Hepatocyte gene therapy in a large animal: A neonatal bovine model of citrullinemia

The development of gene-replacement therapy for inborn errors of metabolism has been hindered by the limited number of suitable large-animal models of these diseases and by inadequate methods of assessing the efficacy of treatment. Such methods should provide sensitive detection of expression in vivo and should be unaffected by concurrent pharmacologic and dietary regimens. We present the results of studies in a neonatal bovine model of citrullinemia, an inborn error of urea-cycle metabolism characterized by deficiency of argininosuccinate synthetase and consequent life-threatening hyperammonemia. Measurements of the flux of nitrogen from orally administered 15NH4 to [15N]urea were used to determine urea-cycle activity in vivo. In control animals, these isotopic measurements proved to be unaffected by pharmacologic treatments. Systemic administration of a first-generation E1-deleted adenoviral vector expressing human argininosuccinate synthetase resulted in transduction of hepatocytes and partial correction of the enzyme defect. The isotopic method showed significant restoration of urea synthesis. Moreover, the calves showed clinical improvement and normalization of plasma glutamine levels after treatment. The results show the clinical efficacy of treating a large-animal model of an inborn error of hepatocyte metabolism in conjunction with a method for sensitively measuring correction in vivo. These studies will be applicable to human trials of the treatment of this disorder and other related urea-cycle disorders.

The cellulose-binding domain of the major cellobiohydrolase of Trichoderma reesei exhibits true reversibility and a high exchange rate on crystalline cellulose.

Cellulose-binding domains (CBDs) bind specifically to cellulose, and form distinct domains of most cellulose degrading enzymes. The CBD-mediated binding of the enzyme has a fundamental role in the hydrolysis of the solid cellulose substrate. In this work we have investigated the reversibility and kinetics of the binding of the CBD from Trichoderma reesei cellobiohydrolase I on microcrystalline cellulose. The CBD was produced in Escherichia coli, purified, and radioactively labeled by reductive alkylation with 3H. Sensitive detection of the labeled CBD allowed more detailed analysis of its behavior than has been possible before, and important novel features were resolved. Binding of the CBD was found to be temperature sensitive, with an increased affinity at lower temperatures. The interaction of the CBD with cellulose was shown to be fully reversible and the CBD could be eluted from cellulose by simple dilution. The rate of exchange measured for the CBD-cellulose interaction compares well with the hydrolysis rate of cellobiohydrolase I, which is consistent with its proposed mode of action as a processive exoglucanase.

Microscale mesoarrays created by dip-pen nanolithography for screening of protein-protein interactions

Using microarrays to probe protein-protein interactions is becoming increasingly attractive due to their compatibility with highly sensitive detection techniques, selectivity of interaction, robustness and capacity for examining multiple proteins simultaneously. The major drawback to using this approach is the relatively large volumes and high concentrations necessary. Reducing the protein array spot size should allow for smaller volumes and lower concentrations to be used as well as opening the way for combination with more sensitive detection technologies. Dip-Pen Nanolithography (DPN) is a recently developed technique for structure creation on the nano to microscale with the capacity to create biological architectures. Here we describe the creation of miniaturised microarrays, 'mesoarrays', using DPN with protein spots 400× smaller by area compared to conventional microarrays. The mesoarrays were then used to probe the ERK2-KSR binding event of the Ras/Raf/MEK/ERK signalling pathway at a physical scale below that previously reported. Whilst the overall assay efficiency was determined to be low, the mesoarrays could detect KSR binding to ERK2 repeatedly and with low non-specific binding. This study serves as a first step towards an approach that can be used for analysis of proteins at a concentration level comparable to that found in the cellular environment.

Quantitative Analysis and Determination of Microcystin in water by Capillary Electrophoresis Mass Spectrometry

The presence of harmful algal blooms (HAB) is a growing concern in aquatic environments. Among HAB organisms, cyanobacteria are of special concern because they have been reported worldwide to cause environmental and human health problem through contamination of drinking water. Although several analytical approaches have been applied to monitoring cyanobacteria toxins, conventional methods are costly and time-consuming so that analyses take weeks for field sampling and subsequent lab analysis. Capillary electrophoresis (CE) becomes a particularly suitable analytical separation method that can couple very small samples and rapid separations to a wide range of selective and sensitive detection techniques. This paper demonstrates a method for rapid separation and identification of four microcystin variants commonly found in aquatic environments. CE coupled to UV and electrospray ionization time-of-flight mass spectrometry (ESI-TOF) procedures were developed. All four analytes were separated within 6 minutes. The ESI-TOF experiment provides accurate molecular information, which further identifies analytes.

