Ratiometric electrochemical aptasensor based on split aptamer and Au-rGO for detection of aflatoxin M1.

A novel ratiometric electrochemical aptasensor based on split aptamer and Au-reduced graphene oxide (Au-rGO) nanomaterials was proposed to detect aflatoxin M1 (AFM1). In this work, Au-rGO nanomaterials were coated on the electrode through the electrodeposition method to increase the aptamer enrichment. We split the aptamer of AFM1 into 2 sequences (S1 and S2), where S1 was immobilized on the electrode due to the Au-S bond, and S2 was tagged with methylene blue (MB) and acted as a response signal. A complementary strand to S1 (CS1) labeled with ferrocene (Fc) was introduced as another reporter. In the presence of AFM1, CS1 was released from the electrode surface due to the formation of the S1-AFM1-S2 complex, leading to a decrease in Fc and an increase in the MB signal. The developed ratiometric aptasensor exhibited a linear range of 0.03 µg L-1 to 2.00 µg L-1, with a detection limit of 0.015 µg L-1 for AFM1 detection. The ratiometric aptasensor also showed a linear relationship from 0.2 µg L-1 to 1.00 µg L-1, with a detection limit of 0.05 µg L-1 in natural milk after sample pretreatment, indicating the successful application of the developed ratiometric aptasensor. Our proposed strategy provides a new way to construct aptasensors with high sensitivity and selectivity.

A novel electrochemical immunosensor based on Fe3O4@graphene nanocomposite modified glassy carbon electrode for rapid detection of Salmonella in milk.

Foods contaminated by foodborne pathogens have always been a great threat to human life. Herein, we constructed an electrochemical immunosensor for Salmonella detection by using a Fe3O4@graphene modified electrode. Because of the excellent electrical conductivity and mechanical stability of graphene and the large specific surface area of Fe3O4, the Fe3O4@graphene nanocomposite exhibits an excellent electrical signal, which greatly increased the sensitivity of the immunosensor. Gold nanoparticles were deposited on Fe3O4@graphene nanocomposite by electrochemical technology for the immobilization of the antibody. Cyclic voltammetry was selected to electrochemically characterize the construction process of immunosensors. The microstructure and morphology of related nanocomposites were analyzed by scanning electron microscopy. Under optimized experimental conditions, a good linear relationship was achieved in the Salmonella concentration range of 2.4 × 102 to 2.4 × 107 cfu/mL, and the limit of detection of the immunosensor was 2.4 × 102 cfu/mL. Additionally, the constructed immunosensor exhibited acceptable selectivity, reproducibility, and stability and provides a new reference for detecting pathogenic bacteria in milk.

Electrochemical enzyme-linked immunosorbent assay (e-ELISA) for parasitic nematode Ostertagia ostertagi (brown stomach worm) infections in dairy cattle.

A sensitive electrochemical immunoassay (e-ELISA) has been developed for the detection of the gastrointestinal parasitic nematode Ostertagia ostertagi (brown stomach worm) in infected and control serum samples. An antigen-indirect immunoassay format was employed to detect the presence of O. ostertagi antibodies, coupled with an anti-species monoclonal horseradish peroxidase (HRP) conjugate. ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) and TMB (3,3',5,5'-tetramethylbenzidine/hydrogen peroxide) were investigated as both chromogenic visualising reagents for optical ELISA and electroactive substrates for electrochemical ELISA in the HRP catalysed oxidation reaction. Coulometry was applied for the detection of O. ostertagi antibodies (via TMB electrochemistry) and compared with the commercial optical ELISA (ABTS based SVANOVIR® O. ostertagi-Ab ELISA kit). Cost-effective in-house sensors were designed and fabricated using polyester and chemical adhesive materials with the aid of stencil printing and laser machining techniques. The performance of the electrochemical ELISA and sensor was evaluated by investigating redox mediators (ABTS vs. TMB), stop solutions (sodium dodecyl sulfate vs. sulfuric acid) and incubation times (150 min vs. 70 min vs. 25 min). For a total assay incubation time of 70 minutes, the TMB/H2SO4 based e-ELISA was able to differentiate between positive (P) and negative (N) control serum samples, with a P/N70 control ratio 1.6 times higher than that of optical ELISA (TMB/H2SO4 combination) and 2.9 times higher than that of the commercial ELISA kit (ABTS/SDS combination). Furthermore, the e-ELISA approach is quicker and required only 25 min (total incubation time) with even better response (P/N25 = 14.7), which is approximately 4-fold higher than the optical immunoassay (P/N25 = 3.8). The proposed e-ELISA is specific (selective Ab-Ag interactions) and highly sensitive - capable of detecting up to 16-fold dilutions of a positive control serum sample. The electrochemical ELISA approach has the potential for rapid sample screening in a portable, disposable format, contributing to the quest for effective prevention and control of parasitic Ostertagia ostertagi infections in cattle.

A PVC/polypyrrole sensor designed for beef taste detection using electrochemical methods and sensory evaluation.

