h内完成。本检测方法的建立对于快速筛查鼠伤寒沙门氏菌具有重要意义。     

团聚状纳米AuPd修饰的H2O2生物传感器研究

采用微胶束法室温条件下制备团聚状的AuPd合金纳米粒子,使用紫外可见光谱(UV-vis),透射电镜(TEM),X-射线粉末衍射(XRD)和X-射线能量色散谱(EDS)表征团聚结构AuPd合金纳米粒子的形貌、尺寸、结构和组成。用制备的AuPd纳米粒子修饰辣根过氧化物酶玻碳电极,制备无电子媒介的过氧化氢生物传感器HRP/AuPd/GCE。使用循环伏安法和计时电流法表征了HRP/AuPd/GCE对H2O2的检测性能。实验结果表明:该传感器对H2O2具有良好的检测性能和稳定性,在H2O2浓度为1×10-7mol/L～5×10-3mol/L范围内检测电流与H2O2浓度有线性关系,线性相关系数R2=0.995 01,检出限为7.6×10-7mol/L。     

A Biotin–Streptavidin Amplified Enzyme-Linked Immunosorbent Assay with Improved Sensitivity for Rapid Detection of Ractopamine in muscular tissue Development and Nonspecific Adsorption Reduction

An effective biotin–streptavidin amplified enzyme-linked immunosorbent assay (BA-ELISA) was optimized and characterized for the rapid detection of Ractopamine (RAC) residue in muscular tissue. Purification of the RAC antiserum by protein A-Sepharose 4B followed with bovine serum albumin (BSA)-Sepharose 4B affinity chromatography enhanced the sensitivity and reduce the background adsorption. Blocking with 0.5% skimmed milk power and diluting streptavidin–HRP conjugates with 0.5% BSA/phosphate-buffered saline (PBS) effectively remove the nonspecific adsorption in biotin–streptavidin amplified ELISA system. The established method allowed RAC determination with an IC₅₀ value of 0.3 ± 0.02 ng ml⁻¹ and a limit of detection of 0.02 ± 0.003 ng ml⁻¹, more sensitive than the other reported methods. The variation coefficients of intra-assay and inter-assay were all below 7%. RAC residue in pig muscular tissue could be quantified without matrix effects after a 5-fold extraction and 2-fold dilution with PBS. Recoveries of RAC in pig muscular tissue ranged from 75% to 82.75%. The results were also compared with those from HPLC and a good correlation was obtained (r ² = 0.9822). The characters show that the established biotin–streptavidin amplified ELISA could be potentially useful in rapid detection of RAC in animal-derived foods.     

Structural and thermodynamic features of covalent conjugates of sodium caseinate with maltodextrins underlying their functionality

The sodium caseinate (SCN)-maltodextrin (MD) covalent conjugates were prepared by a food-grade process involving the first step of the Maillard reaction. The covalent conjugates were prepared with different weight ratios of biopolymers (R(MD?:?SCN) = 0.4; 1; 2; 5) in the system using maltodextrins of strongly different dextrose equivalents (DE), i.e., DE = 2 and 10. We have observed that the covalent conjugation of SCN with MD, in contrast to their simple mixing, improved the protein solubility in an aqueous medium in a wide pH range that was more pronounced in the vicinity of the SCN isoelectric point (pH 3.8-4.4). The extent of SCN solubility was mainly governed by the weight/molar ratio of the biopolymers in the covalent conjugates, R(MD?:?SCN). Data of static multiangle laser light scattering showed that the revealed increase in the solubility of the conjugates could be predominantly attributable to the dramatic increase in their thermodynamic affinity for an aqueous medium. Which was most pronounced for the maltodextrin with the higher DE (DE = 10). The direct relationship between the increase in the solubility of the conjugates and the increase in their foaming ability, as compared against SCN, has been revealed as a rule both at neutral pH and at the pI. In addition, the found improvement in the protein foaming ability was also defined by both the weight/molar ratio (R(MD?:?SCN)) and the dextrose equivalent of the maltodextrins attached to the protein.     