Electrochemical RNA genosensors for toxic algal species: enhancing selectivity and sensitivity

Harmful algal blooms (HABs) are becoming more frequent as climate changes, with tropical species moving northward. Monitoring programs detecting the presence of toxic algae before they bloom are of paramount importance to protect aquatic ecosystems, aquaculture, human health and local economies. Rapid and reliable species identification methods using molecular barcodes coupled to biosensor detection tools have received increasing attention over the past decade as an alternative to the impractical standard microscopic counting-based techniques. This work reports on a PCR amplification-free electrochemical genosensor for the enhanced selective and sensitive detection of RNA from multiple Mediterranean toxic algal species. For a sandwich hybridization (SHA), we designed longer capture and signal probes for more specific target discrimination against a single base-pair mismatch from closely related species and for reproducible signals. We optimized experimental conditions, viz., minimal probe concentration in the SHA on a screen-printed gold electrode and selected the best electrochemical mediator. Probes from 13 Mediterranean dinoflagellate species were tested under optimized conditions and the format further tested for quantification of RNA from environmental samples. We not only enhanced the selectivity and sensitivity of the state-of-the-art toxic algal genosensors but also increased the repertoire of toxic algal biosensors in the Mediterranean, towards an integral and automatic monitoring system.

Electrochemical RNA genosensors for toxic algal species: enhancing selectivity and sensitivity

Harmful algal blooms (HABs) are becoming more frequent as climate changes, with tropical species moving northward. Monitoring programs detecting the presence of toxic algae before they bloom are of paramount importance to protect aquatic ecosystems, aquaculture, human health and local economies. Rapid and reliable species identification methods using molecular barcodes coupled to biosensor detection tools have received increasing attention over the past decade as an alternative to the impractical standard microscopic counting-based techniques. This work reports on a PCR amplification-free electrochemical genosensor for the enhanced selective and sensitive detection of RNA from multiple Mediterranean toxic algal species. For a sandwich hybridization (SHA), we designed longer capture and signal probes for more specific target discrimination against a single base-pair mismatch from closely related species and for reproducible signals. We optimized experimental conditions, viz., minimal probe concentration in the SHA on a screen-printed gold electrode and selected the best electrochemical mediator. Probes from 13 Mediterranean dinoflagellate species were tested under optimized conditions and the format further tested for quantification of RNA from environmental samples. We not only enhanced the selectivity and sensitivity of the state-of-the-art toxic algal genosensors but also increased the repertoire of toxic algal biosensors in the Mediterranean, towards an integral and automatic monitoring system.

Preparation of an Immunosorbent and its use in the Solid Phase Extraction of Benzodiazepines

The use of capillary electrophoresis (CE) has been restricted to applications having high sample concentrations because of its low sensitivity caused by small injection volumes and, when ultraviolet (UV) detection is used, the short optical path length. Sensitivity in CE can be improved by using more sensitive detection systems, or by preconcentration techniques which are based on chromatographic and/or electrophoretic principles. One of the promising strategies to improve sensitivity is solid phase extraction (SPE). Solid Phase Extraction utilizes high sample volumes and a variety of complex matrixes to facilitate trace detection. To increase the specificity of the SPE a selective solid phase must be chosen. Immunosorbents, which are a combination of an antibody and a solid support, have proven to be an excellent option because of high selectivity of the antibody. This thesis is an exploratory study of the application of immunosorbent-SPE combined with CE for trace concentration of benzodiazepines. This research describes the immobilization and performance evaluation of an immunosorbent prepared by immobilizing a benzodiazepine-specific antibody on aminopropyl silica. The binding capacity of the immunosorbent, measured as µg of benzodiazepine/ gram of immunosorbent, was 39 ± 10. The long term stability of the prepared immunosorbent has been improved by capping the remaining aminopropyl groups by reaction with acetic anhydride. The capped immunosorbent retained its binding capacity after several uses.

Electrochemical Immunosensing of Cortisol in an Automated Microfluidic System Towards Point-of-Care Applications