An electrochemical sensor for detection of beef taste was designed in this study. This sensor was based on the structure of polyvinyl chloride/polypyrrole (PVC/PPy), which was polymerized onto the surface of a platinum (Pt) electrode to form a Pt-PPy-PVC film. Detecting by electrochemical methods, the sensor was well characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The sensor was applied to detect 10 rib-eye beef samples and the accuracy of the new sensor was validated by sensory evaluation and ion sensor detection. Several cluster analysis methods were used in the study to distinguish the beef samples. According to the obtained results, the designed sensor showed a high degree of association of electrochemical detection and sensory evaluation, which proved a fast and precise sensor for beef taste detection.

Distribution and concentration of maternal progesterone in the yolk of Greater Rhea eggs (Rhea americana).

Progesterone is the most concentrated maternal yolk steroid characterized to date in birds; however, no information about it is available in ratite eggs. We collected freshly laid eggs from zoo-housed Greater Rhea females (Rhea americana) bred under similar rearing conditions during two breeding seasons to characterize concentration and distribution of maternal yolk progesterone. After high-performance liquid chromatography analysis, yolk hormone was measured using a commercial electrochemiluminescence immunoassay. Progesterone concentrations were found to vary significantly among the yolk layers, supporting a follicular origin for this steroid in Greater Rhea eggs. Additionally, highly similar mean absolute yolk progesterone concentrations were detected between 2013 and 2015 breeding seasons (1,332.98 ± 82.59 and 1,313.59 ± 85.19 ng/g, respectively). These values are also comparable to those found in some domestic carinate species. Findings suggest that at population level, when rearing conditions are similar, mean absolute yolk maternal progesterone concentrations also appear bounded. Future research on the factors and mechanisms that regulate progesterone deposition in Greater Rhea eggs is needed to better understand whether its levels depend on different rearing conditions.

Use of experimental design to optimize a triple-potential waveform to develop a method for the determination of streptomycin and dihydrostreptomycin in pharmaceutical veterinary dosage forms by HPLC-PAD.

An HPLC-PAD method using a gold working electrode and a triple-potential waveform was developed for the simultaneous determination of streptomycin and dihydrostreptomycin in veterinary drugs. Glucose was used as the internal standard, and the triple-potential waveform was optimized using a factorial and a central composite design. The optimum potentials were as follows: amperometric detection, E1=-0.15V; cleaning potential, E2=+0.85V; and reactivation of the electrode surface, E3=-0.65V. For the separation of the aminoglycosides and the internal standard of glucose, a CarboPac™ PA1 anion exchange column was used together with a mobile phase consisting of a 0.070 mol L(-1) sodium hydroxide solution in the isocratic elution mode with a flow rate of 0.8 mL min(-1). The method was validated and applied to the determination of streptomycin and dihydrostreptomycin in veterinary formulations (injection, suspension and ointment) without any previous sample pretreatment, except for the ointments, for which a liquid-liquid extraction was required before HPLC-PAD analysis. The method showed adequate selectivity, with an accuracy of 98-107% and a precision of less than 3.9%.

Development of an immunosensor for the detection of Francisella tularensis antibodies.

Tularemia, also known as rabbit fever, is a highly infectious zoonotic disease caused by a non-motile and non-spore-forming Gram-negative coccoid rod bacterium, Francisella tularensis. It occurs naturally in lagomorphs (rabbits and hares), but many animals have been reported to be susceptible. Transmission to humans is mostly caused by inhalation of aerosolised bacteria, handling of infected animals, arthropod stings, and ingestion of contaminated foods and water. At present, pathogenic isolation, molecular detection, and serology are the most commonly used methods to confirm the diagnosis of tularemia. In this work, an electrochemical immunosensor for the detection of anti-F. tularensis antibodies was developed, consisting of gold-based self-assembled monolayers of a carboxylic-group-terminated bipodal alkanethiol that is covalently linked to a lipopolysaccharide (LPS) that can be found in the outer membrane of the bacteria F. tularensis. The presence of anti-F. tularensis antibodies was measured using horseradish peroxidase-labelled protein A (HRP-protein A) from Staphylococcus aureus, and the developed immunosensor gave a stable quantitative response to different anti-F. tularensis FB11 antibody concentrations after 30 min with a limit of detection of 15 ng/mL, RSD of 9%, n = 3. The developed immunosensor was tested with serum from animals infected with tularemia and was compared to the results obtained using ELISA showing an excellent degree of correlation.

Accuracy of capillary blood 3-ß-hydroxybutyrate determination for the detection and treatment of canine diabetic ketoacidosis.

