Surface plasmon resonance analysis of staphylococcal enterotoxin B in food

Surface plasmon resonance (SPR) biosensors are electro-optical instruments used for analyzing real-time protein-protein interactions. This work evaluates an SPR biosensor (Biacore 3000) in the detection of staphylococcal enterotoxin B (SEB) in foods. A sandwich SPR immunosensor involving two antibodies was used. The capturing antibody, bound covalently to the surface of the biosensor chip, performs the initial binding of the antigen and a second antibody binds to the captured antigen. The second antibody makes antigen verification possible and amplifies the signal. Pure SEB as well as SEB in spiked foods (milk and meat) were detected with little interference from the food matrix. In the control experiments with uncontaminated food samples no significant signal was detected. The SPR biosensor assay detects SEB at approximately10 ng/ml rapidly, with initial binding within 2 min. The entire measurement cycle (including washing and chip regeneration) may take 5 min using one antibody or 8 min using two antibodies. These results suggest that the SPR biosensor may be a useful tool for real-time analysis of toxin in foods.     

SPR生物传感器高通量检测多种食源性 致病菌的研究

目的 建立一种基于表面等离子共振技术 (Surface Plasmon Resonance technology,SPR)原理的生物传感器方法,实现11种常见致病菌的高通量检测。方法 用表面自组装技术(self-assembled monolayer,SAM)在金膜表面引入羧基,然后通过氨-羧基反应将5’端标记有氨基的探针固定在芯片表面,经多重PCR扩增出的产物变性、杂交、生物素-亲和素系统信号放大等步骤完成目的片段的检测。对所建立的多重PCR-SPR检测方法的灵敏性、特异性等技术指标进行评价。 结果 本研究建立的多重PCR-SPR检测方法具有良好的灵敏性,该芯片检测11种常见致病菌的灵敏性均达到103~104,能满足现有检测要求。通过对实验室保存的14株标准菌株和沙门氏菌等90份样品分离株进行检测,结果显示该检测方法具有较好的特异性,无交叉反应。结论 本研究建立的多重PCR-SPR 检测系统具有良好的灵敏性和特异性,完全适用于11种致病菌的高通量检测。该检测方法检测时间短、检测成本低、减少了人为因素的影响,具有良好的应用前景。     

Quick detection technique for clenbuterol hydrochloride by using surface plasmon resonance biosensor

Clenbuterol hydrochloride (CLB) is a kind of lean meat powder which can increase the conversion rate of lean meat. Though it is allowed in human and veterinary medicine, CLB is harmful for human body and, therefore, is restricted to its use as a growth promoter. In this paper, a quick detection technique for clenbuterol hydrochloride is proposed. The technique, which is based on surface plasmon resonance (SPR) and immune reaction, includes two methods, the direct method and the suppression method. The direct method can be applied, to screen an antibody and in the study of the dynamic reaction. For the immune suppression method, the concentration of CLB is inversely proportional to the change of SPR angle. The detection limit of this method is smaller than 2 μg/L, and the pork extraction is detected. The concentration of CLB in the sample is 2.75 μg/L, and the concentration of antibody is 250 μg/L. The detection of CLB by SPR is quick, highly sensitive, and label free. And the utilized equipment is cheap, simple, environmental friendly, and easy to control. The proposed technique could achieve the real-time detection of a large number of samples and be widely used to detect CLB in all fields of food processing. Therefore, this method is expected to be used in the inspection process for foods in the supermarket, bazaar, and factory.     

Immunomagnetic flow cytometric detection of staphylococcal enterotoxin B in raw and dry milk

A rapid and sensitive method for detection of staphylococcal enterotoxin B (SEB) in raw and dry milk samples with the use of antibody-based immunomagnetic separation (IMS) in conjunction with flow cytometry (FCM) was developed. Sheep anti-SEB immunoglobulin G (IgG) was immobilized on Dynabeads M-280. The SEB initially binds to the capturing antibody, which is bound on the magnetic beads. The rabbit anti-SEB IgG binds to the captured toxin and is further labeled with a Cy5-labeled goat anti-rabbit IgG antibody. The percentage of the beads that were fluorescent was measured by FCM. FCM was carried out for 1 min, and the data obtained were expressed as histograms for particle size (forward light scatter) and histograms for fluorescence intensity. A peak corresponding to the magnetic beads was clearly distinguished from a peak derived from contaminating particles in the sample solution. In the absence of SEB, about 10% of the beads emitted fluorescence. The percentage of fluorescent beads and the fluorescence intensity increased with increasing SEB concentrations. For this IMS-FCM assay, the lower limits of detection for SEB were estimated to be 0.01 and 0.25 ng/ml for buffer and milk samples, respectively.     

