Development of a novel immunobiosensor method for the rapid detection of okadaic acid contamination in shellfish extracts

The mouse bioassay is the methodology that is most widely used to detect okadaic acid (OA) in shellfish samples. This is one of the best-known toxins, and it belongs to the family of marine biotoxins referred to as the diarrhetic shellfish poisons (DSP). Due to animal welfare concerns, alternative methods of toxin detection are being sought. A rapid and specific biosensor immunoassay method was developed and validated for the detection of OA. An optical sensor instrument based on the surface plasmon resonance (SPR) phenomenon was utilised. A polyclonal antibody to OA was raised against OA-bovine thyroglobulin conjugate and OA-N-hydroxy succinimide ester was immobilised onto an amine sensor chip surface. The assay parameters selected for the analysis of the samples were: antibody dilution, 1/750; ratio of antibody to standard, 1:1; volume of sample injected, 25 mu l min(-1); flow rate, 25 mu l min(-1). An assay action limit of 126 ng g(-1) was established by analysing of 20 shellfish samples spiked with OA at the critical concentration of 160 ng g(-1), which is the action limit established by the European Union (EU). At this concentration of OA, the assay delivered coefficient of variations (CVs) of

Nucleic acid hybridizations as radioactive tool for rapid detection of banana bunchy top virus

Banana bunchy top disease (BBTD) caused by banana bunchy top virus (BBTV) was radioactively detected by nucleic acid hybridization techniques. Results showed that, 32P-labelled insert of pBT338 was hybridized with nucleic acid extracts from BBTV-infected plants from Egypt and Australia but not with those from CMV-infected plants from Egypt. Results revealed that BBTV was greatly detected in midrib, roots, meristem, corm, leaves and pseudostem respectively. BBTV was also detected in symptomless young plants prepared from diseased plant materials grown under tissue culture conditions but was not present in those performed from healthy plant materials. The sensitivity of dot blot and Southern blot hybridizations for the detection of BBTV was also performed for the detection of BBTV.

Lactic acid bacteria and their antimicrobial peptides : Induction,detection,partial characterization, and potential applications

Bacteriocin-producing lactic acid bacteria and their isolated peptide bacteriocins are of value to control pathogens and spoiling microorganisms in foods and feed. Nisin is the only bacteriocin that is commonly accepted as a food preservative and has a broad spectrum of activity against Gram-positive organisms including spore forming bacteria. In this study nisin induction was studied from two perspectives, induction from inside of the cell and selection of nisin inducible strains with increased nisin induction sensitivity. The results showed that a mutation in the nisin precursor transporter NisT rendered L. lactis incapable of nisin secretion and lead to nisin accumulation inside the cells. Intracellular proteolytic activity could cleave the N-terminal leader peptide of nisin precursor, resulting in active nisin in the cells. Using a nisin sensitive GFP bioassay it could be shown, that the active intracellular nisin could function as an inducer without any detectable release from the cells. The results suggested that nisin can be inserted into the cytoplasmic membrane from inside the cell and activate NisK. This model of two-component regulation may be a general mechanism of how amphiphilic signals activate the histidine kinase sensor and would represent a novel way for a signal transduction pathway to recognize its signal. In addition, nisin induction was studied through the isolation of natural mutants of the GFPuv nisin bioassay strain L. lactis LAC275 using fl uorescence-activated cell sorting (FACS). The isolated mutant strains represent second generation of GFPuv bioassay strains which can allow the detection of nisin at lower levels. The applied aspect of this thesis was focused on the potential of bacteriocins in chicken farming. One aim was to study nisin as a potential growth promoter in chicken feed. Therefore, the lactic acid bacteria of chicken crop and the nisin sensitivity of the isolated strains were tested. It was found that in the crop Lactobacillus reuteri, L. salivarius and L. crispatus were the dominating bacteria and variation in nisin resistance level of these strains was found. This suggested that nisin may be used as growth promoter without wiping out the dominating bacterial species in the crop. As the isolated lactobacilli may serve as bacteria promoting chicken health or reducing zoonoosis and bacteriocin production is one property associated with probiotics, the isolated strains were screened for bacteriocin activity against the pathogen Campylobacter jejuni. The results showed that many of the isolated L. salivarius strains could inhibit the growth of C. jejuni. The bacteriocin of the L. salivarius LAB47 strain, with the strongest activity, was further characterized. Salivaricin 47 is heat-stable and active in pH range 3 to 8, and the molecular mass was estimated to be approximately 3.2 kDa based on tricine SDS-PAGE analysis.

