Biosensor analysis of beta-lactams in milk using the carboxypeptidase activity of a bacterial penicillin binding protein

The applicability of a beta-lactam receptor protein for detection of beta-lactam antibiotics in milk using surface plasmon resonance (SPR) biosensor technology was investigated. The advantage of using a receptor protein instead of antibodies for detection of beta-lactams is that a generic assay, specific for the active form of the beta-lactam structure, is obtained. Two assays based on the enzymatic activity of the DD-carboxypeptidase from Actinomadura R39 were developed, using a Biacore SPR biosensor. The carboxypeptidase converts a tri-peptide into a di-peptide, a reaction which is inhibited in the presence of beta-lactams. Polyclonal antibodies against the 2 peptides were developed and used to measure the amount of enzymatic product formed (di-peptide assay) or the amount of remaining enzymatic substrate (tri-peptide assay), respectively. The 2 assays showed similar performances with respect to detection limits (1.2 and 1.5 microg/kg, respectively) and precision (coefficient of variation <5%) for penicillin G in milk. Several other beta-lactams were detected at or near their respective maximum residue limit. Furthermore, the 2 peptide assays were evaluated against 5 commercial kit tests in the screening of 195 producer milk samples. The biosensor assays showed 0% false-negative and 27% false-positive results, whereas the figures were 0% false-negative and 27-53% false-positive results for other screening tests investigated.     

Semiempirical calculations of the hydrolysis of penicillin G

A theoretical study of the gas-phase alkaline hydrolysis of penicillin G on the assumption of a B_AC2 mechanism is reported. Various semiempirical methods were used to determine the influence of different parameterizations on the process. Among the most salient results obtained, the standard AM1 method predicted opening of the thiazolidine ring to yield the corresponding imine and enamine structures.     

Development of an optical biosensor assay for detection of β-lactam antibiotics in milk using the penicillin-binding protein 2x*

An assay was developed for the detection of residues of penicillins and cephalosporins in milk using a surface plasmon resonance (SPR) biosensor. The assay was based on the inhibition of the binding of digoxigenin-labelled ampicillin (DIG-AMPI) to a soluble penicillin-binding protein 2x derivative (PBP 2x*) of Streptococcus pneumoniae. Samples were incubated with PBP 2x* in a first step, whereby β-lactams in positive samples would bind to the PBP 2x*. Non-complexed PBP 2x* was then allowed to form a complex with DIG-AMPI in a second incubation step. The formed DIG-AMPI/PBP 2x*-complexes were detected in a SPR-based biospecific interaction assay (BIA) for digoxigenin with an antibody against digoxigenin immobilised on the sensor chip. Although binding of matrix components to the sensor chip (non-specific binding) occurred, benzylpenicillin, ampicillin, amoxicillin, cloxacillin, cephalexin and cefoperazone could be detected in defatted bulk raw milk samples at concentrations corresponding to the maximum residue limits (MRL) set by the European Union. The influence of matrix components on the performance of the assay was examined in more detail by analysing individual raw milk samples from 19 cows. Compared to bulk raw milk samples, individual samples showed a higher level and variation of matrix interferences. Non-specific binding could be reduced to a lower and more constant level by a heat-treatment step, a centrifugation step and the addition of carboxymethylated dextran to the samples. With this sample preparation, benzylpenicillin could be detected at MRL (4 μg kg⁻¹) in individual raw milk samples. Thus, the assay could be the basis for a screening test for routine use.     

Time-resolved fluoroimmunoassay for quantitative determination of ampicillin in cow milk samples with different fat contents

In this paper, we have reported an immunoassay with time-resolved revelation system for ampicillin in raw milk samples. Immunological methods appear to be a promising approach in the analysis of β-lactam compounds, because they do not need previous sample pre-treatments. In fact, β-lactam ring is not very stable in extensive sample pre-treatment procedures requested in conventional analytical techniques. Specimens were collected from lactating cows bred in various conditions and assayed for the fat contents. Ampicillin was assayed in samples with different fat concentrations. The assay was performed using ampicillin-specific polyclonal antibody raised in rabbit; the immunogen was synthesized using bovine thyroglobulin conjugated to ampicillin by glutaraldehyde reaction; as fluorescent marker we used goat anti-rabbit IgG conjugated with a chelating molecule complexed with Eu³⁺. Bovine serum albumin (BSA) conjugated with ampicillin was synthesized and used to prepare a solid phase on polystyrene microtiter plates. The use of a lanthanide chelate as label allowed to achieve 1 ng mL⁻¹ sensitivity, which is four times more sensitive than limits requested from European Community. Fat contents did not affect the assay performance.     

