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Nous dissenys biomoleculars en genosensors i immunosensors per a la seguretat alimentàriaLermo Soria, Ana Isabel 29 June 2009 (has links)
Al camp alimentari hi ha una creixent demanda de metodologies analítiques que siguin ràpides, selectives i econòmiques, que permetin la detecció de diferents contaminants alimentaris per a garantir la seguretat del producte al llarg de la cadena alimentària de producció. En aquest context, els biosensors electroquímics s'alcen com a candidats ideals com alternativa a la instrumentació analítica convencional per a l'anàlisi fora de l'àmbit del laboratori. En aquesta tesi es van desenvolupar nous dissenys biomoleculars basats en biosensors electroquímics per a la seguretat alimentària abordant tres aspectes crítics dels dispositius biosensors, que convergeixen cap a la simplificació metodològica: la immobilització orientada del biomaterial, la marcació i la transducció del senyal electroquímic. Donada l'experiència del nostre grup d'investigació en la fabricació de compòsits de grafit-epoxi (GEC) i els avantatges electroquímics demostrats d'aquest material, es van construir nous transductors electroquímics basats en compòsits rígids per a la immobilització orientada de biomolècules per aconseguir la simplificació metodològica en la detecció de DNA i immunoespècies. Els nous transductors construïts es van basar en: i) biocompòsits de grafit-epoxi modificats en volum amb la proteïna avidina (Av-GEB), i ii) compòsits amb un element magnètic integrat (m-GEC). Els biocompòsits d'avidina constitueixen una plataforma universal per a la immobilització directa orientada de material biològic biotinilat, mitjançant la forta unió avidina-biotina. El propi transductor actua com a reservori de material biològic i es pot renovar amb un simple procediment de polit, obtenint una nova superfície per cada assaig. Els magneto elèctrodes permeten la integració de partícules magnètiques als procediments biosensors per tal de realitzar les reaccions biològiques de biorreconeixement en solució, reduint la complexitat de la matriu de les mostres i preconcentrant l'anàlit, obtenint així, una detecció més sensible. Es va dissenyar una nova estratègia de detecció de DNA provinent de mostres reals dels bacteris patògens Salmonella i Escherichia coli, mitjançant la incorporació d'encebadors marcats a la mescla de reacció de la PCR. A part d'amplificar la quantitat de DNA, es va aconseguir marcar doblement el producte amplificat amb biotina i digoxigenina, per aconseguir la immobilització posterior a partícules magnètiques recobertes d'estreptavidina o Av-GEB i la unió d'un conjugat enzimàtic (antiDIG-HRP) per a detectar el producte amplificat. Així es van obtenir uns protocols simples, ràpids i molt sensibles. Aquests protocols van proporcionar límits de detecció més baixos en comparació amb la tècnica clàssica de gel d'electroforesi i la PCR quantitativa amb sistema TaqMan. També es van obtenir resultats molt prometedors incorporant a la PCR un encebador magnètic, encebador unit a partícules magnètiques. Així, es va aconseguir l'amplificació directa a la superfície de les partícules magnètiques, simplificant la metodologia. Aquesta estratègia presenta potencial aplicabilitat per a la detecció directa electroquímica de productes amplificats al termociclador de la PCR. A l'últim bloc de la tesi es va desenvolupar una estratègia immunosensora simplificada amb detecció electroquímica, en la que l'haptè es va immobilitzar a partícules magnètiques recobertes de grups químics tosil, formant un enllaç covalent amb la molècula de naturalesa haptènica. Amb aquesta estratègia es va aconseguir quantificar l'àcid fòlic en mostres cegues de llet enriquida, obtenint valors de recuperació propers al 100%; i quantificar l'àcid fòlic en mostres reals de llet enriquida, obtenint valors molt propers als indicats a la composició dels productes. / The control of food quality along the food production chain has become of growing interest since the increasing incidence of food poisoning is a significant public health concern for customers worldwide. The development of new analytical methodologies with the advantages of rapid response, selectivity and low-cost is still a challenge for food hygiene inspection. Biosensors offer an exciting alternative to the traditional methods allowing decentralised analysis. Novel biomolecular approaches in electrochemical biosensing were developed in this thesis. The main features of these novel biosensing devices were focused on the integration of processes and the