A microarray biosensor for multiplexed detection of microbes using grating-coupled surface plasmon resonance imaging

Grating-coupled surface plasmon resonance imaging (GCSPRI) utilizes an optical diffraction grating embossed on a gold-coated sensor chip to couple collimated incident light into surface plasmons. The angle at which this coupling occurs is sensitive to the capture of analyte at the chip surface. This approach permits the use of disposable biosensor chips that can be mass-produced at low cost and spotted in microarray format to greatly increase multiplexing capabilities. The current GCSPRI instrument has the capacity to simultaneously measure binding at over 1000 unique, discrete regions of interest (ROIs) by utilizing a compact microarray of antibodies or other specific capture molecules immobilized on the sensor chip. In this report, we describe the use of GCSPRI to directly detect multiple analytes over a large dynamic range, including soluble protein toxins, bacterial cells, and viruses, in near real-time. GCSPRI was used to detect a variety of agents that would be useful for diagnostic and environmental sensing purposes, including macromolecular antigens, a nontoxic form of Pseudomonas aeruginosa exotoxin A (ntPE), Bacillus globigii, Mycoplasma hyopneumoniae, Listeria monocytogenes, Escherichia coli, and M13 bacteriophage. These studies indicate that GCSPRI can be used to simultaneously assess the presence of toxins and pathogens, as well as quantify specific antibodies to environmental agents, in a rapid, label-free, and highly multiplexed assay requiring nanoliter amounts of capture reagents.     

Quantification of calpastatin using an optical surface plasmon resonance biosensor

An immunological biosensor for calpastatin was developed on a surface plasmon resonance based system (Biacore Q). The performance of the biosensor assay was evaluated using ovine and bovine muscle and heart extracts with known calpastatin activity. In addition, the relationship between immunologically detectable calpastatin at 1 day postmortem and shear force at 14 days postmortem was investigated for bovine longissimus dorsi. Calpastatin biosensor results for several experiments were linearly related to calpastatin activity measurements with correlation coefficients ranging from 0.51 to 0.99. The intra- and inter-assay CVs were <6% (n=12). During postmortem storage, the amount of immunologically detectable calpastatin decreased faster than the inhibitory activity in the enzymatic assay. Probably, the epitope recognized by the antibody is degraded faster than the inhibitory sites of calpastatin during postmortem storage. Calpastatin content at 1 day postmortem was correlated to shear force at 14 days postmortem (r=0.75). It is anticipated that developments in the near future will allow for at-line calpastatin determinations in beef plants. At present, the calpastatin biosensor assay appears suitable for research purposes where large numbers of samples need to be processed for breed evaluation or selection programs because this assay requires less labor than other methods.     

金黄色葡萄球菌肠毒素B检测方法的研究进展

金黄色葡萄球菌分泌的肠毒素B(staphylococcal enterotoxin B,SEB)作为与食品中毒相关的重要毒素之一而被广泛报道。同时由于其具有热稳定性、易制备、高毒及易传播等特点引起科研人员的高度重视,因此,建立SEB高效的检测方法非常重要。本文对SEB的检测方法进行了综述,主要讨论了生物学检测、免疫凝集实验、琼脂糖扩散法、酶联免疫检测技术、放射性免疫检测技术、免疫荧光检测技术、胶体金试纸条检测技术、基因探针、仪器分析、生物传感检测等检测方法的原理及相关国内外研究进展的应用和局限,这对提前预防金黄色葡萄球菌肠毒素B引起的食物中毒具有重要意义,同时也为今后开发新型检测方法提供理论参考和技术支持。     

Gold nanoparticle-based lateral flow assay for detection of staphylococcal enterotoxin B

The lateral flow assay (LFA), a rapid, sensitive, and reproducible technique, was successfully applied to detect staphylococcal enterotoxin B (SEB). The assay was based on a double-antibody sandwich format on a porous nitrocellulose membrane. When SEB-containing samples were applied to the LFA-device, the toxin initially reacted with polyclonal antibody (Pab)-coated colloidal gold particles and then reacted with the fixed Pab on the membrane. These reactions resulted in a red line at the detection zone, with intensity proportional to the SEB concentration (under 100 ng/ml). With this method, 1 ng/ml of SEB can be detected in less than 5 min and was highly reproducible. Signal can be amplified to 10 pg/ml by silver enhancement. This assay also showed no cross-reaction with other SEs, such as SEA, SEC, SED and SEE. The assay was significantly faster than the ELISA or real-time PCR assay and should facilitate early and rapid SEB detection in clinical and food samples.     