Biosensor for determination of hydrogen peroxide based on catalase activity of human erythrocytes

A hydrogen peroxide biosensor based on human erythrocytes is described. Erythrocytes are retained on the surface of an oxygen electrode by a semipermeable membrane. The response is based on the catalase activity of the erythrocytes. The sensitivity of 10^?4 mol 1^?1 and linearity from 1.5×10^?4 to 5×10^?3 mol^?1 are comparable to those of analogous enzyme biosensors for hydrogen peroxide determination. The greatest advantages of this biosensor are its easy preparation and a lifetime of 2 months together with good reproducibility (relative standard deviation <5%) and selectivity; only ascorbic acid appeared to interfere with the measurements.     

Sensitive monitoring of penicillin antibiotics in milk and honey treated by stir bar sorptive extraction based on monolith and LC‐electrospray MS detection

In the present study, a convenient and sensitive method for determination of six penicillin antibiotics (amoxicillin, ampicillin, penicillin G, oxacillin, cloxacillin, and dicloxacillin) in milk and honey samples was developed. Milk and honey samples were diluted with water, then directly treated by stir bar sorptive extraction based on poly (vinylimidazole‐divinylbenzene) monolithic material as coating. The analytes were analyzed by LC/ESI‐ MS/MS. Several extraction parameters including extraction and desorption time, pH value, and ionic strength in sample matrix were investigated in detail. Under the optimized extraction conditions, the calculated detection limits for the target compounds were as low as 0.23–2.66 ng/kg in milk and 0.18–1.42 ng/kg in honey, respectively. Good linearity was obtained for analytes with the correlation coefficients (R²) above 0.997. Excellent method reproducibility was achieved in terms of intraday and interday precisions, indicated by the RSDs of <5.0 and <10.0%, respectively. Finally, the proposed method was successfully applied to the determination of penicillin antibiotics residues in different milk and honey samples.     

TIRF-based biosensor for sensitive detection of progesterone in milk based on ultra-sensitive progesterone detection in water

We report on recent advances of our immunoassay for the hormone progesterone in cows milk. Detection is based on total internal reflectance fluorescence (TIRF), the binding-inhibition assay with an immobilized progesterone derivative, and a commercially available monoclonal antibody to progesterone as biological recognition element. The fully automated River Analyzer (RIANA) biosensor for unattended, cost-effective, and continuous monitoring of environmental pollution therefore was adapted for sensitive determination of progesterone in milk. First, the sensitivity and robustness of the existing progesterone assay for water analysis were improved, resulting in a detection limit (LOD) of only 0.2 pg mL^–1 and a quantification limit (LOQ) of only 2.0 pg mL^–1. These extraordinary results are the lowest detection and quantification limits for progesterone determination using biosensors yet reported in the literature. Second, the accurate indicator of ovulation was calibrated and detected in three different types of milk (UHT milk, fresh milk, and raw milk). For commercial milk and randomly procured raw milk nominal levels of progesterone are typically in the range 5–15 ng mL^–1. Limits of detection (LOD) achieved for added progesterone (i.e. spiked samples) were between 45.5 and 56.1 pg mL^–1 depending on milk type. Having in mind the 1:10 dilution factor, these results are still a success. For the first time a commercially available antibody was incorporated into an immunoassay for progesterone detection in bovine milk, giving a detection limit below 1 ng mL^–1 for a fully automated biosensor. Thus the outstanding progress made with this biosensor in environmental monitoring and water analysis has now been successfully adapted to milk analysis for use in the field of reproduction management.Dedicated to the memory of Wilhelm Fresenius.     

Biosensor based on laccase and an ionic liquid for determination of rosmarinic acid in plant extracts

Novel biosensors based on laccase from Aspergillus oryzae and the ionic liquids (ILs) 1-n-butyl-3-methylimidazolium hexafluorophosphate (BMIPF₆) and 1-n-butyl-3-methylimidazolium tetrafluoroborate (BMIBF₄) were constructed for determination of rosmarinic acid by square-wave voltammetry. The laccase catalyzes the oxidation of rosmarinic acid to the corresponding o-quinone, which is electrochemically reduced back to rosmarinic acid at +0.2 V vs. Ag/AgCl. The biosensor based on BMIPF₆ showed a better performance than that based on BMIBF₄. The best performance was obtained with 50:20:15:15% (w/w/w/w) of the graphite powder:laccase:Nujol:BMIPF₆ composition in 0.1 mol L⁻¹ acetate buffer solution (pH 5.0). The rosmarinic acid concentration was linear in the range of 9.99 × 10⁻⁷ to 6.54 × 10⁻⁵ mol L⁻¹ (r = 0.9996) with a detection limit of 1.88 × 10⁻⁷ mol L⁻¹. The recovery study for rosmarinic acid in plant extract samples gave values from 96.1 to 105.0% and the concentrations determined were in agreement with those obtained using capillary electrophoresis at the 95% confidence level. The BMIPF₆-biosensor demonstrated long-term stability (300 days; 920 determinations) and reproducibility, with a relative standard deviation of 0.56%.     

