Kairi Kivirand

Biosensors for Biogenic Amines: The Present State of Art Mini-Review

The analysis of biogenic amines in fresh and processed food is of great importance not only for the potential risk these compounds have on human health, but also because these amines can perform as chemical indicators of food quality and enable the assessment of food processing conditions and/or microbial contamination. A good option for a rapid detection of biogenic amines is the application of biosensors, as these devices enable the obtainment of results in a few minutes without pretreatment of the analyzed material. Biosensors for biogenic amines comprise various combinations of different enzymes for selective biorecognition and signal transduction systems and are based on different signal mechanisms. The present review gives a condensed overview of the recent developments and issues in the construction of biosensors for the detection of most common biogenic amines found in food and other protein-containing material.

Interference of the Simultaneous Presence of Different Biogenic Amines on the Response of an Amine Oxidase–Based Biosensor

Abstract The application of an amine oxidase–based biosensor, which was selective for several biogenic amines, was examined for the simultaneous detection of cadaverine, putrescine, and histamine. The biosensor response parameters in mixtures containing cadaverine or putrescine were 1.14 times less than the sum of corresponding parameters in single-substrate solutions. Histamine did not cause any change of the biosensor response in the presence of cadaverine and/or putrescine. This “screening” of biosensor signal parameters by putrescine and cadaverine should be considered during calibration of amine oxidase–based biosensors used for the detection of biogenic amines.

Analyzing the biosensor signal in flows: Studies with glucose optrodes

Responses of enzymatic bio-optrodes in flow regime were studied and an original model was proposed with the aim of establishing a reliable method for a quick determination of biosensor signal parameters, applicable for biosensor calibration. A dual-optrode glucose biosensor, comprising of a glucose bio-optrode and a reference oxygen optrode, both placed into identical flow channels, was developed and used as a model system. The signal parameters of this biosensor at different substrate concentrations were not dependent on the speed of the probe flow and could be determined from the initial part of the biosensor transient phase signal, providing a valuable tool for rapid analysis. In addition, the model helped to design the biosensor system with reduced impact of enzyme inactivation to the system stability (20% decrease of the enzyme activity lead to only a 1% decrease of the slope of the calibration curve) and hence significantly prolong the effective lifetime of bio-optrodes.

Biosensors for the detection of antibiotic residues in milk

Milk and dairy products are important nutrients for all age groups. However, the use of antibiotics for the treatment of food-producing animals generates the risk to human health, as these compounds and their metabolites can be transferred into milk. Rapid testing of the presence of antibiotics in raw milk to grant its quality has become a major task for farmers and dairy industry. The conventional analytical methods are either too slow or do not enable quantitative detection of antibiotic residues, so alternative methods that are rapid, cost effective, and easy to perform should be considered. The present chapter gives an overview of the recent developments and issues of the construction of different biosensors for the detection of antibiotic residues in milk.

Signal Analysis and Calibration of Biosensors for Biogenic Amines in the Mixtures of Several Substrates

In real life we should often conduct analyses, where several compounds with similar properties are simultaneously present in solutions. In traditional analytical chemistry, this problem is usually solved by the pre-treatment of probes, enabling to eliminate the interference effect of different compounds. This process requires skilled labour, resources and time and eliminates the possibility to carry out on-line analyses. A promising option for the conduction of on-line analyses is the application of biosensors, which are considered to provide reliable results, at least concerning the issues of selectivity. As biosensors are based on a selective bio-recognition of assessable compounds, there are typically only a restricted number of molecules (besides the ones of the analyte) present in a probe, which can induce measurable signals. However, in cases we have several competing compounds which can generate identical measurable effects, the selectivity of a biosensor can be quite poor and the results illusory. This phenomenon occurs clearly in the studies with enzyme inhibition-based biosensors (Luque de Castro & Herrera, 2003), but can be also well observed with biosensors, based on enzymes having activity towards several substrates, e.g. biosensors measuring biogenic amines (Kivirand & Rinken, 2009), different sugars etc. Biogenic amines (BAs) are natural nitrogenous compounds formed mainly in the process of decarboxylation and aging of free amino acids. The detection of these compounds is a valuable tool for assessing the freshness and quality of a wide variety of protein-containing products like fish, meat, cheese, wine etc. (Yano et al, 1996;Vinci & Antonelli, 2002;Onal, 2007). The most common biogenic amines, used for the indication of food quality are histamine, putrescine and cadaverine (Kivirand & Rinken, 2011). Other BAs, commonly determined in foodstuff are trimethylamine (Mitsubayashi et al, 2004), spermidine, spermine and tyramine (Alonso-Lomillo et al, 2010). At present, regulations have been established only for the intake of histamine, but no accordant limits are set for other BA-s, including putrescine and cadaverine, although several studies have indicated that putrescine and cadaverine could increase the toxicity of histamine by inhibiting the enzymes involved in histamine biodegradation (Niculescu et al, 2000). The allowed maximum residue level of histamine in food according to EEC regulations is 100 mg/kg (EEC, 2001); the international food safety organization FDA has established the histamine level to 50 mg/kg (FDA, 2001).

Effect of spermidine and its metabolites on the activity of pea seedlings diamine oxidase and the problems of biosensing of biogenic amines with this enzyme.

