Assay development for identifying inhibitors of the mycobacterial FadD32 activity

FadD32, a fatty acyl-AMP ligase (FAAL32) involved in the biosynthesis of mycolic acids, major and specific lipid components of the mycobacterial cell envelope, is essential for the survival of Mycobacterium tuberculosis, the causative agent of tuberculosis. The protein catalyzes the conversion of fatty acid to acyl-adenylate (acyl-AMP) in the presence of adenosine triphosphate and is conserved in all the mycobacterial species sequenced so far, thus representing a promising target for the development of novel antituberculous drugs. Here, we describe the optimization of the protein purification procedure and the development of a high-throughput screening assay for FadD32 activity. This spectrophotometric assay measuring the release of inorganic phosphate was optimized using the Mycobacterium smegmatis FadD32 as a surrogate enzyme. We describe the use of Tm (melting temperature) shift assay, which measures the modulation of FadD32 thermal stability, as a tool for the identification of potential ligands and for validation of compounds as inhibitors. Screening of a selected library of compounds led to the identification of five novel classes of inhibitors.

Remote, subsurface detection of the algal toxin domoic acid onboard the Environmental Sample Processor: Assay development and field trials

The ability to detect harmful algal bloom (HAB) species and their toxins in real- or near real-time is a critical need for researchers studying HAB/toxin dynamics, as well as for coastal resource managers charged with monitoring bloom populations in order to mitigate their wide ranging impacts. The Environmental Sample Processor (ESP), a robotic electromechanical/fluidic system, was developed for the autonomous, subsurface application of molecular diagnostic tests and has successfully detected several HAB species using DNA probe arrays during field deployments. Since toxin production and thus the potential for public health and ecosystem effects varies considerably in natural phytoplankton populations, the concurrent detection of HAB species and their toxins onboard the ESP is essential. We describe herein the development of methods for extracting the algal toxin domoic acid (DA) from Pseudonitzschia cells (extraction efficiency >90%) and testing of samples using a competitive ELISA onboard the ESP. The assay detection limit is in the low ng/mL range (in extract), which corresponds to low ng/L levels of DA in seawater for a 0.5 L sample volume acquired by the ESP. We also report the first in situ detection of both a HAB organism (i.e., Pseudo-nitzschia) and its toxin, domoic acid, via the sequential (within 2-3 h) conduct of species- and toxin-specific assays during ESP deployments in Monterey Bay, CA, USA. Efforts are now underway to further refine the assay and conduct additional calibration exercises with the aim of obtaining more reliable, accurate estimates of bloom toxicity and thus their potential impacts. Published by Elsevier B.V.

Microarray-formatted clinical biomarker assay development using peptide aptamers to anterior gradient-2

Anterior gradient-2 protein was identified using proteomic technologies as a p53 inhibitor which is overexpressed in human cancers, and this protein presents a novel pro-oncogenic target with which to develop diagnostic assays for biomarker detection in clinical tissue. Combinatorial phage-peptide libraries were used to select 12 amino acid polypeptide aptamers toward anterior gradient-2 to determine whether methods can be developed to affinity purify the protein from clinical biopsies. Selecting phage aptamers through four rounds of screening on recombinant human anterior gradient-2 protein identified two classes of peptide ligand that bind to distinct epitopes on anterior gradient-2 protein in an immunoblot. Synthetic biotinylated peptide aptamers bound in an ELISA format to anterior gradient-2, and substitution mutagenesis further minimized one polypeptide aptamer to a hexapeptide core. Aptamers containing this latter consensus sequence could be used to affinity purify to homogeneity human anterior gradient-2 protein from a single clinical biopsy. The spotting of a panel of peptide aptamers onto a protein microarray matrix could be used to quantify anterior gradient-2 protein from crude clinical biopsy lysates, providing a format for quantitative screening. These data highlight the utility of peptide combinatorial libraries to acquire rapidly a high-affinity ligand that can selectively bind a target protein from a clinical biopsy and provide a technological approach for clinical biomarker assay development in an aptamer microarray format.

