Desenvolvimento de imunossensor eletroquímico para a detecção de Salmonella sp. em leite a partir da goma do cajueiro carboximetilada.

Tese (Doutorado em Biotecnologia) - Rede Nordeste de Biotecnologia, Universidade Estadual do Ceará, Fortaleza. Co-orientadora: Roselayne Ferro Furtad

Bacterial sensors and controllers based on organic bioelectronics

Bacterial infections and contaminations are worldwide problems, leading to morbidity and mortality, food waste and economic losses in a variety of industries. The situation is aggravated by the increased occurrence of antibiotic-resistant strains, identified by the WHO as one of the biggest threats to development, food security and public health today. The solution to this problem is complex and requires efforts from several different layers of the society, and different disciplines. The knowledge about microbiology has greatly advanced in the last decades and several powerful methods were introduced. However, in most clinical microbiology laboratories, culture-based techniques are still standard practice, representing a bottleneck in the diagnostic workflow. In this thesis, we prototype novel methods to detect and identify bacteria, aiming to reduce the time and workload for future microbiology research and diagnostics. Furthermore, a new methodology is devised to evaluate antimicrobial surface properties for relevant high-touch surfaces. In Paper I, we investigated whether conducting polymers can be applied for label-free electrochemical detection of bacteria. Employing a poly(3,4-ethylenedioxythiophene): polystyrenesulfonate (PEDOT:PSS)-based two-electrode sensor we demonstrated that potentiometric detection and quantification of Salmonella Enteritidis is possible within 15 min, without any sample pre-treatment. We show that the reduction of PEDOT:PSS electrode occurs by low molecular weight species secreted by Salmonella Enteritidis. To evaluate the genericity of the sensor, several uropathogenic strains were tested and we found that they could all be detected using the sensor. In its current form, the sensor is a prototype, and we aim to improve its sensitivity and introduce specificity. Electroactivity was shown to be a rather common characteristic of bacteria and consequentially, electrochemical methods for detection and characterization of microbes are gaining momentum. We envision that this field will provide novel diagnostic devices but also contribute to discoveries in basic science. Luminescent conjugated oligothiophenes, called optotracers, have previously been applied in microbiology to visualize extracellular matrix components in biofilms of Salmonella and Escherichia coli. In Paper II, we investigated the use of optotracers for detection and visualization of Staphylococcus aureus (S. aureus). We show that the optotracer HS-167 selectively binds to Staphylococci and can be used for fluorometric detection and quantification of S. aureus, as well as for staining and visualization using confocal microscopy. HS-167 displays an on-switch of fluorescence upon binding and it does not affect bacterial growth, which enabled us to develop a high-throughput assay where the fluorescence was plotted against bacterial density, measured as an increase in turbidity. The resulting slope was a quantifiable variable that we employed to compare binding of HS-167 to different species and strains. Diverse approaches collectively pointed to the cell envelope as the target for HS-167 binding. Finally, we showed that binding is highly dependent on the environmental conditions and those can be adjusted to tune the selectivity of HS-167. To improve optotracer design for detection of S. aureus, a better insight into the structure- function relationship is needed. In Paper III, we set out to establish a structured approach to optotracer screening that would enable us to compare optotracer performance. As we compared a library of ten different optotracers, we identified the length to be positively correlated and the total negative charge to be negatively correlated with the ability to detect S. aureus. A balance between the two was necessary to achieve the highest signal while maintaining selectivity. Selected optotracers were added to S. aureus and visualized under the confocal microscope. All localized in the cell envelope of the bacterium, as was previously observed for HS-167 (Paper II). We foresee that further insight into the binding mechanism will enable targeted optotracer design, and together with optimized assay conditions, specific detection of different bacterial species. Copper is known to possess antimicrobial properties, yet studies have reported discrepant results on its efficacy, especially in the clinical settings. Disagreeing results were ascribed to the lack of a standardized approach to evaluate the antimicrobial properties of copper surfaces. In Paper IV, we establish a multifaceted approach to address the effect of human touch, which we simulate by applying artificial sweat, on surface corrosion and antimicrobial properties of copper. We found that artificial sweat accelerates corrosion, leading to changes in surface appearance and wettability. Corrosion did not negatively affect the antimicrobial properties of copper as these surfaces killed bacteria within minutes, regardless of ageing or corrosion product formation. The antimicrobial effect is ascribed in part to copper ions released from the surface and in part to direct surface contact. To further validate the results of this study, other bacterial species need to be tested. Since high touch surfaces are likely to collect a lot of microbes over time, it would be of interest to determine how the bacterial load affects the antimicrobial properties

Electrochemical biosensor based on microfabricated electrode arrays for life sciences applications