This dissertation describes the development of a label-free, electrochemical immunosensing platform integrated into a low-cost microfluidic system for the sensitive, selective and accurate detection of cortisol, a steroid hormone co-related with many physiological disorders. Abnormal levels of cortisol is indicative of conditions such as Cushing’s syndrome, Addison’s disease, adrenal insufficiencies and more recently post-traumatic stress disorder (PTSD). Electrochemical detection of immuno-complex formation is utilized for the sensitive detection of Cortisol using Anti-Cortisol antibodies immobilized on sensing electrodes. Electrochemical detection techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) have been utilized for the characterization and sensing of the label-free detection of Cortisol. The utilization of nanomaterial’s as the immobilizing matrix for Anti-cortisol antibodies that leads to improved sensor response has been explored. A hybrid nano-composite of Polyanaline-Ag/AgO film has been fabricated onto Au substrate using electrophoretic deposition for the preparation of electrochemical immunosening of cortisol. Using a conventional 3-electrode electrochemical cell, a linear sensing range of 1pM to 1µM at a sensitivity of 66µA/M and detection limit of 0.64pg/mL has been demonstrated for detection of cortisol. Alternately, a self-assembled monolayer (SAM) of dithiobis(succinimidylpropionte) (DTSP) has been fabricated for the modification of sensing electrode to immobilize with Anti-Cortisol antibodies. To increase the sensitivity at lower detection limit and to develop a point-of-care sensing platform, the DTSP-SAM has been fabricated on micromachined interdigitated microelectrodes (µIDE). Detection of cortisol is demonstrated at a sensitivity of 20.7µA/M and detection limit of 10pg/mL for a linear sensing range of 10pM to 200nM using the µIDE’s. A simple, low-cost microfluidic system is designed using low-temperature co-fired ceramics (LTCC) technology for the integration of the electrochemical cortisol immunosensor and automation of the immunoassay. For the first time, the non-specific adsorption of analyte on LTCC has been characterized for microfluidic applications. The design, fabrication technique and fluidic characterization of the immunoassay are presented. The DTSP-SAM based electrochemical immunosensor on µIDE is integrated into the LTCC microfluidic system and cortisol detection is achieved in the microfluidic system in a fully automated assay. The fully automated microfluidic immunosensor hold great promise for accurate, sensitive detection of cortisol in point-of-care applications.

Rapid and sensitive measurements of nitrate ester explosives using microchip electrophoresis with electrochemical detection

This article describes an effective microchip protocol based on electrophoretic-separation and electrochemical detection for highly sensitive and rapid measurements of nitrate ester explosives, including ethylene glycol dinitrate (EGDN), pentaerythritol tetranitrate (PETN), propylene glycol dinitrate (PGDN) and glyceryl trinitrate (nitroglycerin, NG). Factors influencing the separation and detection processes were examined and optimized. Under the optimal separation conditions obtained using a 15 mM borate buffer (pH 9.2) containing 20 mM SDS, and applying a separation voltage of 1500 V, the four nitrate ester explosives were separated within less than 3 min. The glassy-carbon amperometric detector (operated at -0.9 V vs. Ag/AgCl) offers convenient cathodic detection down to the picogram level, with detection limits of 0.5 ppm and 0.3 ppm for PGDN and for NG, respectively, along with good repeatability (RSD of 1.8-2.3%; n = 6) and linearity (over the 10-60 ppm range). Such effective microchip operation offers great promise for field screening of nitrate ester explosives and for supporting various counter-terrorism surveillance activities.

Sensitive and specific detection of Clavibacter xyli subsp. xyli, causal agent of ratoon stunting disease of sugarcane, with a polymerase chain reaction-based assay

A sensitive, specific polymerase chain reaction-based assay was developed for the detection of the causal agent of ratoon stunting disease of sugarcane, Clavibacter xyli subsp. xyli. This assay uses oligonucleotide primers derived from the internal transcribed spacer region between the 16S and 23S rRNA genes of the bacterial rRNA operon. The assay is specific for C. xyli subsp. xyli and does not produce an amplification product from the template of the closely related bacterium C. xyli subsp. cynodontis, nor from other bacterial species. The assay was successfully applied to the detection of C. xyli subsp. xyli in fibrovascular fluid extracted from sugarcane and was sensitive to approximately 22 cells per PCR assay. A multiplex PCR test was also developed which identified and differentiated C. xyli subsp. xyli and C. xyli subsp. cynodontis in a single PCR assay.

Detection of FRET signals with a wavelength sensitive device based on a-SiC:H

Glucose sensing is an issue with great interest in medical and biological applications. One possible approach to glucose detection takes advantage of measuring changes in fluorescence resonance energy transfer (FRET) between a fluorescent donor and an acceptor within a protein which undergoes glucose-induced changes in conformation. This demands the detection of fluorescent signals in the visible spectrum. In this paper we analyzed the emission spectrum obtained from fluorescent labels attached to a protein which changes its conformation in the presence of glucose using a commercial spectrofluorometer. Different glucose nanosensors were used to measure the output spectra with fluorescent signals located at the cyan and yellow bands of the spectrum. A new device is presented based on multilayered a-SiC:H heterostructures to detect identical transient visible signals. The transducer consists of a p-i'(a-SiC:H)-n/p-i(a-Si:H)-n heterostructure optimized for the detection of the fluorescence resonance energy transfer between fluorophores with excitation in the violet (400 nm) and emissions in the cyan (470 nm) and yellow (588 nm) range of the spectrum. Results show that the device photocurrent signal measured under reverse bias and using appropriate steady state optical bias, allows the separate detection of the cyan and yellow fluorescence signals. (C) 2013 Elsevier B.V. All rights reserved.