Development of a rapid low cost fluorescent biosensor for the detection of food contaminants

A prototype fluorescent based biosensor has been developed for the antibody based detection of food related contaminants. Its performance was characterised and showed a typical antibody binding signal of 200-2000 mV, a short term noise of 9.1 mV, and baseline slope of -0.016 mV/s over 4 h. Bulk signal detection repeatability (n=23) and reproducibility (n=3) were less than 2.4%CV. The biosensor detection unit was evaluated using two food related model systems proving its ability to monitor both binding using commercial products and inhibition through the development of an assay. This assay development potential was evaluated by observing the biosensor's performance whilst appraising several labelled antibody and glass slide configurations. The molecular interaction between biotin and an anti-biotin antibody was shown to be inhibited by 41% due to the presence of biotin in a sample. A food toxin (domoic acid) calibration curve was produced, with %CVs ranging from 2.7 to 7.8%, and a midpoint of approximately 17 ng/ml with further optimisation possible. The ultimate aim of this study was to demonstrate the working principles of this innovative biosensor as a potential portable tool with the opportunity of interchangeable assays. The biosensor design is applicable for the requirements of routine food contaminant analysis, with respect to performance, functionality and cost. (C) 2012 Elsevier B.V. All rights reserved.

Development and optimisation of photonic crystal based nanosensors

This thesis involved the development of two Biosensors and their associated assays for the detection of diseases, namely IBR and BVD for veterinary use and C1q protein as a biomarker to pancreatic cancer for medical application, using Surface Plasmon Resonance (SPR) and nanoplasmonics. SPR techniques have been used by a number of groups, both in research [1-3] and commercially [4, 5] , as a diagnostic tool for the detection of various biomolecules, especially antibodies [6-8]. The biosensor market is an ever expanding field, with new technology and new companies rapidly emerging on the market, for both human [8] and veterinary applications [9, 10]. In Chapter 2, we discuss the development of a simultaneous IBR and BVD virus assay for the detection of antibodies in bovine serum on an SPR-2 platform. Pancreatic cancer is the most lethal cancer by organ site, partially due to the lack of a reliable molecular signature for diagnostic testing. C1q protein has been recently proposed as a biomarker within a panel for the detection of pancreatic cancer. The third chapter discusses the fabrication, assays and characterisation of nanoplasmonic arrays. We will talk about developing C1q scFv antibody assays, clone screening of the antibodies and subsequently moving the assays onto the nanoplasmonic array platform for static assays, as well as a custom hybrid benchtop system as a diagnostic method for the detection of pancreatic cancer. Finally, in chapter 4, we move on to Guided Mode Resonance (GMR) sensors, as a low-cost option for potential use in Point-of Care diagnostics. C1q and BVD assays used in the prior formats are transferred to this platform, to ascertain its usability as a cost effective, reliable sensor for diagnostic testing. We discuss the fabrication, characterisation and assay development, as well as their use in the benchtop hybrid system.

Large-scale interlaboratory study to develop, analytically validate and apply highly multiplexed, quantitative peptide assays to measure cancer-relevant proteins in plasma

There is an increasing need in biology and clinical medicine to robustly and reliably measure tens-to-hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma, and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and 7 control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to sub-nanogram/mL sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and inter-laboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy isotope labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an inter-laboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality c`ontrol measures, enables sensitive, specific, reproducible and quantitative measurements of proteins and peptides in complex biological matrices such as plasma.

Drug discovery approaches utilizing three-dimensional cell culture

Historically, two-dimensional (2D) cell culture has been the preferred method of producing disease models in vitro. Recently, there has been a move away from 2D culture in favor of generating three-dimensional (3D) multicellular structures, which are thought to be more representative of the in vivo environment. This transition has brought with it an influx of technologies capable of producing these structures in various ways. However, it is becoming evident that many of these technologies do not perform well in automated in vitro drug discovery units. We believe that this is a result of their incompatibility with high-throughput screening (HTS). In this study, we review a number of technologies, which are currently available for producing in vitro 3D disease models. We assess their amenability with high-content screening and HTS and highlight our own work in attempting to address many of the practical problems that are hampering the successful deployment of 3D cell systems in mainstream research.