In developing a biosensor, the utmost important aspects that need to be emphasized are the specificity and selectivity of the transducer. These two vital prerequisites are of paramount in ensuring a robust and reliable biosensor. Improvements in electrochemical sensors can be achieved by using microelectrodes and to modify the electrode surface (using chemical or biological recognition layers to improve the sensitivity and selectivity). The fabrication and characterisations of silicon-based and glass-based gold microelectrode arrays with various geometries (band and disc) and dimension (ranging from 10 μm-100 nm) were reported. It was found that silicon-based transducers of 10 μm gold microelectrode array exhibited the most stable and reproducible electrochemical measurements hence this dimension was selected for further study. Chemical electrodeposition on both 10 μm microband and microdisc were found viable by electro-assisted self-assembled sol-gel silica film and nanoporous-gold electrodeposition respectively. The fabrication and characterisations of on-chip electrochemical cell was also reported with a fixed diameter/width dimension and interspacing variation. With this regard, the 10 μm microelectrode array with interspacing distance of 100 μm exhibited the best electrochemical response. Surface functionalisations on single chip of planar gold macroelectrodes were also studied for the immobilisation of histidine-tagged protein and antibody. Imaging techniques such as atomic force microscopy, fluorescent microscopy or scanning electron microscope were employed to complement the electrochemical characterisations. The long-chain thiol of self-assembled monolayer with NTA-metal ligand coordination was selected for the histidine-tagged protein while silanisation technique was selected for the antibody immobilisation. The final part of the thesis described the development of a T-2 labelless immunosensor using impedimetric approach. Good antibody calibration curve was obtained for both 10 μm microband and 10 μm microdisc array. For the establishment of the T-2/HT-2 toxin calibration curve, it was found that larger microdisc array dimension was required to produce better calibration curve. The calibration curves established in buffer solution show that the microelectrode arrays were sensitive and able to detect levels of T-2/HT-2 toxin as low as 25 ppb (25 μg kg-1) with a limit of quantitation of 4.89 ppb for a 10 μm microband array and 1.53 ppb for the 40 μm microdisc array

Biosensor development for the analysis of food quality

This thesis describes the development and evaluation of a number of biosensors for food applications. The first part of this thesis deals with the development of Surface Plasmon Resonance (SPR) biosensor systems, coupled with Polymerase Chain Reaction (PCR) for the detection of GMO related amplified nucleic acids in foodstuffs. The first SPR Biosensor described, used streptavidin-biotin linkage chemistry to attach a P35S nucleic acid probe on dextran-coated SPR transducer chips. Methodologies were developed for both the PCR stage and post-PCR sample preparation for the sensitive, rapid and cost-effective detection of GMO-specific amplified DNA sequences. The final embodiment of the method was an asymmetric PCR amplification system with a simple sample processing step (0.3 M NaOH for 30 min in 20 % v/v formamide). The developed PCR-SPR system was successfully applied to the screening of samples of GMO origin. The second SPR biosensor reported herein, is based on a SPR chip immobilised single-stranded thiolated DNA. The thiolated probe exhibited a hybridisation capacity of 95 RU (Resonance Units) for 100 nM of complementary DNA target and a detection limit of 5 nM. The potential of the current probe system for the detection of symmetrically amplified DNA sequences of short length was subsequently confirmed. The second part of this thesis involved preliminary studies into the development of simple, disposable screen-printed electrodes for the electrochemical determination of glucose and L-amino acids in horticultural products. The dynamic range of the developed biosensors was up to 10 mM for glucose and up to 1 mM for L-leucine determination. The developed glucose biosensor exhibited encouraging analytical performance in fresh fruit samples. However, the L-amino acid oxidase electrodes consistently underestimated the amino acid content of the fruit samples. The latter observation was found to be primarily due to inhibitory components in the matrix.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

A novel gold nanoparticle-based approach for the rapid diagnosis of meningococcal infection

