Direct optical biosensor analysis of folate-binding protein in milk

An automated, rapid, sensitive, and label-free biosensor-based assay for folate-binding protein (FBP) in bovine milk utilizing surface plasmon resonance optical detection is described. The active concentration of FBP is estimated from its specific interaction with a pteroyl-l-glutamic (folic) acid derivative immobilized on the sensor surface in a direct binding assay format. Milk, colostrum, and milk powders are prepared for analysis by dilution into buffer. Analysis conditions, including ligand immobilization, flow rate, contact time, and regeneration, have been defined, and nonspecific binding considerations were evaluated. Performance parameters include a working range for FBP in buffer of 0-200 ng/mL, a method detection limit of 0.13 microg/mL in fluid milk, overall instrument response RSD(R) of 0.64%, a mean interassay RSD(R) of 7.3% for skim milk powder, and surface stability over ca. 200 samples. The technique was applied to the measurement of active FBP content of consumer milks, the heat classification of skim milk powders manufactured under a wide range of thermal processing protocols, and change during early bovine lactation.     

Luminex-based triplex immunoassay for the simultaneous detection of soy, pea, and soluble wheat proteins in milk powder

An automated fluorescent microsphere-based flow cytometric triplex immunoassay, using the Luminex 100 flow analyzer with MultiAnalyte Profiling (xMAP) technology, was developed for the simultaneous detection of proteins from three vegetable sources as potential fraudulent adulterants in milk powder. In the final triplex inhibition immunoassay, soluble wheat proteins (SWP) and proteins from soy and pea were coupled to three different microsphere sets. A mixture of these microsphere sets was transferred to a microtiter plate well together with the sample and a mixture of three affinity-purified polyclonal antibodies raised against the proteins and labeled with a fluorophore (Alexa 532). After incubation for 1.5 h at room temperature in the dark, the fluorescence intensities on the microspheres were directly measured (no wash procedure) in the Luminex during 10 s per well (100 microspheres per set). The sensitivities of the three assays for plant protein extracts were determined as 0.5-0.6 microg/mL at 50% inhibition. For the detection of the vegetable proteins in milk powder, the samples were dissolved in buffer (0.1 g in 10 mL) and further diluted (20 times) to create a 50% inhibition at approximately 0.5% of the vegetable proteins in the total protein content of milk powder. With the help of calibration standards, prepared under conditions comparable to those for sample materials, the triplex immunoassay proved to be quantitative above 0.1%, although concentrations in high-heated milk powders were underestimated. Due to the xMAP technology, in which 100 different microsphere sets can be distinguished, this triplex immunoassay can easily be extended to detect other possible adulterants.     

Determination of choline in milk, milk powder, and soy lecithin hydrolysates by flow injection analysis and amperometric detection with a choline oxidase based biosensor

A fast-response and interference-free amperometric biosensor based on choline oxidase immobilized onto an electropolymerized polypyrrole film for flow injection determination of choline in milk, milk powder, and soy lecithin hydrolysates is described. The sensor displayed an Imax value of 1.9 +/- 0.2 microA and an apparent Michaelis-Menten constant, k'M, equal to 1.75 +/- 0.07 mM. Detection limits of 0.12 microM could be obtained. Because even a slight deterioration of the anti-interference membrane can adversely affect measurement accuracy, a real time monitoring of the biosensor selectivity has been achieved by a dual Pt electrode flow-through cell where the enzyme modified electrode is coupled to an enzyme-free electrode in a parallel configuration. Finally, bracketing technique (alternate injections of sample and standards) allows a two-point calibration to be performed in real-time, correcting for any drift in sensor response.     

Disposable biosensor test for organophosphate and carbamate insecticides in milk

A highly sensitive and rapid biosensor test based on disposable screen-printed thick-film electrodes was developed, which is suitable for monitoring organophosphate and carbamate residues in foods of animal origin with increased fat contents such as milk. The wild-type enzyme was combined with three engineered variants of Nippostrongylus brasiliensis acetylcholinesterase (NbAChE), to obtain enhanced sensitivity. The sample pretreatment could be reduced to a minimum. There was no extraction or fat removal necessary. With the biosensor test paraoxon concentrations down to 1 microg/L could be detected in milk. The detection limit for carbaryl was 20 microg/L. Recovery rates for paraoxon and carbaryl in milk samples lay between 89 and 107%. Ten milk samples from local markets were tested both with the biosensor test and with standard chromatographic multiresidue methods. Two milk samples caused AChE inhibition rates of >50%. Accordingly, 4 microg/L tebufenpyrad, 4 microg/L tetraconazole, and 2 microg/L bifenthrin were detected in one of these milk samples. The other milk sample contained 2 microg/L tebufenpyrad.     