Mechanistic studies of reactions at the single-molecule level using microfluidics with applications in molecular diagnostics

Motivated by needs in molecular diagnostics and advances in microfabrication, researchers started to seek help from microfluidic technology, as it provides approaches to achieve high throughput, high sensitivity, and high resolution. One strategy applied in microfluidics to fulfill such requirements is to convert continuous analog signal into digitalized signal. One most commonly used example for this conversion is digital PCR, where by counting the number of reacted compartments (triggered by the presence of the target entity) out of the total number of compartments, one could use Poisson statistics to calculate the amount of input target.

However, there are still problems to be solved and assumptions to be validated before the technology is widely employed. In this dissertation, the digital quantification strategy has been examined from two angles: efficiency and robustness. The former is a critical factor for ensuring the accuracy of absolute quantification methods, and the latter is the premise for such technology to be practically implemented in diagnosis beyond the laboratory. The two angles are further framed into a “fate” and “rate” determination scheme, where the influence of different parameters is attributed to fate determination step or rate determination step. In this discussion, microfluidic platforms have been used to understand reaction mechanism at single molecule level. Although the discussion raises more challenges for digital assay development, it brings the problem to the attention of the scientific community for the first time.

This dissertation also contributes towards developing POC test in limited resource settings. On one hand, it adds ease of access to the tests by incorporating massively producible, low cost plastic material and by integrating new features that allow instant result acquisition and result feedback. On the other hand, it explores new isothermal chemistry and new strategies to address important global health concerns such as cyctatin C quantification, HIV/HCV detection and treatment monitoring as well as HCV genotyping.

Paralytic shellfish poisoning (PSP) toxin binders for optical biosensor technology: problems and possibilities for the future: a review

This review examines the developments in optical biosensor technology, which uses the phenomenon of surface plasmon resonance, for the detection of paralytic shellfish poisoning (PSP) toxins. Optical biosensor technology measures the competitive biomolecular interaction of a specific biological recognition element or binder with a target toxin immobilised onto a sensor chip surface against toxin in a sample. Different binders such as receptors and antibodies previously employed in functional and immunological assays have been assessed. Highlighted are the difficulties in detecting this range of low molecular weight toxins, with analogues differing at four chemical substitution sites, using a single binder. The complications that arise with the toxicity factors of each toxin relative to the parent compound, saxitoxin, for the measurement of total toxicity relative to the mouse bioassay are also considered. For antibodies, the cross-reactivity profile does not always correlate to toxic potency, but rather to the toxin structure to which it was produced. Restrictions and availability of the toxins makes alternative chemical strategies for the synthesis of protein conjugate derivatives for antibody production a difficult task. However, when two antibodies with different cross-reactivity profiles are employed, with a toxin chip surface generic to both antibodies, it was demonstrated that the cross-reactivity profile of each could be combined into a single-assay format. Difficulties with receptors for optical biosensor analysis of low molecular weight compounds are discussed, as are the potential of alternative non-antibody-based binders for future assay development in this area.

First report of the use of a saxitoxin-protein conjugate to develop a DNA aptamer to a small molecule toxin

Saxitoxin (STX) is a low molecular weight neurotoxin mainly produced by certain marine dinoflagellates that, along with its family of similarly related paralytic shellfish toxins, may cause the potentially fatal intoxication known as paralytic shellfish poisoning. Illness and fatality rates are low due to the effective monitoring programs that determine when toxins exceed the established regulatory action level and effectuate shellfish harvesting closures accordingly. Such monitoring programs rely on the ability to rapidly screen large volumes of samples. Many of the screening assays currently available employ antibodies or live animals. This research focused on developing an analytical recognition element that would eliminate the challenges associated with the limited availability of antibodies and the use of animals. Here we report the discovery of a DNA aptamer that targets STX. Concentration-dependent and selective binding of the aptamer to STX was determined using a surface plasmon resonance sensor. Not only does this work represent the first reported aptamer to STX, but also the first aptamer to any marine biotoxin. A novel strategy of using a toxin-protein conjugate for DNA aptamer selection was successfully implemented to overcome the challenges associated with aptamer selection to small molecules. Taking advantage of such an approach could lead to increased diversity and accessibility of aptamers to low molecular weight toxins, which could then be incorporated as analytical recognition elements in diagnostic assays for foodborne toxin detection. The selected STX aptamer sequence is provided here, making it available to any investigator for use in assay development for the detection of STX.