Preparation of CIT-protein conjugates using a novel citrinin derivative as hapten

In this paper, a novel citrinin (CIT) derivative, 8-MeO-CIT, was synthesized by the method of methylation, with the protection of hydroxyl in CIT using methoxyl for the first time. The overall yield of 8-methoxyl-citrinin (8-MeO-CIT) was about 49.8%. 8-MeO-CIT was then purified by two-dimensional TLC, and its characteristics were analyzed by HPLC and ESI–MS. 8-MeO-CIT-BSA and CIT-BSA were prepared using the NHS-ester method and the Mannich-type method, respectively. Characteristics of both CIT-protein conjugates were determined by UV–Vis and FT-IR spectroscopy. Results showed that the conjugate molar ratio and utilized ratio of CIT in 8-MeO-CIT-BSA increased by 4.1 and 18.4% compared to CIT-BSA. It suggested that NHS-ester method using novel 8-MeO-CIT as hapten might prefer to traditional Mannich-type method for preparing CIT-protein conjugates.     

基于核酸适配体的镉离子可视化检测方法

利用适配体对镉离子的特异性识别作用,以镉离子适配体及互补链为生物识别单元,建立基于核酸适配体的镉离子可视化检测方法,实现对自来水中镉离子痕量检测。该方法将镉离子适配体固定在微孔板上,再与适配体互补链及-辣根过氧化物酶(HRP)复合物进行竞争性结合,通过HRP对底物的催化水解反应引起450nm处特征峰的变化来定量检测镉离子。结果表明:该检测体系在0.1~5.0ng/mL时具有线性关系(R~2=0.995 8),检测限低至0.5ng/mL。该方法选择性良好、操作简单、灵敏度高,并且具有较好的实用性,可用于食品安全分析和环境监测中金属离子镉的检测。     

Determination of aflatoxins by enzyme-linked immunosorbent assay with special reference to aflatoxin M1 in raw milk

There is an increasing requirement for the screening of animal feedstuffs, plant products and edible animal tissues for human consumption, for the presence of contamination by aflatoxins. An enzyme-linked immunosorbent assay is described which may be used to determine aflatoxins in raw milk at 0.1μg litre^?1 and in extracted feedstuffs at the level of 5μgkg^?1. The method includes a novel conjugation procedure allowing heterologous conjugates to be prepared resulting in increased sensitivity without interference by sample matrix effects. Suitably cross-reactive antisera were produced using the cyclopentanone ring of the aflatoxin molecule as the principal immunogenic determinant.     

AuRu纳米粒子修饰的辣根过氧化物酶传感器研究

采用反向胶束法在室温下合成出AuRu纳米粒子,使用X射线衍射(XRD)、能量色散X射线光谱 (EDS)、高分辨透射电镜(HRTEM)表征其结构、组成和形貌,制备其修饰辣根过氧化物酶传感器(HRP/AuRu/GC)。使用循环伏安法(CV)和计时电流法(i-t)研究该传感器对H2O2的检测性能。结果表明：HRP/AuRu/GC对H2O2检测具有高的灵敏度和稳定性,在H2O2浓度为1×10-7～1×10-3mol/L范围内检测电流与浓度的负对数成线性关系,线性相关系数R2＝0.9959,检出限为5.02×10-8mol/L。电极使用1个月后其响应电流为原来的91.54%。     