Monitoring of melamine contamination in fat watery milk by the photoluminescence analysis

A photoluminescence method to detect the toxic melamine contamination in fat watery milk has been proposed. Despite the quite different luminescence origins of milk and melamine patterns, their wide emission spectra under UV excitation are similar and in the range of 2.2-3.5 eV. The complex milk photoluminescence spectrum composed of riboflavin, furosine, lactulose, Vitamin E and tryptophan emitting species can be modified if milk pattern is undergone by acid treatment (for example, in vinegar). At the same time the melamine emission is not subjected to any modification in vinegar. It allows quantitatively discriminating the melamine contamination in milk in linear range, at least, 0.05-7 g/l from different photoluminescence spectra of milk (water) with and without melamine. Limit of melamine detection achieves 0.01 g/l.     

Simultaneous determination of penicillin and ampicillin by spectral fibre-optical enzyme optodes and multivariate data analysis based on transient signals obtained by flow injection analysis

A multicomponent detection system using optical biosensors and flow injection analysis is described. The analysis of mixtures containing penicillin and ampicillin was realised by evaluating dynamic measurements of Phenol Red spectra in penicillinase optodes in combination with a diode array spectrometer. A variety of optodes has been produced by changing the composition of the receptor gel and the working pH. A set of characteristic quantities (describing dynamic and static features) could be obtained for each optode. These were used to compare the predictivity of classical multivariate calibration methods as well as of an artificial neural network. In addition, different algorithms were applied for the evaluation of the spectral data in order to select the most appropriate method for feature extraction. In consequence, the information obtained from the multivariate calibration models was used to set up an optimal sensor array consisting of four optodes with different types of penicillinase at different working pH.     

利用溶胶-凝胶生物传感器测定饱和苯系物废水中酚类化合物

研制了用溶胶-凝胶包埋酪氨酸酶碳糊生物电极。其生物电极的工作条件为:电位为-100mV(vs.SCE),溶液pH5.40,响应时间为3min。本文方法对苯酚的检出限为1.00×10-6mol/L,线性范围为1.00×10-6~1.00×10-4mol/L,相对标准偏差为1.04%。实验结果表明,本电极对邻甲酚、对苯二酚、邻苯二酚、对氯苯酚都有良好的响应,但对邻氨基酚、间苯二酚、对甲苯酚、邻硝基酚、2.4二甲基酚响应较差。用本法对化工废水中酚类进行了测定,有机干扰物苯、甲苯、二甲苯对测定结果无影响。     

A rapid and simple HPLC-UV method for the determination of inhibition characteristics of farnesyl transferase inhibitors

Ras proteins play an important role in the development of cancer. Farnesyl transferase inhibitors (FTIs) block the first obligatory post-translational step for activation, prenylation, of Ras proteins. To find new potent FTIs, rapid enzyme activity assays are required to reduce FTI development time. Most assays to date are based on radioactive labelled substrates. We developed a new, in vitro, farnesyl transferase assay based on gradient chromatography coupled to UV detection. Unfarnesylated and farnesylated H-Ras proteins were resolved on a C18 wide-pore HPLC column and their concentrations were determined with use of a calibration curve of unfarnesylated H-Ras. The assay was used to investigate inhibition characteristics of FTIs. The IC50 values of the FTIs L778,123 and SCH66336 were 4.2 nm and 78 microm, respectively. This assay could support the screening and development of FTIs to obtain rapid insights into their inhibitory properties.     