Detection of glycated hemoglobin using 3-Aminophenylboronic acid modified graphene oxide

This paper presents the chemical synthesis of 3-Aminophenylboronic acid (APBA) modified graphene oxide (GO) and its application to the electrochemical detection of glycated hemoglobin (GHb). The compound (GO-APBA) was synthesized by forming an amide linkage between the amino group (-NH2) of APBA and the carboxylic group (-COOH) of GO. The compound was characterized using IR spectroscopy. Detection of GHb was carried out using Electrochemical Impedance Spectroscopic (EIS) measurements with GO-APBA modified glassy carbon electrode as the working electrode.

A fluorescent organic cage for picric acid detection

Dynamic covalent imine chemistry has been utilized to synthesize a fluorescent 3+2] self-assembled nanoscopic organic cage. The fluorescent nature of the reduced analogue of the cage was further exploited for the highly selective detection of the explosive picric acid (PA).

Electron-Rich Triphenylamine-Based Sensors for Picric Acid Detection

This paper demonstrates the role of solvent in selectivity and sensitivity of a series of electron-rich compounds for the detection of trace amounts of picric acid. Two new electron-rich fluorescent esters (6, 7) containing a triphenylamine backbone as well as their analogous carboxylic acids (8, 9) have been synthesized and characterized. Fluorescent triphenylamine coupled with an ethynyl moiety constitutes p-electron-rich selective and sensitive probes for electron-deficient picric acid (PA). In solution, the high sensitivity of all the sensors toward PA can be attributed to a combined effect of the ground-state charge-transfer complex formation and resonance energy transfer between the sensor and analyte. The acids 8 and 9 also showed enhanced sensitivity for nitroaromatics in the solid state, and their enhanced sensitivity could be attributed to exciton migration due to close proximity of the neighboring acid molecules, as evident from the X-ray diffraction study. The compounds were found to be quite sensitive for the detection of trace amount of nitroaromatics in solution, solid, and contact mode.

Boron and Nitrogen Co-doped Carbon Nanoparticles as Photoluminescent Probes for Selective and Sensitive Detection of Picric Acid

Here, we report the synthesis of boron and nitrogen Co-doped carbon nanoparticles (BN-CNPs) by a hydrothermal method using sucrose, boric acid, and urea as the precursors. The BN-CNPs show excellent photoluminescence with a quantum yield of similar to 14.2% in aqueous solution and can be used as photoluminescent probes for selective and sensitive detection of picric acid (PA). PA quenches the photoluminescence signal remarkably, while other explosives cause a little quenching confirming the high selectivity of BN-CNPs. The sensitivity toward PA sensing is high at pH 7 and increases with temperature. The detection limit as well as the sensitivity are shown to improve by adding NaCl to the PA. The low detection limit can be as low as 10 nM at room temperature and pH 7, which indicates the BN-CNPs are superior as compared to other luminescent probes reported in the literature.

Superior Performance of a MoS2-RGO Composite and a Borocarbonitride in the Electrochemical Detection of Dopamine, Uric Acid and Adenine

A MoS2-RGO composite and borocarbonitride (BC5N) have been used as electrodes to selectively detect dopamine and uric acid in the presence of ascorbic acid. Both the electrodes show excellent eletrocatalytic activity towards the detection of dopamine, the detection limits being 0.55 mu M and 2.1 mu M in the case of MoS2-RGO and BCN respectively. MoS2-RGO shows a linear range of current over the 1-110 mu M concentrations of dopamine, while BCN shows over the 2.3-20 mu M range. BCN also exhibits satisfactory performance in the oxidation of uric acid with a detection limit of 3.8 mu M and the linear range from 4 to 40 mu M. The MoS2-RGO has also been used to detect adenine as well.

Non-enzymatic electronic detection of glucose using aminophenylboronic acid functionalized reduced graphene oxide

We report the non-enzymatic electronic detection of glucose using field effect transistor (FET) devices made of aminophenylboronic acid (APBA) functionalized reduced graphene oxide (RGO). Detection of glucose molecules was carried out over a wide dynamic range of concentration varying from 100 pM to 100 mM with a detection limit of similar to 2 nM using both covalently and non-covalently functionalized APBA-RGO complex. The normalized change in electrical conductance data shows that the FET devices made of non-covalently functionalized APBA-RGO complex (nc-APBA-RGO) exhibited a linear response to glucose aqueous solution of concentrations varying from 1 nM to 10 mM and showed 4 times enhanced sensitivity over the devices made of covalently functionalized APBA-RGO complex (c-APBA-RGO). Specificity of APBA-RGO complex to glucose is confirmed from the observation of negligible change in electrical conductance after exposure to 0.1 mM of lactose and other interfering factors. (C) 2015 Elsevier B.V. All rights reserved.