小麦粉中黄曲霉毒素B_1现场检测用纳米修饰传感器

在印刷电极表面涂覆一层壳聚糖(Chit)膜,在膜上同时固定四羧基酞菁铁(Ⅲ)(FePc)和HRP酶标黄曲霉毒素B_1抗体(HRP-Ab-AFB_1)包被纳米金,制备了可用于小麦粉中黄曲霉素B_1(AFB_1)快速检测的新型安培免疫传感器(SPCI Chit/FePc/Au/HRP-Ab-AFB_1).FePc对H_2O_2的还原具有催化作用,可作为HRP酶与电极之间电子传递媒介体.当该传感器在含AFB_1样品的30℃溶液中温育8 min后,AFB_1与Ab-AFB_1的免疫结合导致HRP的活性中心与FePc之间的电子传递被部分阻碍,使HRP对H_2O_2电催化氧化电流I_0降低.△I_0与AFB_1浓度在1.0～200μg/L成线性关系,标准样品的组内和组间变异系数<3.5%;回收率97%～104%,检测限为0.3μg/L.该传感器检测AFB_1温育时间短,一步测定免分离,为小麦粉中的AFB_1现场分析提供了新技术.     

制备方法对乳清分离蛋白-绿原酸共价接枝物结构和功能性质的影响

目的：研究制备方法对乳清分离蛋白（whey protein isolate，WPI）-绿原酸（chlorogenic acid，CA）共价接枝物结构和功能性质的影响。方法：分别以碱法、自由基法和酶法制备WPI-CA共价接枝物，对其反应基团含量、CA接枝量、分子结构、表面疏水性、热稳定性、抗氧化活性和乳化性等进行分析。结果：游离氨基、色氨酸和巯基均参与了反应，游离氨基为主要反应基团。碱法、自由基法和酶法接枝物的CA接枝量分别为（52.70±1.81）、（42.57±1.85）μmol/g和（63.75±2.50）μmol/g。CA的共价接枝使WPI二级结构中α-螺旋相对含量减少，无规卷曲结构相对含量增加，分子内源荧光强度降低。碱法和酶法接枝会降低WPI表面疏水性，增强其热稳定性，自由基法的影响则与两者相反。与WPI相比，WPI-CA共价接枝物的抗氧化活性和乳化性显著增强（P＜0.05），且抗氧化活性与CA接枝量成正比。结论：酶法接枝效率最高，且所得WPI-CA接枝物热稳定性和抗氧化活性最强。本研究可为蛋白质-多酚共价接枝物类抗氧化性载体材料的制备及应用提供参考。     

Formation of soy protein isolate–dextran conjugates by moderate Maillard reaction in macromolecular crowding conditions

BACKGROUND: Several methods have been reported for the conjugation of proteins with polysaccharides. Protein–polysaccharide conjugates can be formed by traditional dry heating, but this process is not attractive from an industrial viewpoint, and no commercial conjugates have been manufactured in this way. In the present study, in order to develop a more practical reaction method, macromolecular crowding was used to attach polysaccharides to proteins. RESULTS: Soy protein isolate–dextran conjugates (SDCs) were prepared via the initial stage of the Maillard reaction in macromolecular crowding conditions. The impact of various processing conditions on the formation of SDCs was investigated. The optimal conditions chosen from the experiments were a soy protein isolate/dextran ratio of 1:1 (w/w), a pH of 6.5, a reaction temperature of 60 °C and a reaction time of 30 h. Circular dichroism spectroscopy showed that the secondary and tertiary structures of the conjugates were changed significantly. Structural flexibility increased, allowing better display of their functional characteristics. The conjugates had a composition with various sizes, especially macromolecules, according to gel permeation chromatography. Thermal analysis showed that the thermal stability of the conjugates was improved. CONCLUSION: The production of SDCs under macromolecular crowding conditions appears to be an effective and promising technique, representing an advance over classic protein glycosylation methods. Copyright © 2012 Society of Chemical Industry     