methodological simplification. These tasks were achieved considering the main critical aspects of biosensors design: the orientated immobilization of the biorecognition element, the labelling process to achieve the analytical signal and finally, the electrochemical transduction. Novel electrochemical transducers based on graphite-epoxy composites (GEC) were developed. The enhanced electrochemical properties of GEC material has been demonstrated previously in our research group. The novel transducers based on rigid composites were used for the simplified and oriented immobilization of biomolecules such as DNA and immunospecies. The new transducers developed were: i) graphite-epoxy biocomposites bulk-modified with avidin (Av-GEB), and ii) magneto electrodes based on graphite-epoxy composite (m-GEC). Avidin biocomposites can be considered as universal platforms for the oriented immobilization of biotinilated biomolecules, through the strong interaction between avidin and biotin. This biocomposite material acts not only as the electrochemical transducer but also as a reservoir for the biological material, being able to be easily renewed by a polish treatment, obtaining a new surface for each assay. The m-GEC magneto electrodes allow the integration of magnetic particles in biosensing procedures. The biorecognition reaction can thus be effectively performed in solution, reducing the matrix effect of the sample and preconcentrating the analyte, as a result these factors enhance the sensitivity of the detection. A sensitive, rapid and user-friendly strategy for the genetic detection of the main food pathogens - Salmonella and Escherichia coli- was designed. This approach was based on a double-tagging PCR by using two labelled primers, followed by electrochemical genosensing of the double-tagged amplicon. During PCR, not only the amplification of the genetic material was achieved, but also the double-labelling of the amplicon ends with both biotin and digoxigenin tags, in order to achieve the immobilization of the amplicon on streptavidin-modified beads or Av-GEB electrodes as well as the binding of an enzymatic conjugate (antiDIG-HRP) for the electrochemical detection. Better results were achieved in terms of limit of detection compared with the classic gel electrophoresis as well as quantitative PCR based on TaqMan probes. Promising results were also obtained with the use of a novel magnetic primer, primer bound to magnetic beads. This approach allows the DNA direct amplification on streptavidin magnetic beads surface. This strategy showed potential applicability for the direct electrochemical detection of the amplified products in the PCR termocycler. Finally, a simple immunosensing strategy with electrochemical detection was developed. In this case, the haptenic molecule was covalently immobilised on tosylactivated magnetic beads. The utility of this strategy was demonstrated for folic acid detection in spiked milk samples, obtaining recovery values around 100%. Moreover, folic acid was also quantified in real enriched milk samples showing results according to the expected labelled values.
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Development of an Acoustic Wave Based Biosensor for Vapor Phase Detection of Small MoleculesStubbs, Desmond Dion 03 November 2005 (has links)
For centuries scientific ingenuity and innovation have been influenced by Mother Natures perfect design. One of her more elusive designs is that of the sensory olfactory system, an array of highly sensitive receptors responsible for chemical vapor recognition. In the animal kingdom this ability is magnified among canines where ppt (parts per trillion) sensitivity values have been reported. Today, detection dogs are considered an essential part of the US drug and explosives detection schemes. However, growing concerns about their susceptibility to extraneous odors have inspired the development of highly sensitive analytical detection tools or biosensors known as electronic noses.
In general, biosensors are distinguished from chemical sensors in that they use an entity of biological origin (e.g. antibody, cell, enzyme) immobilized onto a surface as the chemically-sensitive film on the device. The colloquial view is that the term biosensors refers to devices which detect the presence of entities of biological origin, such as proteins or single-stranded DNA and that this detection must take place in a liquid. Our biosensor utilizes biomolecules, specifically IgG monoclonal antibodies, to achieve molecular recognition of relatively small molecules in the vapor phase.