Evaluation of matrix effect in isotope dilution mass spectrometry based on quantitative analysis of chloramphenicol residues in milk powder

In the present study, we developed a comprehensive strategy to evaluate matrix effect (ME) and its impact on the results of isotope dilution mass spectrometry (IDMS) in analysis of chloramphenicol (CAP) residues in milk powder. Stable isotope-labeled internal standards do not always compensate ME, which brings the variation of the ratio (the peak area of analyte/the peak area of isotope). In our investigation, impact factors of this variation were studied in the extraction solution of milk powder using three mass spectrometers coupled with different ion source designs, and deuterium-labeled chloramphenicol (D5-CAP) was used as the internal standard. ME from mobile phases, sample solvents, pre-treatment methods, sample origins and instruments was evaluated, and its impact on the results of IDMS was assessed using the IDMS correction factor (θ). Our data showed that the impact of ME of mobile phase on the correction factor was significantly greater than that of sample solvent. Significant ion suppression and enhancement effects were observed in different pre-treated sample solutions. The IDMS correction factor in liquid–liquid extraction (LLE) and molecular imprinted polymer (MIP) extract with different instruments was greater or less 1.0, and the IDMS correction factor in hydrophilic lipophilic balance (HLB) and mix-mode cation exchange (MCX) extract with different instruments was all close to 1.0. To the instrument coupled with different ion source design, the impact of ME on IDMS quantitative results was significantly different, exhibiting a large deviation of 11.5%. Taken together, appropriate chromatographic conditions, pre-treatment methods and instruments were crucial to overcome ME and obtain reliable results, when IDMS methods were used in the quantitative analysis of trace target in complex sample matrix.     

利用流动注射型乙酰胆碱酯酶传感器监测海水中马拉硫磷

将乙酰胆碱酯酶(AChE)传感器引入流动注射系统中，研究了传感器连续监测海水中马拉硫磷的可行性。系统中的载液为不含马拉硫磷的海水。传感器适宜的工作条件为：载液和样品的流速为0．39mL／min，进样时间为20min；底物(碘化硫代乙酰胆碱)溶液的浓度为41．6mmol／L，注射量为40μL。利用次氯酸钠溶液对含马拉硫磷的海水样品进行预氧化处理，可以大大提高传感器的灵敏度，对马拉硫磷的检出限达到0．05μg/L；而不进行氧化时的检出限为1．3μg/L。另外，海水样品经过预氧化处理后，传感器对其中0．1-10μg／L的马拉硫磷具有良好的线性响应关系(r=0．991)。测定含马拉硫磷的海水样品后，向传感器持续通入0．5mmol／L的2-PAM溶液15min，可以完全恢复受到100μg／L马拉硫磷抑制的固定化酶活性。结果表明：所设计的酶传感器适于海水中马拉硫磷的连续、灵敏和准确监测。     

Biosensor for determination of carboxylic acids in wines based on the inhibition of sarcosine oxidase

The flow-through amperometric biosensor is presented for determination of carboxylic acids. It is based on two sensor layers that are deposited on a platinum electrode. The inner layer serves to eliminate interferences by limiting diffusion of electrochemically active substances such as ascorbic acid. This layer is electro-polymerized using an equimolar mixture of o-phenylenediamine and resorcinol. The outer layer is prepared by cross-linking the enzyme sarcosine oxidase and bovine serum albumin using glutaraldehyde. The formation of enzymatically produced hydrogen peroxide is monitored at 600 mV vs. an Ag/AgCl reference electrode. The addition of carboxylic acids causes competitive inhibition of the enzyme and a decrease in signal. The assay was optimized for determination of carboxylic acids in wine samples. Following 10-fold dilution, most samples contain 1–10 mM individual carboxylic acids and thus a 5 mM concentration of sarcosine was chosen as being optimal for competition. In case of real samples, the biosensor measures the sum of all carboxylic acids, which serves as a parameter describing the quality of wines. Results from testing several wine samples are reported.     

Visual detection of melamine in milk samples based on label-free and labeled gold nanoparticles

Melamine that can cause serious damage to the organs of animal or human beings was found to bind to polythymine via hydrogen bonding. With this novel discovery, colorimetric detection of melamine based on label-free and labeled gold nanoparticles was developed, respectively. Both of the methods revealed good selectivity for melamine over other components that may exist in milk and good anti-influence ability. The raw milk samples were pretreated according to the National standard method combined with a solid phase extraction monolithic column. The accurate quantification of melamine as low as 41.7 nM and 46.5 nM was obtained, respectively. It also guarantees fast and reliable readout with naked eyes, making visual detection possible. Further comparison between label-free and labeled based methods was discussed in this paper.     