智舌对奶粉中抗生素残留的检测研究

本文利用智舌对奶粉中相同浓度的六种抗生素进行了辨识,并对新霉素检测浓度进行了初步研究。采用铂、金、钯、钨、钛和银6个电极组成的传感器阵列和1、10和100Hz三个脉冲频率进行检测,并通过主成分分析、线性判别分析和偏最小二乘法进行数据分析。结果显示：智舌对不同种抗生素和不同浓度的新霉素具有较好的辨识能力,定性分析能够达到国家最高残留限量标准;利用PLS建立模型定量分析,新霉素最适检测浓度范围在200-1000ug/kg附近。     

检测食品沙门氏菌的生物传感器持久性研究

为了研究温度对生物传感器检测的持久性的影响,制备了用于检测食品中沙门氏菌的磁致伸缩生物传感器,以磁致伸缩膜片作为物理传感器,多克隆抗体作为生物识别元件,采用Langmuir-Blodgett(LB)技术将多克隆抗体固定在磁致伸缩膜片表面。当食品中沙门氏菌吸附在生物传感器上时,将引起其共振频率漂移。通过测试与分析磁致伸缩生物传感器共振频率漂移值,并利用扫描电子显微镜(scanning electron microscope)观察吸附了沙门氏菌的生物传感器表面,对生物传感器在25(室温)、45及65℃的持久性进行研究。结果表明:多克隆抗体磁致伸缩生物传感器与沙门氏菌的结合能力随着时间的延长逐渐降低;且温度越高,传感器的持久性越差;在25、45及65℃时,多克隆抗体磁致伸缩生物传感器的持久期分别为30、8和5 d,由此获得了多克隆抗体生物传感器在常用温度下的持久性。并结合阿伦尼乌斯方程,计算得到该生物传感器的激活能为13.024 kJ/mol。进一步证实了磁致伸缩生物传感器可用于定量检测实际溶液中沙门氏菌的浓度,表明生物传感器可应用于食品中细菌的实时快速定量检测。     

线扫描式拉曼高光谱成像技术无损检测奶粉三聚氰胺

为了实现颗粒状样本的大面积无损快速检测,该研究结合拉曼光谱和高光谱技术搭建了一套线扫描式拉曼高光谱检测系统,对奶粉和三聚氰胺颗粒混合样本进行了检测研究。研究通过高斯窗平滑法和air PLS基线校正方法分别消除了拉曼光谱中的噪声信号和荧光背景,选取三聚氰胺主要特征峰(671.71 cm-1)处的单波段图像作为是否含有三聚氰胺颗粒的判断依据。研究首先对三聚氰胺产生的拉曼信号在奶粉颗粒中的穿透深度进行了检测,随后完成了10种不同浓度的三聚氰胺奶粉混合样本的拉曼高光谱采集,对特征单波段图像中各像素点的拉曼强度平均值进行一元线性分析,并对单波段图像进行二值化处理。结果显示,在三聚氰胺特征单波段图像中,感兴趣区域内所有像素点的拉曼强度平均值与三聚氰胺浓度之间线性度较高,其决定系数R2达到了0.995 4。在二值图像中,三聚氰胺颗粒的位置信息能够直观的展现。研究结果表明,拉曼高光谱成像技术具有快速、无损和大面积检测的特点,在实际应用中具有巨大潜力。     