An evaluation of the capability of a biolayer interferometry biosensor to detect low-molecular-weight food contaminants

The safety of our food is an essential requirement of society. One well-recognised threat is that of chemical contamination of our food, where low-molecular-weight compounds such as biotoxins, drug residues and pesticides are present. Low-cost, rapid screening procedures are sought to discriminate the suspect samples from the population, thus selecting only these to be forwarded for confirmatory analysis. Many biosensor assays have been developed as screening tools in food contaminant analysis, but these tend to be electrochemical, fluorescence or surface plasmon resonance based. An alternative approach is the use of biolayer interferometry, which has become established in drug discovery and life science studies but is only now emerging as a potential tool in the analysis of food contaminants. A biolayer interferometry biosensor was assessed using domoic acid as a model compound. Instrument repeatability was tested by simultaneously producing six calibration curves showing replicate repeatability (n = 2) ranging from 0.1 to 6.5 % CV with individual concentration measurements (n = 12) ranging from 4.3 to 9.3 % CV, giving a calibration curve midpoint of 7.5 ng/ml (2.3 % CV (n = 6)). Reproducibility was assessed by producing three calibration curves on different days, giving a midpoint of 7.5 ng/ml (3.4 %CV (n = 3)). It was further shown, using assay development techniques, that the calibration curve midpoint could be adjusted from 10.4 to 1.9 ng/ml by varying assay parameters before the simultaneous construction of three calibration curves in matrix and buffer. Sensitivity of the assay compared favourably with previously published biosensor data for domoic acid. © 2013 Springer-Verlag Berlin Heidelberg.

An optimized reverse-phase high performance liquid chromatographic method for evaluating percutaneous absorption of glucosamine hydrochloride

A relatively simple, selective, precise and accurate high performance liquid chromatography (HPLC) method based on a reaction of phenylisothiocyanate (PITC) with glucosamine (GL) in alkaline media was developed and validated to determine glucosamine hydrochloride permeating through human skin in vitro. It is usually problematic to develop an accurate assay for chemicals traversing skin because the excellent barrier properties of the tissue ensure that only low amounts of the material pass through the membrane and skin components may leach out of the tissue to interfere with the analysis. In addition, in the case of glucosamine hydrochloride, chemical instability adds further complexity to assay development. The assay, utilising the PITC-GL reaction was refined by optimizing the reaction temperature, reaction time and PITC concentration. The reaction produces a phenylthiocarbamyl-glucosamine (PTC-GL) adduct which was separated on a reverse-phase (RP) column packed with 5 microm ODS (C18) Hypersil particles using a diode array detector (DAD) at 245 nm. The mobile phase was methanol-water-glacial acetic acid (10:89.96:0.04 v/v/v, pH 3.5) delivered to the column at 1 ml min-1 and the column temperature was maintained at 30 degrees C. Galactosamine hydrochloride (Gal-HCl) was used as an internal standard. Using a saturated aqueous solution of glucosamine hydrochloride, in vitro permeation studies were performed at 32+/-1 degrees C over 48 h using human epidermal membranes prepared by a heat separation method and mounted in Franz-type diffusion cells with a diffusional area 2.15+/-0.1 cm2. The optimum derivatisation reaction conditions for reaction temperature, reaction time and PITC concentration were found to be 80 degrees C, 30 min and 1% v/v, respectively. PTC-Gal and GL adducts eluted at 8.9 and 9.7 min, respectively. The detector response was found to be linear in the concentration range 0-1000 microg ml-1. The assay was robust with intra- and inter-day precisions (described as a percentage of relative standard deviation, %R.S.D.) <12. Intra- and inter-day accuracy (as a percentage of the relative error, %RE) was <or=-5.60 and <or=-8.00, respectively. Using this assay, it was found that GL-HCl permeates through human skin with a flux 1.497+/-0.42 microg cm-2 h-1, a permeability coefficient of 5.66+/-1.6x10(-6) cm h-1 and with a lag time of 10.9+/-4.6 h.