Construction of a simple optical sensor based on air stable lipid film with incorporated urease for the rapid detection of urea in milk

This work describes the construction of a simple optical sensor for the rapid, selective and sensitive detection of urea in milk using air stable lipid films with incorporated urease. The lipid film is stabilized on a glass filter by polymerization using UV (ultra-violet) radiation prior its use. Methacrylic acid was the functional monomer, ethylene glycol dimethacrylate was the crosslinker and 2,2′-azobis-(2-methylpropionitrile) was the initiator. Urease is incorporated within this mixture prior to the polymerization. The presence of the enzyme in these films quenched this fluorescence and the colour became similar to that of the filters without the lipid films. A drop of aqueous solution of urea provided a “switching on” of the fluorescence which allows the rapid detection of this compound at the levels of 10⁻⁸ M concentrations. The investigation of the effect of potent interferences included a wide range of compounds usually found in foods and also of proteins and lipids. These lipid membranes were used for the rapid detection of urea in milk.     

Influence of the fat characteristics on the physicochemical behavior of oil-in-water emulsions based on milk proteins-glycerol esters mixtures

Oil-in-water emulsions based on 10% milk protein preparation, 0.3% mono-di-glycerides (MDG) and 8% vegetable oil were prepared for models typifying ice cream formulations. Two MDG (saturated and partially unsaturated) and four fats (oleic oil, hydrogenated and refined coconut oils, refined palm oil) were chosen to investigate the interactions occurring between the oil phase, the MDG and the milk proteins. Influence of temperature (4 °C) and ageing (24 h at 4 °C) was also tested. The emulsions were characterized for protein desorption, particle size distribution and rheological properties. The dynamic surface activity of the milk proteins and the MDG at the oil-water interface was also determined. At 20 °C, emulsions were mostly stabilized by proteins although the protein load at the globule surface strongly depended on the emulsifier and the oil phase natures. A displacement of the proteins adsorbed at the oil droplet interface by the lipid surfactant was a consequence of the temperature decrease and/or ageing step, suggesting a disruption of the interfacial protein interactions. This disruption was more or less marked depending on the physicochemical characteristics of the surfactant and the oil used (amount of crystallized matter, fatty acid chain length and unsaturation degree). In parallel, the variation of the apparent viscosity of the various emulsions upon temperature was well correlated with the solid fat content. On the whole, the results obtained suggested that not only the surfactant molecules, i.e. emulsifiers and proteins, but also the fat used in the emulsion formulation participated in the development of the interface characteristics and rheological properties.     

Amperometric Enzyme‐Based Biosensor for Direct Detection of Formaldehyde in the Gas Phase: Dependence on Electrolyte Composition

An enzymatic sensor detecting the analyte formaldehyde directly from the gas phase is under investigation. In contrast to existing systems, it enables the quantification of the analyte without prior sampling or accumulation and thus can be used as an online system to monitor the formaldehyde concentration in ambient air. The amperometric sensor depends on the enzymatic conversion of the analyte using formaldehyde dehydrogenase from P. putida [EC. 1.2.1.46] as the recognition element. It shows a linear response curve up to 15 ppm, with a detection limit of 0.03 pm (S/N=3). In order to optimize the sensor performance the electrolyte composition within the sensor was varied with respect to pH value, buffer concentration and the addition of Ca²⁺ and Mg²⁺ ions. To elucidate the influence of the mediator and the enzyme on the sensor performance the stability and activity of the electrochemical mediator and the enzyme alone was examined separately in these different electrolytes.     

Dipstick based immunochemiluminescence biosensor for the analysis of vitamin B12 in energy drinks: A novel approach

In this article, we describe a dipstick based immunochemiluminescence (immuno-CL) biosensor for the detection of vitamin B₁₂ in energy drinks. The method is a direct competitive type format involving the immobilization of vitamin B₁₂ antibody on nitrocellulose membrane (NC) followed by treatment with vitamin B₁₂ and vitamin B₁₂–alkaline phosphatase conjugate to facilitate the competitive binding. The dipstick was further treated with substrate disodium 2-chloro-5-(4-methoxyspiro {1,2-dioxetane-3,2¢-(5¢-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)-1-phenyl phosphate (CDP-Star) to generate chemiluminescence (CL). The number of photons generated was inversely proportional to the vitamin B₁₂ concentration. After systematic optimization, the limit of detection was 1 ng mL⁻¹. The coefficient of variation was below 0.2% for both intra- and inter-assay precision. Vitamin B₁₂ was extracted from energy drinks with recovery ranged from 90 to 99.4%. Two different energy drinks samples were analyzed, and a good correlation was observed when the data were compared with a reference enzyme linked immuno sorbent assay (ELISA) method. The developed method is suitable for an accurate, sensitive, and high-throughput screening of vitamin B₁₂ in energy drinks samples. The dipstick technique based on immuno-CL is suitable for the detection of several analyte in food and environmental samples.