挤压温度对挤压大米蛋白与葡萄糖复合物功能和结构特性的影响

为获得一种快速、有效的工业化生产方法,本研究在大米蛋白与葡萄糖发生美拉德反应前对大米蛋白进行挤压改性。大米蛋白在不同温度（80、90、100、110、120、130 ℃）下挤压,然后在pH 10.5条件下与葡萄糖接合30 min。分析不同挤压温度对其功能性质（溶解度、乳化活性和乳化稳定性）的影响,利用扫描电子显微镜、傅里叶变换红外光谱和十二烷基硫酸钠-聚丙稀酰胺凝胶电泳对挤压后的大米蛋白和葡萄糖复合物进行结构表征。结果表明：与天然大米蛋白质和葡萄糖（native rice protein and glucose,NRPG）复合物相比,挤压大米蛋白和葡萄糖（extruded rice protein and glucose,ERPG）复合物在90 ℃的糖基化程度最高。与NRPG相比,ERPG（90～120 ℃）的溶解度降低,ERPG（80～90 ℃）的乳化活性、乳化稳定性和表面疏水性升高,而100～130 ℃的时候缓慢降低。红外光谱结果表明,与NRPG相比,ERPG具有较高的α-螺旋、β-转角和无规卷曲,β-折叠结构相对含量较低。十二烷基硫酸钠-聚丙稀酰胺凝胶电泳显示蛋白质通过挤压聚合成更大的颗粒。扫描电子显微镜显示,ERPG具有更多的无序结构和不规则碎片。     

免疫磁纳米粒子对肠炎沙门氏菌的富集分离

采用化学共沉淀法制备了羧基化磁纳米粒子,分别对磁纳米粒子-沙门氏菌多克隆抗体复合物（免疫磁纳米粒子）的偶合条件和免疫磁纳米粒子富集分离肠炎沙门氏菌的条件进行了优化,为肠炎沙门氏菌的富集分离和检测提供一种更为快捷、高效的方法。结果表明,当51.7 μg/mL羧基化磁纳米粒子与碳二亚胺/N-羟基丁二酰亚胺（0.4 mol/L/0.1 mol/L）、1.0 mg/mL的多克隆抗体的体积比为1∶2∶2时,37 ℃水浴加热40 min,两者的偶合效果最佳。应用上述优化条件制备的免疫磁纳米粒子吸附104 CFU/mL肠炎沙门氏菌,当两者的体积比为4∶5,孵育时间为40 min时,免疫磁纳米粒子对肠炎沙门氏菌的吸附效率可达到94.36%。     

α-乳白蛋白单克隆抗体的制备及斑点印迹定性检测方法的建立

以α-乳白蛋白为抗原免疫Balb/c小鼠,采用淋巴细胞杂交瘤技术制备抗α-乳白蛋白单克隆抗体;采用碳二亚胺法,并通过工艺优化,将单抗与量子点高效偶联;在此基础上,建立了基于斑点印迹技术定性检测乳制品中过敏原α-乳白蛋白的方法。结果表明:采用杂交瘤技术制备的单克隆抗体特异性好,与牛奶中其他蛋白无交叉反应;当量子点、1-（3-二甲氨基丙基）-3-乙基碳二亚胺盐酸盐（EDC）、N-羟基丁二酰亚胺（NHS）、单抗摩尔比为1∶10∶6∶4时,量子点与单抗有较好的偶联效果;以量子点标记单抗建立的斑点印迹方法可直观、快速的定性分析乳制品中过敏原α-乳白蛋白。因此,该方法具有一定的实用价值。      

Technical note: Development of a quantitative PCR method for monitoring strain dynamics during yogurt manufacture

Yogurt starter cultures may consist of multiple strains of Lactobacillus delbrueckii ssp. bulgaricus (LB) and Streptococcus thermophilus (ST). Conventional plating methods for monitoring LB and ST levels during yogurt manufacture do not allow for quantification of individual strains. The objective of the present work was to develop a quantitative PCR method for quantification of individual strains in a commercial yogurt starter culture. Strain-specific primers were designed for 2 ST strains (ST DGCC7796 and ST DGCC7710), 1 LB strain (DGCC4078), and 1 Lactobacillus delbrueckii ssp. lactis strain (LL; DGCC4550). Primers for the individual ST and LB strains were designed to target unique DNA sequences in clustered regularly interspersed short palindromic repeats. Primers for LL were designed to target a putative mannitol-specific IIbC component of the phosphotransferase system. Following evaluation of primer specificity, standard curves relating cell number to cycle threshold were prepared for each strain individually and in combination in yogurt mix, and no significant differences in the slopes were observed. Strain balance data was collected for yogurt prepared at 41 and 43°C to demonstrate the potential application of this method.     