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Theoretical and Experimental Characterization of Time-Dependent Signatures of Acoustic Wave Based BiosensorsLee, Sang Hun 13 July 2006 (has links)
The object of this thesis research is to facilitate the appraisal and analysis of the signatures of the modern acoustic wave biosensors, as well as to improve the experimental methodology to enhance sensor performance. For this purpose, both theoretical characterization of acoustic wave sensor signatures and experimental studies for the most frequently used acoustic wave biosensors, the liquid phase QCM (quartz crystal microbalance) and the vapor phase SAW (surface acoustic wave) sensors, are presented. For the study of SAW vapor phase detection, the author fabricated different types of two-port SAW resonator sensors on quartz substrates and designed and performed a significant number of detection experiments. These were conducted both with calibrated or known target samples under laboratory conditions at Georgia Tech Hunt Lab and with samples of unknown concentrations such as seized crack cocaine (courtesy of Georgia Bureau of Investigation, GBI) to see the sensors capability to work in the field conditions. In addition, the dependence of the SAW sensor signatures on specific locations of the surface perturbation was investigated to account for some observed abnormal responses. Finally, a novel approach to classify and visualize chemically analogous substances is introduced.
The author expects that the thesis work herein may contribute to the study of the modern acoustic wave biosensors which includes but is not limited to: the establishment of underpinning theory that will aid in the evaluation of the signatures; the practical aspects of design and fabrication of SAW devices specific to the vapor phase immunoassay; the development of efficient experimental methodologies; the strategic immobilization of a biolayer on SAW resonator based biosensors; and, the acquisition of reference data for the development of commercial acoustic wave sensors.
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Design, Synthesis and Characterization of Zinc(II)-Selective Ratiometric Fluorescent SensorsWu, Yonggang 14 November 2007 (has links)
Zinc is an important micronutrient but the biological function of its labile form is poorly understood. Zinc selective fluorescence sensors, recognized as the major tool to gain information about the role of zinc in living systems, have been attracting more and more interest.
The most promising solution currently being studied comes in the form of ratiometric sensors. Unlike sensors based on the switch-on mechanism, ratiometric sensors determine the free metal concentration directly from the ratio of the emission intensities at two wavelengths. The major restriction on the design of this type of sensor is from the necessity for a spectral-shift upon binding metal ions. To develop novel ratiometric sensors, we have developed designs based on excited-state intramolecular proton transfer (ESIPT).
In the absence of ZnII at neutral pH, the 2-(2 -sulfonamidophenyl)benzimidazole family undergoes ESIPT to yield a highly Stokes-shifted emission from the proton-transfer tautomer. Coordination of ZnII inhibits the ESIPT process and yields a significant hypsochromic shift of the fluorescence emission maximum. By implementing structural modifications, we were able to gauge free ZnII concentrations in the millimolar to picomolar range.
To tune the peak excitation towards lower energy, a property that is of particular importance in the light of biological applications, we modified the platform molecule with extended pi-conjugation and by substituent engineering. The position of the modification and the nature of the substituents strongly influenced the photophysical properties of the investigated derivatives. Several fluorophores revealed emission ratiometric properties with a large dynamic range combined with a peak absorption beyond 350 nm, rendering these probes promising candidates for applications.
To further understand the origin of the substituent effect, we studied five derivatives for the solvatochromic shift analysis and quantum chemical studies. The results showed that the negative solvatochromic shift behavior was most pronounced in protic solvents presumably due to specific hydrogen-bonding interactions. The extrapolated gas-phase emission energies correlated qualitatively with the trends in Stokes shifts, suggesting that solute-solvent interactions do not play a significant role in explaining the divergent emission energy shifts. Detailed quantum chemical calculations not only confirmed the moderately polarized nature of the ESIPT tautomers but also provided a rationale for the observed emission shifts based on the differential change in the HOMO and LUMO energies.