Boron doped diamond biosensor for detection of Escherichia coli

o-Nitrophenol, released from o-nitrophenyl-beta-D-galactopyranose as catalyzed by beta-galactosidase, a tetramer of Escherichia coli, has been exploited for the detection of E. coli contamination in foodstuffs. This reaction was detected using a boron doped diamond electrode poised at +0.93 V, without any surface modification. The enzyme was effectively induced by isopropyl-beta-D-thiogalacto-pyranoside with the maximum enzyme activity observed with sodium dodecyl sulfate at 50 degrees C. A biphasic calibration plot was observed: 4 x 10(4) to 2 x 10(5) cells/mL and 2 x 10(5) to 6 x 10(6) cells/mL for the first and second region, respectively. The detection limit was 4 x 10(4) cells/mL with a total analysis time of <1.5 h. Electrode fouling was easily overcome by approximately 40 rapid CV scans, and the method was applicable for detecting E. coli in artificially spiked samples of beef, pork, chicken, milk, and tap water.     

Determination of beta-lactams in milk using a surface plasmon resonance-based biosensor

Two surface plasmon resonance (SPR)-based biosensor assays for detection of beta-lactam antibiotics in milk are reported. The assays are based on the enzymatic activity of a carboxypeptidase converting a 3-peptide into a 2-peptide, a reaction that is inhibited in the presence of beta-lactams. Antibodies were used to measure either the amount of formed enzymatic product or the amount of remaining enzymatic substrate. Both assays detected different beta-lactams at or below European maximum residue limits (MRLs), and the detection limit for penicillin G was 1.2 microg/kg and 1.5 microg/kg for the 2- and 3-peptide assays, respectively. The precision (CV) was < 5%, both within and between assays at the penicillin G MRL (4 microg/kg). The biosensor results obtained upon analysis of incurred milk samples were compared with results obtained by liquid chromatography (HPLC), and the method agreements were, in general, good.     

Direct competitive enzyme-linked immunosorbent assay for sulfamethazine residues in milk

A direct competitive enzyme-linked immunosorbent assay (ELISA) is described for the detection and estimation of sulfamethazine residues in milk. Samples are cleaned up rapidly by acidifying and centrifuging the milk, adjusting the supernatant liquid to pH 7.0, and centrifuging again. The supernate is then assayed using set points to estimate sulfamethazine levels in the sample in the range of 1 ppb to 1 ppm. Multiple samples of milk can be screened in 1.5-2 h by this ELISA method.     

Development of milk powder reference materials certified for aflatoxin M1 content (Part II): Certification of milk powder RM 283

The development of a full cream milk powder reference material, certified for its aflatoxin M1 content (target concentration: 0.1 microgram/kg), is described. The material (RM 283) was prepared and certified within the Reference Material Programme of the Community Bureau of Reference, along with other members of a series of milk powder reference materials. Homogeneity, evaluated by determining the aflatoxin M1 content of 30 units, was found to be acceptable (coefficient of variation of analysis results: 9.1%); stability has been demonstrated in a long-term study. The certification exercise involved 7 laboratories. Calibration, control of recoveries, blank values, and independence of the replicate measurements were emphasized. All sets of results of the certification exercise were accepted for statistical evaluation. A certified value for the aflatoxin M1 content: 0.09(+0.04)(-0.02) micrograms/kg was derived. The certification of RM 283 completes the series of 4 milk powder reference materials having certified aflatoxin M1 contents.     

Detection and identification of beta-lactam residues in milk using a hybrid biosensor

A novel application of a hybrid biosensor is here employed as an analytical method for the detection and presumptive identification of beta-lactam residues in milk. The method is based on measurements of carbon dioxide (CO2), the production of which is related to the microbial growth of the test microorganism Bacillus stearothermophilus var. calidolactis. The presence of beta-lactams in milk inhibits microbial growth and, consequently, the CO2 production rate. The analysis is based on the variation of CO2 between a milk sample spiked with beta-lactams and a twin milk sample containing beta-lactams plus a broad spectrum beta-lactamase, using an electrochemical device of biosensor. A blank milk sample is included as control. The result is obtained starting from the first 120 min. Moreover, the ability to recognize all of the beta-lactams speeds the total time of analysis when chemical identification and quantification are required. The analytical method appears to be adequate for milk control for qualitative screening purposes, complying with the requirements stated in Decision 2002/657/EC.     