玉米纤维胶-乳清分离蛋白Maillard共聚物对姜黄素乳液稳定性和消化特性的影响

以玉米纤维胶（corn fiber gum,CFG）、乳清分离蛋白（whey protein isolate,WPI）为基质制备Maillard反应产物,探究不同热处理时间下CFG-WPI Maillard共聚物对乳液稳定性的影响;并进一步采用不同乳化剂制备包载姜黄素的乳液体系,并探究其消化特性。结果表明,CFG-WPI Maillard共聚物最佳制备条件为加热温度60 ℃、相对湿度79%、反应时间96 h。与单一CFG、WPI以及CFG-WPI混合物相比,CFG-WPI Maillard共聚物制备的姜黄素乳液粒径以及澄清指数较小,且对pH值、Ca2＋浓度等外界环境因子表现出较强的抵抗力,说明CFG-WPI Maillard共聚物制备的乳液稳定性最高。姜黄素乳液消化特性结果表明,CFG的加入有助于减缓脂质水解速率,与单一WPI相比,CFG以及CFG-WPI混合物制备的乳液中姜黄素的生物有效性较高。而对于共聚物构建的乳液体系而言,粒径是影响姜黄素乳液消化特性以及生物有效性的一个重要因素。本研究为了解CFG-WPI Maillard共聚物乳液的形成规律及消化特性提供了一定的参考,同时为CFG的增值利用提供了新途径。     

Flavan-3-ol-cysteine and acetylcysteine conjugates from edible reagents and the stems of Cynomorium songaricum as potent antioxidants

Flavan-3-ol derivatives, including 3 cysteine conjugates and 3 acetylcysteine conjugates, were prepared using Cynomorium songaricum and edible reagents. The structures were determined, based on NMR and MS spectra. All compounds had stronger radical-scavenging activity than catechin and epicatechin. Moreover, the cysteine conjugates were active against α-glucosidase, whereas catechin and epicatechin were not. Two acetylcysteine flavan-3-ol conjugates, 4β-(l-acetylcysteinyl)-epicatechin 3-O-gallate and 4β-(l-acetylcysteinyl)-epiafzelechin, are novel compounds with better log P values than their cysteine counterparts. These conjugates can be prepared from purified flavan-3-ol polymers or directly from herbal material.     

Comparative studies on the physicochemical properties of peanut protein isolate–polysaccharide conjugates prepared by ultrasonic treatment or classical heating

Peanut protein isolate (PPI) was glycated with dextran or gum Arabic through ultrasonic treatment or classical heating. The physicochemical properties of PPI–polysaccharide conjugates prepared by ultrasonic treatment were compared to those prepared by classical heating. Compared with classical heating, ultrasound could accelerate the graft reaction between PPI and polysaccharides. PPI could glycate more dextran than gum Arabic under both ultrasonic treatment and classical heating. Despite the higher degree of graft, ultrasonic treatment was able to prepare PPI–polysaccharide conjugates with higher color lightness as well as lower yellow tones than classical heating. During glycation, high molecular weight component was formed, and conarachin mainly participated in glycation reaction instead of arachin. Solubility and emulsifying properties of the conjugates prepared through ultrasonic treatment were both improved as compared to conjugates obtained by classical heating and PPI. The solubility of PPI–dextran was improved for pH range 3 to 9, while that of PPI–gum Arabic was improved for pH > 7. Meanwhile, the emulsifying properties of PPI–gum Arabic were better than those of PPI–dextran conjugates. Decrease of lysine and arginine contents suggested these two amino acids attended the glycation between PPI and polysaccharides. Structural feature analyses suggested that conjugates obtained by ultrasonic treatment had less α-helix and more β-structures, higher surface hydrophobicity and less compact tertiary structure as compared to conjugates obtained by classical heating and PPI.