This study revealed the great potential of 2-(2 -arylsulfonamidophenyl)- benzimidazoles, such as tunable peak absorption and emission, a very wide dynamic range regarding to zinc binding, very little solvent polarity dependence, and especially, the emission ratiometric property. All these properties make this system a unique candidate to tackle the problems in the research of zinc biology.
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Novel immobilization chemistry for bioaffinity sensors /Yan, Fei. January 2001 (has links)
Thesis (Ph. D.)--State University of New York at Binghamton, Chemistry Department, 2001. / Includes bibliographical references (leaves 209-237).
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Sequential injection analysis for the investigation of biomolecular interactions /Connors, Wendy Lee. January 2000 (has links)
Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 129-140).
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Chemical and biomolecular functionalization of silicon surfaces for biosensing applicationsLapin, Norman A., January 2009 (has links)
Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Biomedical Engineering." Includes bibliographical references.
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Micro-fabrication of a Mach-Zehnder interferometer combining laser direct writing and fountain pen micropatterning for chemical/biological sensing applicationsKallur, Ajay. Wang, Shuping, January 2009 (has links)
Thesis (M.S.)--University of North Texas, May, 2009. / Title from title page display. Includes bibliographical references.
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Immobilization and Characterization of Physisorbed Antibody Films Using Pneumatic Spray as Deposition TechniqueFigueroa, Jhon J. 01 January 2013 (has links)
The immobilization of antibodies on silica surfaces has been a wide and common practice via cross-linking with the formation of covalent bonds between surface and antibody. The formation of antibody thin films on solid surfaces using pneumatic spray (PS) as the deposition technique and the analysis of the surface morphology of these films were investigated during this study. The pneumatic spray method was compared with the covalent bonding method Avidin-Biotin Bridge (ABB). The intensities and capture efficiency tests showed similar results for both techniques with a lower signal-to-noise ratio (SNR) for the PS deposited films. Specificity tests suggested that the bio-sensitivity of the antibody films that were pneumatically sprayed maintained their capture abilities after the immobilization process. Analysis obtained from the attenuated total reflectance Fourier transform infrared ATR-FTIR support these results indicating that the antibodies retained their native structure and chemical stability thorough the induced physisorption process. The pneumatic spray films also preserve mechanical stability by adhering to the surface after the rinsing procedures.
Capture efficiency was tested for both immobilization techniques, the results of which were similar. The pneumatic spray technique was also tested using a diverse range of deposition times. It was shown that a 2 minute deposition time was sufficient to produce a film with similar capture efficiency to the avidin-biotin bridge technique. The surface density obtained for the 2 minute deposition was 9.05 ng/mm2, which is higher than the range of 2.2 to 4.74 ng/mm2 reported for the avidin-biotin bridge technique[1-3]. The contact angle measurements for the pneumatic spray films showed a higher hydrophobicity compared with the avidin-biotin bridge films. This is due to the higher surface roughness obtained for the avidin-biotin bridge films, a higher surface density for the PS and the random orientation of the antibodies in the pneumatic spray films. A study of shelf life showed that the pneumatic spray technique produces stable films that can be used for as long 100 days (study performed only up to 100 days) with similar capture efficiency to those prepared in the same day.
To further understand the improvement in capture efficiency of the pneumatic spray films, the surface morphology was investigated to determine its influence in the cell adhesion process. The surface was characterized by several different techniques: ellipsometry to determine the thickness of the films, atomic force microscopy (AFM) to calculate the surface roughness, optical microscopy to identify particle formation during antibody immobilization process, fluorescent microscopy and sandwich fluorescent immunoassay to observe the immobilization patterns of antibodies and antigens on the surface, contact angle measurements to analyze the wettability of the antibody films and X-ray photoelectron spectroscopy (XPS) to confirm the presence of antibody on both deposition methods and to propose a growth model for the pneumatic spray deposition technique.