Spermidine is one of the several biogenic amines, produced during the microbial decarboxylation of proteins. Individual biogenic amines in the formed mixtures are frequently analyzed with oxygen sensor based biosensors, as their content serves as a good biomarker for the determination of food quality. In these biosensors, diamine oxidase from pea seedlings (PSAO), catalyzing the oxidation of various biogenic amines by dissolved oxygen is commonly used for the bio-recognition of amines. However, in the presence of spermidine and/or its metabolite 1,3-diaminopropane, the activity of PSAO and the sensitivity of PSAO-based biosensors decrease due to inhibition. The inhibition constant of soluble spermidine, acting as an inhibiting substrate toward PSAO, was found to be (40±15) mM in freshly prepared solution and (0.28±0.05) mM in solution, incubated 30 days at room temperature. The inhibition constant of 1,3-diaminopropane, acting as a competitive inhibitor, was (0.43±0.12) mM as determined through the oxidation reaction of cadaverine. The metabolic half-life of soluble spermidine was 7 days at room temperature and 186 days at 4 °C. The kinetic measurements were carried out with an oxygen sensor; the composition of the solution of degraded spermidine was analyzed with MS.

Modulation of enzyme catalytic properties and biosensor calibration parameters with chlorides: studies with glucose oxidase.

We studied the modulation of calibration parameters of biosensors, in which glucose oxidase was used for bio-recognition, in the presence of different chlorides by following the transient phase dynamics of oxygen concentration with an oxygen optrode. The mechanism of modulation was characterized with the changes of the glucose oxidase catalytic constant and oxygen diffusion constant. The modulation of two biosensor calibration parameters were studied: the maximum calculated signal change was amplified for about 20% in the presence of sodium and magnesium chlorides; the value of the kinetic parameter decreased along with the addition of salts and increased only at sodium chloride concentrations over 0.5 mM. Besides glucose bioassay, the amplification of calibration parameters was also studied in cascaded two-enzyme lactose biosensor, where the initial step of lactose bio-recognition, the β-galactosidase - catalyzed lactose hydrolysis, was additionally accelerated by magnesium ions.

Immunosensing system for rapid multiplex detection of mastitis-causing pathogens in milk

Mastitis, an inflammation of the mammary gland and udder tissue, is the major endemic disease of dairy cattle. In addition to causing health problems to the animals, mastitis leads to the reduction of milk production and quality, representing a significant economic burden for farmers. To enable timely treatment of infected animals with pathogen-specific antibiotics, the development of automated analytical methods for rapid on-site identification and quantification of mastitis-causing pathogens in milk is particularly important. An immunosensing system for multiplex detection of the two most common mastitis-causing pathogens Staphylococcus aureus and Escherichia coli is proposed in the present study. This immunosensor combines Bead Injection Analysis (BIA), attachment of pathogens onto renewable micro-column, biorecognition of bound pathogens by specific antibodies, conjugated with different fluorescence markers and the measurement of fluorescence signals. The analysis takes 20min and exhibits detection limits of < 50 CFU mL-1 for E. coli and 100 CFU mL-1 for S. aureus in milk. The applicability of the immunosensor was demonstrated by analyzing milk samples from cows, who were suffering from acute clinical mastitis.

Design and production of antibodies for the detection of Streptococcus uberis

Streptococcus uberis (S. uberis) is an important environmental pathogen causing mastitis in dairy cattle. Mastitis or mammary gland infection is the most common disease in milking cows and cause significant economic burden to farmers due to the reduction of the amount of milk and its quality. The development of rapid analytical methods for the determination of mastitis-causing pathogens in milk is of utmost importance for the early identification of the causes of mastitis and the beginning of timely treatment of cows. Combining in silico bioinformatic analysis and solid phase peptide synthesis using Fmoc chemistry, we have made two different peptides to mimic the adhesion protein of S. uberis, which is promoting the attachment of bacteria to epithelial cells. After purification with RP-HPLC, the peptides were conjugated with a larger carrier protein (KLH) and used for immunization of rabbits to produce specific antibodies. The separation of anti-S. uberis antibodies from rabbit blood antisera was carried out with affinity chromatography, using the synthetic peptides as affinity ligands. The purified antibodies showed high affinity and specificity towards S. uberis and were used for rapid bio-recognition and identification of S. uberis with an immunobiosensing system.

Determination of Penicillins in Milk by a Dual-Optrode Biosensor

ABSTRACT Penicillins are the most frequently found antibiotic residues in milk, as they are commonly used for the treatment of bacterial infections in cows. In the present study, we introduce a method for the rapid detection of penicillin residues in raw milk based on the determination of glucose concentration in milk with a dual flow-through biosensor. The molar concentration of glucose in milk is typically over 500 times lower than the concentration of lactose and is highly dependent on the rate of lactose hydrolysis, which is catalyzed by β-galactosidase. Glucose concentrations in milk change with variation in the β-galactosidase activity. β-Galactosidase is an enzyme produced in the microbiota in milk and its activity is inhibited by benzylpenicillin. Spiking milk with benzylpenicillin lowers glucose concentrations in comparison to high-quality milk after short storage intervals. The presence of penicillin in the milk of treated animals resulted in decreased glucose concentrations in comparison with high-quality milk that contained no antibiotics. The glucose concentration in milk samples was followed by the system enabling the elimination of the effects of bacterial respiration in the output with reliable results in less than 1 min.