Sensitive bi-enzymatic biosensor based on polyphenoloxidases–gold nanoparticles–chitosan hybrid film–graphene doped carbon paste electrode for carbamates detection

A bi-enzymatic biosensor (LACC–TYR–AuNPs–CS/GPE) for carbamates was prepared in a single step by electrodeposition of a hybrid film onto a graphene doped carbon paste electrode (GPE). Graphene and the gold nanoparticles (AuNPs) were morphologically characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, dynamic light scattering and laser Doppler velocimetry. The electrodeposited hybrid film was composed of laccase (LACC), tyrosinase (TYR) and AuNPs entrapped in a chitosan (CS) polymeric matrix. Experimental parameters, namely graphene redox state, AuNPs:CS ratio, enzymes concentration, pH and inhibition time were evaluated. LACC–TYR–AuNPs–CS/GPE exhibited an improved Michaelis–Menten kinetic constant (26.9 ± 0.5 M) when compared with LACC–AuNPs–CS/GPE (37.8 ± 0.2 M) and TYR–AuNPs–CS/GPE (52.3 ± 0.4 M). Using 4-aminophenol as substrate at pH 5.5, the device presented wide linear ranges, low detection limits (1.68×10− 9 ± 1.18×10− 10 – 2.15×10− 7 ± 3.41×10− 9 M), high accuracy, sensitivity (1.13×106 ± 8.11×104 – 2.19×108 ± 2.51×107 %inhibition M− 1), repeatability (1.2–5.8% RSD), reproducibility (3.2–6.5% RSD) and stability (ca. twenty days) to determine carbaryl, formetanate hydrochloride, propoxur and ziram in citrus fruits based on their inhibitory capacity on the polyphenoloxidases activity. Recoveries at two fortified levels ranged from 93.8 ± 0.3% (lemon) to 97.8 ± 0.3% (orange). Glucose, citric acid and ascorbic acid do not interfere significantly in the electroanalysis. The proposed electroanalytical procedure can be a promising tool for food safety control.

HIGH SENSITIVE PCR METHOD FOR DETECTION OF PATHOGENIC Leptospira spp. IN PARAFFIN-EMBEDDED TISSUES

This study describes the development and application of a new PCR assay for the specific detection of pathogenic leptospires and its comparison with a previously reported PCR protocol. New primers were designed for PCR optimization and evaluation in artificially-infected paraffin-embedded tissues. PCR was then applied to post-mortem, paraffin-embedded samples, followed by amplicon sequencing. The PCR was more efficient than the reported protocol, allowing the amplification of expected DNA fragment from the artificially infected samples and from 44% of the post-mortem samples. The sequences of PCR amplicons from different patients showed >99% homology with pathogenic leptospires DNA sequences. The applicability of a highly sensitive and specific tool to screen histological specimens for the detection of pathogenic Leptospira spp. would facilitate a better assessment of the prevalence and epidemiology of leptospirosis, which constitutes a health problem in many countries.

Comparative evaluation of a simple and sensitive assay for detection of orthomyxo and paramyxoviruses

Studies were done to evaluate comparatively the traditional HA assay and a more recently introduced lectin-neuraminidase (LN) methodologyin search of a simple and sensitive assay for virus detection during laboratorial diagnosis. The results proved the value of LN assay as a sensitive methodologyfor detection of virus particles, presenting results at least equal to those obtained by HA (hemagglutination) assay, with significant values of accumulated frequencies for LN/HA factors (ratios between LN and HA titers) higher than two. The accumulated values of frequencies for LN/HA factors as high as four were very significant, 72.7 (per cent) for influenzavirus and 60.7 (per cent) for Newcastle disease virus (NDV), moreover accumulated frequencies for LN/HA factors even as high as 32 were due to influenzavirus (45.4 per cent) and NDV (7.2 per cent) samples. After the storage period, most of those concentraded samples that even did not present HA titers could be detected through LN assay, demonstrating a lower threshold for virus detection.

A sensitive, specific and reproducible real-time polymerase chain reaction method for detection of Plasmodium vivaxandPlasmodium falciparum infection in field-collected anophelines

We describe a simple method for detection of Plasmodium vivaxand Plasmodium falciparum infection in anophelines using a triplex TaqMan real-time polymerase chain reaction (PCR) assay (18S rRNA). We tested the assay on Anopheles darlingi and Anopheles stephensi colony mosquitoes fed withPlasmodium-infected blood meals and in duplicate on field collected An. darlingi. We compared the real-time PCR results of colony-infected and field collected An. darlingi, separately, to a conventional PCR method. We determined that a cytochromeb-PCR method was only 3.33% as sensitive and 93.38% as specific as our real-time PCR assay with field-collected samples. We demonstrate that this assay is sensitive, specific and reproducible.