Assessment of Tandem Mass Spectrometry and High Resolution Mass Spectrometry for the Analysis of Bupivacaine in Plasma

Triple quadrupole mass spectrometers coupled with high performance liquid chromatography are workhorses in quantitative bioanalyses. It provides substantial benefits including reproducibility, sensitivity and selectivity for trace analysis. Selected Reaction Monitoring allows targeted assay development but data sets generated contain very limited information. Data mining and analysis of non-targeted high-resolution mass spectrometry profiles of biological samples offer the opportunity to perform more exhaustive assessments, including quantitative and qualitative analysis. The objectives of this study was to test method precision and accuracy, statistically compare bupivacaine drug concentration in real study samples and verify if high resolution and accurate mass data collected in scan mode can actually permit retrospective data analysis, more specifically, extract metabolite related information. The precision and accuracy data presented using both instruments provided equivalent results. Overall, the accuracy was ranging from 106.2 to 113.2% and the precision observed was from 1.0 to 3.7%. Statistical comparisons using a linear regression between both methods reveal a coefficient of determination (R2) of 0.9996 and a slope of 1.02 demonstrating a very strong correlation between both methods. Individual sample comparison showed differences from -4.5% to 1.6% well within the accepted analytical error. Moreover, post acquisition extracted ion chromatograms at m/z 233.1648 ± 5 ppm (M-56) and m/z 305.2224 ± 5 ppm (M+16) revealed the presence of desbutyl-bupivacaine and three distinct hydroxylated bupivacaine metabolites. Post acquisition analysis allowed us to produce semiquantitative evaluations of the concentration-time profiles for bupicavaine metabolites.

An optimized reverse-phase high performance liquid chromatographic method for evaluating percutaneous absorption of glucosamine hydrochloride

A relatively simple, selective, precise and accurate high performance liquid chromatography (HPLC) method based on a reaction of phenylisothiocyanate (PITC) with glucosamine (GL) in alkaline media was developed and validated to determine glucosamine hydrochloride permeating through human skin in vitro. It is usually problematic to develop an accurate assay for chemicals traversing skin because the excellent barrier properties of the tissue ensure that only low amounts of the material pass through the membrane and skin components may leach out of the tissue to interfere with the analysis. In addition, in the case of glucosamine hydrochloride, chemical instability adds further complexity to assay development. The assay, utilising the PITC-GL reaction was refined by optimizing the reaction temperature, reaction time and PITC concentration. The reaction produces a phenylthiocarbarnyl-glucosamine (PTC-GL) adduct which was separated on a reverse-phase (RP) column packed with 5 mu m ODS (C-18) Hypersil particles using a diode array detector (DAD) at 245 nm. The mobile phase was methanol-water-glacial acetic acid (10:89.96:0.04 v/v/v, pH 3.5) delivered to the column at 1 ml min(-1) and the column temperature was maintained at 30 degrees C Using a saturated aqueous solution of glucosamine hydrochloride, in vitro permeation studies were performed at 32 +/- 1 degrees C over 48 h using human epidermal membranes prepared by a heat separation method and mounted in Franz-type diffusion cells with a diffusional area 2.15 +/- 0.1 cm(2). The optimum derivatisation reaction conditions for reaction temperature, reaction time and PITC concentration were found to be 80 degrees C, 30 min and 1 % v/v, respectively. PTC-Gal and GL adducts eluted at 8.9 and 9.7 min, respectively. The detector response was found to be linear in the concentration range 0-1000 mu g ml(-1). The assay was robust with intra- and inter-day precisions (described as a percentage of relative standard deviation, %R.S.D.) < 12. Intra- and inter-day accuracy (as a percentage of the relative error, %RE) was <=-5.60 and <=-8.00, respectively. Using this assay, it was found that GL-HCI permeates through human skin with a flux 1.497 +/- 0.42 mu g cm(-2) h(-1), a permeability coefficient of 5.66 +/- 1.6 x 10(-6) cm h(-1) and with a lag time of 10.9 +/- 4.6 h. (c) 2005 Elsevier B.V. All rights reserved.

Co-digestão dos dejetos de suínos e óleo vegetal de descarte: adição de microrganismos e lipases comercias

Pós-graduação em Zootecnia - FCAV