SPR技术对奶粉中维生素B12检测方法的建立

目的 利用表面等离子共振(surface plasmon resonance, SPR)技术, 建立快速定量测定牛奶中维生素B12的方法。方法 将钴胺素共价偶联到表面等离子共振芯片CM5表面, 并对竞争结合的维生素B12结合蛋白的结合浓度及芯片的再生条件进行优化, 检测芯片的稳定性。在无抗生素牛奶中添加系列质量浓度的维生素B12, 利用免疫竞争抑制原理构建标准曲线, 并对市售10个奶粉样品进行检测。结果 制备的芯片稳定, 50个循环相对标准偏差(relative standard deviation, RSD)小于10%。日间批内同一样品差异为0.54%, 该方法的检测限为0.006 μg/100 g, 回收率为92.1%~104.1%。测得的10个牛奶产品中维生素B12含量与对应商品标签值全部在标准规定的范围内。结论 所建立的方法可以在6 h内完成样品的前处理和检测, 是一种简便、快捷的定量检测方法。     

SPR技术检测奶粉中生物素

目的利用表面等离子共振(surface plasmon resonance,SPR)技术,建立快速定量测定牛奶中生物素的方法。方法将生物素共价偶联到表面等离子共振芯片CM5表面,并对竞争结合的生物素结合蛋白的结合浓度及芯片的再生条件进行优化,检测芯片的稳定性。在无抗生素牛奶中添加系列质量浓度的生物素,利用免疫竞争抑制原理构建标准曲线,并对市售10个奶粉样品进行检测。结果制备的芯片稳定,50个循环相对标准偏差(relative standarddeviation,RSD)小于10%。日间批内同一样品差异为8.75%,该方法的检测限为0.1μg/100g,回收率为80.4%~91.2%。10个牛奶产品中生物素含量全部在固定的允许范围内。所建立的方法可以在4 h内完成样品的前处理和检测。结论该方法是一种简便、快捷的定量检测方法。     

Gold nanoparticle-based colorimetric detection of staphylococcal enterotoxin B using ssDNA aptamers

Staphylococcal enterotoxin (SE) B is one of the most common serotype of SEs to cause staphylococcal food poisoning. To ensure food safety, rapid and low-cost methods have been constantly developed and applied to detect SEB worldwide. In the present investigation, a panel of aptamers of single-stranded DNA molecules against SEB was obtained by optimizing the procedure of systematic evolution of ligands by exponential enrichment, and five of them were selected for further analyzing the characteristics of sequences and second structures. Afterward, an aptamer-based colorimetric method of SEB detection was carried out by employing unmodified gold nanoparticle probes (AuNPs). Results showed that the main second structures of these aptamers were stem loop and hairpin forms. In addition, applying one of these aptamers (No. 15-1), SEB could be detected at the concentration of 10 ng/mL by AuNPs-based colorimetric method. Moreover, the aptamer also possessed a good selectivity toward SEB and SEC₁. Our work demonstrated that aptamers had their potential applications as a bioprobe for the detection of SEs in food products.     

Real time biosensor analysis of staphylococcal enterotoxin A in food.

Currently there is no 'real-time' detection system to identify food borne toxins. In order to develop such a system, we have used a evanescent wave biosensor for real time detection of staphylococcal enterotoxin A (SEA) in foods. The approach used here is sandwich biosensor, a method utilizing two antibodies. The toxin binds initially to a capturing antibody which is bound covalently on the surface of the biosensor detector. The second antibody binds to the captured toxin. We were able to measure SEA in foods with little or no background interference, demonstrating that biosensor-based measurement of SEA was possible not only with purified SEA but also in complex food matrices such as hot dogs, potato salad, milk and mushrooms. Autoclaved samples of SEA did not evoke a positive response. With both purified SEA and SEA-spiked foods, the assay sensitivity is 10-100 ng/g depending on the material tested and the assay is rapid ( <4 min) when a single antibody is used.     

Comparison of a fluoroquinolone surface plasmon resonance biosensor screening assay with established methods

The performance of a previously developed immunochemical biosensor screening method for fluoroquinolone (FQ) antibiotics in poultry muscle, fish and egg was compared with established methods. Blank sample material of the target matrices was individually spiked with the FQs at half maximum residue levels. Homogeneity of the test materials was confirmed by liquid chromatography–mass spectrometry (LC–MS/MS). Identical sets of spiked samples as well as incurred samples from a previous feeding experiment were sent to three independent laboratories and analysed by LC–MS/MS, a microbiological assay and the new biosensor assay. The new method correctly identified all contaminated samples and demonstrated advantages in sensitivity and analysis time compared to the microbiological screening assay.     