In human medicine, diagnosis of diabetic ketoacidosis (DKA) is usually based on measurement of capillary 3-ß-hydroxybutyrate (3-HB) with a hand held ketone sensor. This study was conducted to determine if measurement of capillary 3-HB could be useful for the diagnosis and monitoring of canine DKA. Fifteen dogs with diabetic ketosis and 10 with DKA were evaluated. Paired measurements of 3-HB of capillary and venous blood samples were analysed by the electrochemical sensor and reference method. Use of capillary 3-HB measurement during DKA management was then evaluated through simultaneous measurements of capillary 3-HB, urinary AcAc and venous blood gas analysis. Good agreement between capillary and venous 3-HB measurement was detected by the electrochemical sensor and reference method. Monitoring treatment of DKA revealed a significant correlation between capillary 3-HB and acidosis markers, while no significant correlation was observed between AcAc and acidosis markers. A cut-off value of capillary blood 3-HB > 3.8 mmol/L for diagnosis of DKA resulted in 70% and 92% sensitivity and specificity. The electrochemical sensor accurately measures 3-HB concentration in both capillary and venous blood samples, is accurate in diagnosing canine DKA, and appears to reflect the patient's metabolic status during DKA treatment.

Carbon nanotube enhanced label-free immunosensor for amperometric determination of oocyte maturation-inducing hormone in fish.

Maintaining high-quality fish eggs stably and efficiently is important for aquaculture. We developed a label-free immunosensor system for measuring 17,20ß-dihydroxy-4-pregnen-3-one (DHP). DHP is suddenly secreted before ovulation as a maturation-inducing hormone in fish, and therefore, DHP levels are an indicator for predicting ovulation. The method is based on immunologic reactions and amperometric measurement using cyclic voltammetry (CV). For biomolecular immobilization on the surface of sensing electrode, Au electrode, we used self-assembled monolayers of thiol-containing compounds to fix anti-DHP immunoglobulin. In addition, we used a single-walled carbon nanotube to improve sensitivity. Using this electrode, we were able to determine the CV signal change caused by the antigen-antibody complex. The proposed immunosensor system showed a linear correlation (correlation coefficient: 0.9827) between the anodic peak current of the CV and the DHP level in range from 15.6 to 50,000 pg ml(-1). The sensor system was then applied to monitor DHP of goldfish (Carassius auratus). Blood plasma of fish was collected every 3 h after administering a DHP inducer. In the measurement, the anodic peak current of the CV showed distinct changes depending on DHP levels in the blood plasma. A good relationship was observed between DHP levels determined by our proposed system and the conventional method (correlation coefficient: 0.9351).

Functional epitope core motif of the Anaplasma marginale major surface protein 1a and its incorporation onto bioelectrodes for antibody detection.

Anaplasmosis, a persistent intraerythrocytic infection of cattle by Anaplasma marginale, causes severe anemia and a higher rate of abortion, resulting in significant loss to both dairy and beef industries. Clinical diagnosis is based on symptoms and confirmatory laboratory tests are required. Currently, all the diagnostic assays have been developed with whole antigens with indirect ELISA based on multiple epitopes. In a pioneer investigation we demonstrated the use of critical motifs of an epitope as biomarkers for immunosensor applications. Mimotopes of the MSP1a protein functional epitope were obtained through Phage Display after three cycles of selection of a 12-mer random peptide library against the neutralizing monoclonal antibody 15D2. Thirty-nine clones were randomly selected, sequenced, translated and aligned with the native sequence. The consensus sequence SxSSQSEASTSSQLGA was obtained, which is located in C-terminal end of the 28-aa repetitive motif of the MSP1a protein, but the alignment and sequences' variation among mimotopes allowed us to map the critical motif STSSxL within the consensus sequence. Based on these results, two peptides were chemically synthesized: one based on the critical motif (STSSQL, Am1) and the other based on the consensus sequence aligned with the native epitope (SEASTSSQLGA, Am2). Sera from 24 infected and 52 healthy animals were tested by ELISA for reactivity against Am1 and Am2, which presented sensitivities of 96% and 100%, respectively. The Am1 peptide was incorporated onto a biolectrode (graphite modified with poly-3-hydroxyphenylacetic acid) and direct serum detection was demonstrated by impedance, differential pulse voltammetry, and atomic force microscopy. The electrochemical sensor system proved to be highly effective in discriminating sera from positive and negative animals. These immunosensors were highly sensitive and selective for positive IgG, contaminants did not affect measurements, and were based on a simple, fast and reproducible electrochemical system.

Determination of beta-agonists in pig feed, pig urine and pig liver using capillary electrophoresis with electrochemical detection.

The fast separation capability of a capillary electrophoresis with electrochemical detection (CE-ED) system was demonstrated by determining five beta-agonists including clenbuterol, metoprolol, ractopamine, isoxsuprine and salbutamol in several real samples. Factors influencing the separation and detection were examined and optimized. Under the optimum conditions, the five beta-agonists could be well separated within 15min at a separation voltage of 14kV in a 100mmoL H(3)BO(3)-Na(2)B(4)O(7) running buffer (pH 9.0). This method was successfully used in the analysis of clenbuterol, metoprolol, ractopamine, isoxsuprine and salbutamol in pig feed, pig urine and pig liver, providing a useful method for monitoring the illegal use of beta-agonists from growth promotion in food-producing animals.