Label free electrochemiluminescence protocol for sensitive DNA detection with a tris(2,2'-bipyridyl)ruthenium(II) modified electrode based on nucleic acid oxidation

Label free electrochemiluminescence (ECL) DNA detection based on catalytic guanine and adenine bases oxidation using tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)(3)(2+)] modified glassy carbon (GC) electrode was demonstrated in this work. The modified GC electrode was prepared by casting carbon nanotubes (CNT)/Nafion/Ru(bpy)(3)(2+) composite film on the electrode surface. ECL signals of doublestranded DNA and their thermally denatured counterparts can be distinctly discriminated using cyclic voltammetry (CV) with a low concentration (3.04 x 10(-8) mol/L for Salmon Testes-DNA). Most importantly, sensitive single-base mismatch detection of p53 gene sequence segment was realized with 3.93 x 10(-10) mol/L employing CV stimulation (ECL signal of C/A mismatched DNA oligonucleotides was 1.5-fold higher than that of fully base-paired DNA oligonucleotides). Label free, high sensitivity and simplicity for single-base mismatch discrimination were the main advantages of the present ECL technique for DNA detection over the traditional DNA sensors.

CE with electrochemical detection for investigation of label-free recognition of amino acid amides by guanine-rich DNA aptamers

In this work, we report a simple and effective investigation into adaptive interactions between guanine-rich DNA aptamers and amino acid amides by CE with electrochemical (EC) detection. Argininamide (Arm) and tyrosinamide (Tym) were chosen as model molecules. On a copper electrode, Arm generated a good EC signal in 60 mM NaOH at 0.7 V (vs Ag/ AgCl), while Tym. was detected well on a platinum electrode at 1. 3 V in 20 mM phosphate of pH 7.0. Based on their EC properties, the ligands themselves were used as indicators for the adaptive interactions investigated by CE-EC, making any step of labeling and/or modification of aptamers with indicators exempted. Hydrophilic ionic liquid was used as an additive in running buffer of CE to improve the sensitivity of Arm detection, whereas the additive was not used for Tym. detection due to its negative effect. Two guanine-rich DNA aptamers were used for molecular recognition of Arm and Tym. When the aptamers were incubated with ligands, they bound the model molecules with high affinity and specificity, reflected by obvious decreases in the signals of ligands but no changes in those of the control molecules. However, the ligands were hardly affected by the control ssDNAs after incubation. The results revealed the specific recognition of Arm and Tym. by the aptamers.

Electrochemiluminescent detection of chlorpromazine by selective preconcentration at a lauric acid-modified carbon paste electrode using tris(2,2 '-bipyridine)ruthenium(II)

A new detection scheme for the determination of adsorbable coreactants of Ru(bpy)(3)(2+) electrochemiluminescent reaction is presented. It is based on selective preconcentration of coreactant onto an electrode, followed by Ru(bpy)(3)(2+) electrochemiluminescent detection. The coreactant employed is chlorpromazine. It was sensitively detected after 5-min preconcentration onto a lauric acid-modified carbon paste electrode. The linear concentration range was found to occur from 1 x 10(-8) to 3 x 10(-6) mol L-1 with a detection limit of 3.1 x 10(-9) mol L-1. The total analysis time is less than 10 min. As a result of selective preconcentration and medium exchange, such remarkable selectivity is achieved that reproducible quantitation of chlorpromazine in urine is possible.

Determination of barbituric acid and 2-thiobarbituric acid with end-column electrochemical detection by capillary electrophoresis

Capillary electrophoresis (CE) with end-column electrochemical detection (EC) of barbituric acid (BA) and 2-thiobarbituric acid (TA) has been described. Under optimum condition, BA and TA were separated satisfactorily, and a response of high sensitivity and stability was obtained at a detection potential of 1.25 V versus Ag/AgCl. Optimized end-column detection provides detection limit as low as 0.5 and 0.1 mu M for BA and TA, respectively. The calibration graph was linear over three orders of magnitude. The relative standard deviations (n = 10) of peak currents and migration times obtained for both BA and TA were 3.4, 3.7, and 1.7, 1.2%, respectively. The proposed method has been applied to analyze water sample with satisfactory results. (C) 2000 Elsevier Science B.V. All rights reserved.