A possible explanation for the similar results of capture efficiency for both techniques can be attributed to three main factors. First, the antibodies retained their native structure thorough the induced physisorption process allowing then to capture antigen normally. Second, the lack of orientation of the antibodies in the pneumatic films was compensated by high surface density thereby offering more binding sites to capture antigens. Third, hydrophobic surfaces are favorable to cell adhesion, therefore the high hydrophobicity of the pneumatic spray films increases the capture efficiency. It is important to mention that the time that it takes to produce the immunoassay surfaces was reduced dramatically from more than twenty four hours for the avidin-biotin bridge films to only a few minutes for the pneumatic spray films. In addition, pneumatic spray films significantly reduce the amount of materials and chemicals used in the deposition process. These factors make the pneumatic spray technique an excellent technique for the immobilization of antibodies on glass slides for commercial bio-sensor devices.
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Study of aptamer selection methodologies for developing piezoelectric quartz crystal biosensors to detect albumin in urine, malaria and SARS protein biomarkers in serumAlbano, Dharmatov Rahula Barlongo January 2014 (has links)
Due to the high affinity and selectivity of aptamer selected from DNA library towards target proteins, the use of specific protein biomarkers to assist early diagnosis, and the recent rapid development of piezoelectric quartz crystal (PQC) biosensor technology, the research reported is focused on developing PQC biosensor using aptamers selected from a DNA library as selective coating for the detection of albumin in urine, two protein biomarkers for malaria in sera, and the determination of trace amount of SCV helicase protein from SAR in sera. Three new aptamer-PQC biosensors are developed with research conducted on aptamer selection, optimization of coating methods for aptamers on PQC, and coupling paramagnetic nanoparticle technology with PQC biosensor in flow injection analysis (FIA) to enrich analyte and eliminate sample matrix interference.
A new approach for aptamer selection is successfully attempted in the development of the first aptamer-PQC biosensor with an aim to offer an early detection of microalbuminuria enabled by an increase in detection sensitivity with aptamer selected by its actual binding with albumin-PQC biosensor. The aptamer-PQC sensor developed has shown to give a working range between 0.1 to 10 ug/mL, a detection limit (S/N = 3, n=3) of 0.048 μg/ml, repeatability of RSD = 6.8% (n=3), a response time of 1 minute and a throughput of 60 samples/hour. The developed piezoelectric aptamer sensor is shown to have sufficient sensitivity to detect microgram quantities of albumin in urine.
Aptamer-PQC biosensors are developed for the detection of two protein biomarkers, PfLDH for general diagnosis and PfHRP-2 for a specific diagnosis of Plasmodium falciparum malaria parasitemia, in sera. The results show satisfactory working range from 10-100 ng/mL for both PfLDH and PfHRP-2 protein biomarker, low detection limit of 1.8 and 4.7 ng/mL and satisfactory repeatability (%RSD, n = 3) of 7.4% and 9.2% for PfHRP-2 and PfLDH respectively. It is first report for aptamer based PQC biosensor to detect malarial PfHRP-2 and PfLDH at ppb range to meet the requirement for their diagnosis.
To meet the highly demanding challenge for detecting protein biomarkers at pg/mL level in high-protein sera sample, the coupling of paramagnetic nanoparticle technology with aptamer-PQC biosensor was attempted for developing aptamer-PQC biosensor for selective detection of SCV helicase protein produced from SARS CoV replication. The coupling of aptamer coated paramagnetic nanoparticles for sample pretreatment to aptamer-PQC biosensor has shown to detect helicase protein in one-minute assay with a detection limit of 350 pg/mL. The aptamer-coated crystal exhibits a frequency shift linearly proportional to the concentration of SARS helicase from 1 to 1000 ng/mL with a correlation coefficient of 0.9975 and a repeatability of 6.8% (%RSD, n=3). After the enrichment procedure, recoveries of 102% and 119% were achieved using samples spiked with SARS helicase at concentrations of 10 ng/mL and 1.0 ng/mL respectively. It is the first report to detect for SCV helicase protein using PQC biosensor at pg/mL level after magnetic bead enrichment. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy
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