The bacterial meningitis caused by Neisseria meningitidis is responsible for considerable morbidity and mortality throughout the world. Given the limitations of existing diagnostic tests and the severity of the illness associated with the disease, there is a clear requirement for a rapid and specific diagnostic assay. This thesis describes the development of nanoparticle based tests for the detection of Neisseria meningitidis specific cell surface markers. As an initial target antigen, a recombinant form of highly conserved outer membrane protein, OMP85 was used. Within the OMP85 protein sequence, a predicted antigenic sequence between residues 720 and 745 was identified and found to be unique to this organism. This amino acid sequence was synthesised as peptide (SR1) with a gly-gly-cysteine spacer sequence at the N-terminus using t-boc chemistry. Also, the major virulence factor, capsular polysaccharide of N. meningitidis serogroup B bacteria was purified. Polyclonal antibodies were raised against purified OMP85 antigen in rabbits and against SR1 peptide and also against formalin inactivated N. meningitidis serogroup B whole cell bacteria in sheep. This panel of different antibodies including the commercial anti-capsular monoclonal antibodies were examined for cross reactivity against a range of closely related Gram negative bacteria. Based on these cross-reactivity studies, a highly specific anti-NM antibody was developed following purification of the anti-SR1 antiserum by immuno-affinity chromatography. Purified OMP85 antigen and anti-OMP85 antibody were successfully conjugated on 13, 30, 40, 50 and 60 nm gold nanoparticles by an electrostatic adsorption method. Coupling of the gold nanoparticles results in a shift of the respective surface plasmon peak toward longer wavelengths (in the range of 600-800 nm) resulting in a change of the colour of the colloidal suspension from red to purple to blue. An attempt was made to develop a rapid diagnostic assay based on gold nanoparticle induced colour shift assay for N. meningitidis by utilising the specific interaction of OMP85 and anti-OMP85 antibody conjugated to gold nanoparticles as a model system. However, this system was not reproducible and is likely to be due to problems with stability of gold nanoparticles during the conjugation process. As an alternative approach, a highly selective quartz crystal microbalance (QCM)-based immunosensor was designed using the same OMP85/anti-OMP85 antibody system. A method was developed using polyvinylidene fluoride (PVDF) coated QCM crystals with protein A for the directional orientation of the antibodies. To further enhance the sensitivity of the test, OMP85-conjugated gold nanoparticles were used as signal amplification probes for the reproducible detection of the target down to 300 ng/mL, corresponding to a five fold increase in sensitivity compared to detection of OMP85 antigen alone. Also, this sensor has successfully been employed to detect whole cell bacteria at a concentration as low as 100 cfu/mL. Thus, in this study using the real-time QCM measurements, a novel strategy has been developed for the sensitive detection of both N. meningitidis bacteria and the protein antigen at very low concentrations, using gold nanoparticles as signal amplification probes

Entwicklung elektrochemischer Biosensoren für die Tumordiagnostik

Die vorliegende Arbeit befasst sich mit der Entwicklung und Anwendung elektrochemischer Biosensoren zur Erweiterung oder zum Ersatz herkömmlicher Diagnostikverfahren. Als Basis für die Biosensoren wurden Elektrodenarraychips entworfen und im Reinraum gefertigt. Die als 9WPtE bezeichneten Elektrodenarrays waren aus 3 x 3 Elektrodenpaaren im 96-well-Maßstab (ANSI-Standard) aufgebaut. Jedes Elektrodenpaar bestand aus einer kreisrunden Arbeitselektrode mit einem Durchmesser von 1,9 mm und einer Gegenelektrode als offenem Kreisring um die Arbeitselektrode mit einem Durchmesser von 7 mm. Außerhalb des Reinraums wurden separate Messkammern und Ag/AgCl-Referenzelektroden integriert. Sowohl das Referenzsystem als auch die Signalqualität der 9WPtE-Elektrodenarraychips wurden mittels Zyklovoltammetrie, Impedanzspektroskopie und Rasterkraftmikroskopie analysiert und anhand dieser Untersuchungen optimiert. Das Augenmerk lag hierbei auf den Produktionsprozessen zur Herstellung der Elektrodenarraychips, auf den Elektrolytbedingungen für die elektrochemischen Messungen und auf der Recyclebarkeit der Chips. Die Funktionalisierung der Arbeitselektroden der 9WPtE-Chips erfolgte mit sich selbst-organisierenden Schichten aus Thiolen. An die Thiole wurden mittels Chemoligation die biologischen Erkennungskomponenten kovalent gekoppelt. Mit dem 9WPtE-Elektrodenarray wurde auf diese Weise ein funktionsfähiger kompetitiver Immunosensoren gegen den Tumormarker Tenascin C entwickelt. Außerdem wurden der 9WPtE-Chip und ein zusätzlich entwickelter Durchflusssensor, basierend auf dem Prinzip des 9WPtE, genutzt, um die Möglichkeit der Detektion ganzer eukaryotischer Zellen zu untersuchen

Preparation, Physico-Chemical Properties and Biomedical Applications of Nanoparticles

Nowadays, the impact of nanotechnology on applications in medicine and biomedical sciences has broader societal and economic effects, enhancing awareness of the business, regulatory, and administrative aspects of medical applications. The selected papers included in the present Special Issue gives readers a critical, balanced and realistic evaluation of existing nanomedicine developments and future prospects, allowing practitioners to plan and make decisions.The topics of this book covers the use of nanoparticles and nanotechnology in medical applications including biomaterials for tissue regeneration, diagnosis and monitoring, surgery, prosthetics, drug delivery systems, nanocarriers, and wound dressing. I would like to express my gratitude to all contributors to this issue, who have given so much of their time and effort to help create this collection of high quality papers