Heat-induced redistribution of disulfide bonds in milk proteins. 1. Bovine beta-lactoglobulin

Changes in the structure and chemistry of beta-lactoglobulin (beta-LG) play an important role in the processing and functionality of milk products. In model beta-LG systems, there is evidence that the aggregates of heated beta-LG are held together by a mixture of intermolecular non-covalent association and heat-induced non-native disulfide bonds. Although a number of non-native disulfide bonds have been identified, little is known about the initial inter- and intramolecular disulfide bond rearrangements that occur as a result of heating. These interchange reactions were explored by examining the products of heat treatment to determine the novel disulfide bonds that form in the heated beta-LG aggregates. The native protein and heat-induced aggregates were hydrolyzed by trypsin, and the resulting peptides, before and after reduction with dithiothreitol, were separated by high-performance liquid chromatography and their identities confirmed by electrospray ionization mass spectrometry. Comparisons of these peptide patterns showed that some of the Cys160 was in the reduced form in heated beta-LG aggregates, indicating that the Cys160-Cys66 disulfide bond had been broken during heating. This finding suggests that disulfide bond interchange reactions between beta-LG non-native monomers, or polymers, and other proteins could occur largely via Cys160.     

检测有机磷农药残留生物传感器的温度特性

为了考察检测有机磷农药残留用量热式生物传感器的温度特性,本文采用分光光度法研究了固定化鸡肝酯酶的活力、农药敌敌畏对鸡肝酯酶的抑制等与温度的关系。在生物传感器的实测条件下,研究了不同温度固定化酶活力的操作流失情况。研究显示,温度对酶反应的影响很大,控温精度的提高将增强检测的精度和稳定性。温度55～60℃时,是酶反应速度最快的区域;抑制时间5 min时,农药敌敌畏对鸡肝脂酶产生了明显的抑制作用。在农药浓度小于1 mg/L的区域,随浓度变化的相对抑制关系接近于线性;一定浓度的农药敌敌畏对酶的抑制程度随着温度的变     

A group-specific microbiological test for the detection of tetracycline residues in raw milk

The potentiality of using a luminescent Escherichia coli strain for the specific detection of tetracycline residues in raw bovine milk was investigated. The sensor cells contain a reporter plasmid carrying the bacterial luciferase operon of Photorhabdus luminescens under the control of the tetracycline responsive control region from transposon Tn10. Incubation of the cells with the sample containing tetracyclines increases the light emission of the sensor cells. The most sensitive tetracycline detection was achieved in 120 min and by using CDTA as a chelating agent in the assay. Heat-treatment of milk before the assay decreased the variations in background luminescence signals and in tetracycline-induced luminescence between different milk samples. The detection limits for tetracycline, oxytetracycline, chlortetracycline, doxycycline, methacycline, demeclocycline, and minocycline were between 2 and 35 ng/mL. Nontetracycline antibiotics did not significantly interfere with the detection of tetracyclines.     

Reliable enzyme-linked immunosorbent assay for the determination of coconut milk proteins in processed foods

This study was designed to develop a novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of coconut milk proteins in processed foods. The developed sandwich ELISA was able to detect coconut milk proteins from various coconut milk products and did not show any cross-reactivity with 41 of 42 kinds of popularly used food ingredients, thus reflecting great specificity for coconut milk proteins. In addition, the established ELISA is highly sensitive and allowed the detection of 0.31 μg/g of coconut milk protein in complex food matrices. This proposed assay could serve as a useful tool for the detection of the presence of hidden coconut milk proteins in processed foods.     

Changes in structures of milk proteins upon photo-oxidation

Changes in protein structures as a result of riboflavin-induced photo-oxidation were studied for six milk proteins: alpha-casein, beta-casein, kappa-casein, lactoferrin, alpha-lactalbumin, and beta-lactoglobulin. The milk proteins showed significant variability in sensitivity to photo-oxidation. After photo-oxidation, an increase in carbonyl content because of oxidation of tryptophan, histidine, and methionine, as well as formation of dityrosine, was observed for all proteins studied, although at very different levels. Generally, the increment was highest for alpha- and beta-casein and was lowest for lactoferrin. Loss of tryptophan because of photo-oxidation was well-correlated with the formation of the tryptophan oxidation products, N-formylkynurenine and kynurenine. Changes at the tertiary protein structure level were observed after photo-oxidation of the globular proteins, where tryptophan fluorescence emission indicated unfolding of alpha-lactalbumin and beta-lactoglobulin, whereas lactoferrin achieved a more compact tertiary structure. Changes in secondary structure were observed for alpha-lactalbumin and beta-lactoglobulin, whereas the secondary structure of lactoferrin did not change. Polymerization of alpha- and beta-casein and of lactoferrin was observed, whereas kappa-casein, alpha-lactalbumin, and beta-lactoglobulin showed little tendency to polymerize after photo-oxidation. Lability toward photo-oxidation is discussed according to the structural stabilities of the globular proteins.     