表面等离子体共振免疫传感器用于快速检测微囊藻毒素的研究

针对现有微囊藻毒素（MC-LR）检测方法操作复杂、因标记而污染环境、仪器贵重,不利于现场快速检测等问题,将自行研制的表面等离子体共振（SPR）生物芯片检测仪应用于微囊藻毒素的检测,提出抑制型SPR生物芯片快速检测痕量微囊藻毒素的方法。采用该方法分别对浓度为3.5、2.5、1.5、1、0μg/L的MC-LR样品进行了检测。结果表明:该方法检测限小于1μg/L,可满足世界卫生组织（WHO）对于饮用水和我国地表水环境质量标准中MC-LR最低含量检测的需求。该方法完成一个样品检测耗时约8 min,相比于高效液相色谱法（HPLC）和酶联免疫吸附法（ELISA）等传统检测方法,快速定量是其最大的优势,可用于食品质量监控和现场实时检测。      

表面等离子共振技术快速检测食品中的 玉米赤霉烯酮毒素

目的建立基于表面等离子共振(surface plasmon resonance,SPR)技术快速检测食品中玉米赤霉烯酮毒素(zearalenone,ZEN)的方法。方法采用表面自组装技术(self-assembled monolayer,SAM)在金膜的表面修饰羧基基团,将ZEN抗原与牛血清白蛋白(albumin from bovine serum,BSA)偶联物(ZEN-BSA)通过共价键固定在芯片的表面,采用竞争法检测样品中的玉米赤霉烯酮毒素。结果该方法的检测限为8.2 ng/m L,ZEN单克隆抗体与呕吐毒素、黄曲霉毒素B_1、赭曲霉毒素、伏马毒素等没有交叉反应,与α-玉米赤霉烯醇和β-玉米赤霉烯醇交叉反应率分别为15.3%和11.5%。结论本方法具有简便、快速和高灵敏度等优势,在食品中真菌毒素的快速检测方面具有潜在的应用价值。     

A new strategy for the analysis of tetracycline residues in foodstuffs by a surface plasmon resonance biosensor

A new strategy was developed for the establishment of an indirect tetracycline assay using surface plasmon resonance (SPR). The principle for the assay is based on the most important resistance mechanism against tetracycline in gram-negative bacteria. Tetracyclines release the 46.6 kDa Tet repressor protein (TetR) from the tet operator (tetO), a biotinylated short double strand DNA sequence bound to a streptavidin biosensor chip. Tetracyclines present in a sample solution bind to the repressor protein, inducing a conformational change accompanied with a reduction of the affinity constant of TetR to tetO. We were able to quantify tetracycline residues in spiked raw milk samples in concentrations corresponding to the maximum residue limit (MRL) set by the European Union. Honey samples could also be analysed but with lower sensitivity. The assay detects seven of the most commonly used tetracyclines in veterinary medicine. Nine antibiotics of five other antibiotic classes were tested and no interference with the SPR assay was found. Thus, the described principle forms the basis for screening assays to routinely test for tetracycline residues in foodstuffs.     

Chemiluminescent imaging detection of staphylococcal enterotoxin C1 in milk and water samples

A sensitive, simple and rapid technique for high throughput simultaneous detection of staphylococcal enterotoxin C₁ (SEC₁) has been developed. The proposed method has the advantage of showing the specificity of enzyme-linked immunosorbent assays (ELISA), sensitivity of luminol-based enhanced chemiluminescence (ECL) assay, and high throughput of chemiluminescence (CL) imaging assay. It was based on a standard sandwich immunoassay format; 96-well ELISA plates were used as solid phase material. A commercial high-sensitivity cooled CCD camera has been applied to image the weak CL. Under the optimum conditions, the increased CL intensity was proportional with the concentration of SEC₁ in the range of 8.0–125.0 ng ml⁻¹ and the detection limit was 0.5 ng ml⁻¹ (3σ). The relative standard deviation (RSD) for eight parallel measurements of 25.0 ng ml⁻¹ SEC₁ was 0.06. The proposed method has been successfully applied to the determination of SEC₁ in milk and water samples. The results obtained compared well with those by ELISA.     

表面等离子共振技术在真菌毒素检测中的应用研究进展

真菌毒素污染的食品严重影响人类的健康,对真菌毒素的监测与防控是构建食品安全保障体系的重要一环。表面等离子共振(surface plasmon resonance,SPR)生物传感器以快速、免标记、高通量、高灵敏等优点,已广泛应用在药物筛选、食品检测、环境监测、临床诊断等领域。本文就SPR生物传感器在食品中真菌毒素快速筛查方面的应用研究进行了综述。