Front-face fluorescence spectroscopy allows the characterization of mild heat treatments applied to milk. Relations with the denaturation of milk proteins

Emission and excitation spectra of intrinsic fluorophores present in milk were used to evaluate changes in milk following thermal treatments in the 57-72 degrees C temperature range from 0.5 min up to 30 min. Alternatively, the concentrations of native alkaline phosphatase, lactoferrin, immunoglobulin G, bovine serum albumin, beta-lactoglobulin, and alpha-lactalbumin were determined in the same samples by enzymatic and immunochemical techniques. As principal component analysis applied to the normalized fluorescence spectra successfully discriminated different milk samples according to the temperature and time of thermal treatment, principal component regression was applied to predict the amounts of the native proteins investigated using fluorescence data. The results showed strong correlations between measured and predicted data for alkaline phosphatase and beta-lactoglobulin. This study has demonstrated that front-face fluorescence spectroscopy has a promising potential to become a rapid and nondestructive analytical technique for the evaluation of physicochemical changes in milk induced by low thermal treatment.     

Portable surface plasmon resonance immunosensor for the detection of fluoroquinolone antibiotic residues in milk

An inexpensive and portable surface plasmon resonance (SPR) sensor, SPReeta Evaluation Kit SPR3, has been used to develop a biosensor for the determination of fluoroquinolone antibiotics (FQs) and to demonstrate its performance analyzing FQ residues in milk samples. The SPReeta three-channel gold chips were activated with a mixed self-assembled monolayer (m-SAM) and functionalized with a FQ haptenized protein. Binding of the antibody produced a concentration-dependent increase of the SPR signal as a result of the change in the refraction index. Similarly, the presence of the FQ produced a dose-dependent decrease of the response, which allowed a good limit of detection (LOD) to be obtained (1.0 ± 0.4 μg L(-1) for enrofloxacin in buffer). The response was reproducible in all three channels, on different injections and days, and also between chips. Milk samples could be analyzed after a simple sample treatment involving fat removal by centrifugation and dilution with water. Under these conditions calibration curves were obtained showing that FQ residues can be analyzed in milk samples with an IC(50) value of 26.4 ± 7.2 μg L(-1) and a LOD of 2.0 ± 0.2 μg L(-1) (for enrofloxacin), far below the European Union regulations for this antibiotic family in this matrix. Finally, the paper also demonstrates that the biosensor is able to selectively detect the presence of FQs in milk samples, even in the presence of other antibiotics. Enrofloxacin, ciprofloxacin, and norfloxacin residues were detected in blind samples supplied by Nestle? Co.     

用于农药残留快速检测的生物传感器灵敏度筛选试验(简报)

为了提高乙酰胆碱酯酶生物传感器的灵敏度,使其更有效地应用于有机磷农药的快速检测。以商品乙酰胆碱酯酶（家蝇）和自制的鸡肝酶、鸡脑酶、鲤鱼脑酶为研究对象,比较了这4种乙酰胆碱酯酶的活性;检测了不同浓度的氧乐果、敌百虫和敌敌畏对4种酶的酶活抑制率。结果表明,鸡脑乙酰胆碱酯酶活性抑制率最高,3种农药对其的抑制率分别为：敌敌畏100 μg/L的抑制率为54.1%,敌百虫100 μg/L的抑制率为50.56%,氧化乐果100 μg/L的抑制率为24.16%。鸡脑酶廉价易得,且易于纯化,与自制的鸡肝酶和鲤鱼脑酶相比,乙酰胆碱酯酶的活性抑制率显著较高。因此鸡脑酶可以作为农产品农药残留快速检测用酶。