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Ce travail s’inscrit dans le cadre de la recherche relative au développement de biosenseurs à ADN électrochimiques. Des aspects fondamentaux, ainsi que des aspects d’application de la détection d’hybridation d’ADN sont envisagés. Dans un premier temps, le comportement interfacial et le processus d’hybridation d’oligonucléotides d’ADN linéaires et ADN hairpin (structure en épingle à cheveux) nonmarqués sont étudiés en formant des monocouches auto-assemblées mixtes de monobrins d’ADN (ssADN) thiolés et d’un hydroxyalcanethiol (4-mercaptobutan-1-ol) par coadsorption spontanée sur des électrodes d’or polycristallin. L’immobilisation de monocouches mixtes ssADN/MCB est caractérisée par voie électrochimique et par spectroscopie des photoélectrons X. Des mesures de chronocoulométrie, en présence de [Ru(NH3)6]3+ (RuHex), permettent de déterminer la quantité d’ADN dans la monocouche mixte formée. Les résultats montrent que l’excès superficiel d’ADN linéaire est plus important que l’excès superficiel d’ADN hairpin sous des conditions de formation identiques. La réaction de reconnaissance moléculaire d’hybridation est détectée par des mesures d’impédance en présence de [Fe(CN)6]3-/4-. L’hybridation se traduit dans le cas de l’ADN linéaire par une augmentation de la résistance au transfert d’électron Rct tandis que dans le cas de l’ADN hairpin, Rct diminue. Ces différences sont dues au plus faible recouvrement et au changement de conformation des molécules d’ADN hairpin lors de l’hybridation. Des mesures de réflectivité de neutrons nous ont permis de mettre en évidence l’augmentation de l’épaisseur du film d’ADN hairpin et de confirmer le changement conformationel ces sondes lors de la reconnaissance moléculaire. Dans la seconde partie, nous présentons une nouvelle méthode électrochimique de détection d’hybridation, basée sur les interactions électrostatiques entre le complexe cationique RuHex et les groupements phosphates de l’ADN. Afin d’améliorer la détection des molécules de PNA (peptide nucleic acid) ont été immobilisées comme sondes de reconnaissance moléculaire. Après hybridation des sondes PNA avec le brin complémentaire, RuHex s’adsorbe sur l’ADN hybridé et un signal de réduction de ces complexes redox, enregistré par voltampérométrie alternative, constitue une signature claire de l’hybridation d’ADN à l’interface modifiée. Les interactions RuHex/PNA-ADN ont été étudiées. La constante d’adsorption de RuHex sur l’électrode modifiée PNA/MCB après hybridation est évaluée à 2,9 (±0,3) 105 M-1 en milieu Tris-HCl 0,01M, selon une isotherme de Langmuir. Les performances analytiques de la méthode de détection (sensibilité, sélectivité et reproductibilité) ont été évaluées et optimisées pour la détection des séquences d’ADN du gène de l’ARNr 23S d’Helicobacter pylori. La méthode de détection électrochimique présentée est assez sélective pour permettre de discriminer les mutations ponctuelles A2143G et A2144C de la séquence de type sauvage. La diminution significative des signaux d’admittance enregistrés en présence des séquences mutées est attribuée à la capacité accrue de discrimination de mutations ponctuelles des molécules PNA. La réponse de détection est linéaire en fonction du logarithme de la concentration de la cible d’ADN sur plus de quatre ordres de grandeur (10-6 M à 10-10 M). La limite de détection de l’oligonucléotide d’ADN complémentaire de 80 pM est très bonne. La méthode a été appliquée avec succès à la détection de fragments PCR complémentaires de 100 et 400 paires de bases, amplifiés à partir de souches SS1 d’H.pylori.

We have discovered that the current protocols to assemble Au nanoparticles based on DNA hybridization do not work well with the small metal nanoparticles (e.g. 5 nm Au, 3.6 nm Pt and 3.2 nm Ru particles). Further investigations revealed the presence of strong interaction between the oligonucleotide backbone and the surface of the small metal nanoparticles. The oligonucleotides in this case are recumbent on the particle surface and are therefore not optimally oriented for hybridization. The nonspecific adsorption of oligonucleotides on small metal nanoparticles must be overcome before DNA hybridization can be accepted as a general assembly method. Two methods have been suggested as possible solutions to this problem. One is based on the use of stabilizer molecules which compete with the oligonucleotides for adsorption on the metal nanoparticle surface. Unfortunately, the reported success of this approach in small Au nanoparticles (using K₂BSPP) and Au films (using 6-mercapto-1-hexanol) could not be extended to the assembly of Pt and Ru nanoparticles by DNA hybridization. The second approach is to simply use larger metal particles. Indeed most reports on the DNA hybridization induced assembly of Au nanoparticles have made use of relatively large particles (>10 nm), hinting at a weaker non-specific interaction between the oligonucleotides and large Au nanoparticles. However, most current methods of nanoparticle synthesis are optimized to produce metal nanoparticles only within a narrow size range. We find that core-shell nanoparticles formed by the seeded growth method may be used to artificially enlarge the size of the metal particles to reduce the nonspecific binding of oligonucleotides. We demonstrate herein a core-shell assisted growth method to assemble Pt and Ru nanoparticles by DNA hybridization. This method involves firstly synthesizing approximately 16 nm core-shell Ag-Pt and 21 nm core-shell Au-Ru nanoparticles from 9.6 nm Ag seeds and 17.2 nm Au seeds respectively by the seed-mediated growth method. The core-shell nanoparticles were then functionalized by complementary thiolated oligonucleotides followed by aging in 0.2 M PBS buffer for 6 hours. The DNA hybridization induced bimetallic assembly of Pt and Ru nanoparticles could then be carried out in 0.3 M PBS buffer for 10 hours. / Singapore-MIT Alliance (SMA)

A new format for microfluidic based DNA biosensors is presented in which the biorecognition element (single stranded DNA probes) is immobilized as a continuous density gradient of probes along the length of a microfluidic channel instead of a standard array format commonly used in microarray technologies or DNA based biosensors. The development of continuous density gradients of immobilized probe was achieved by electrokinetically subjecting probes that were terminated with an appropriate functional group for a surface coupling reaction to increasing convective velocity along the length of the microfluidic channel. This gradient format was able to discriminate between a fully complementary target and one containing 3 BPM based on the spatial pattern of hybridization for picomole quantities of DNA targets. Temperature mediated destabilization of DNA hybrids demonstrated that the density of immobilized probes plays an important role in the thermodynamic stability of DNA hybrids. In addition, it was found that efficiency, selectivity and melt temperature of DNA hybrids for surface based hybridization is dependent on the density of the probe molecules.

A new format for microfluidic based DNA biosensors is presented in which the biorecognition element (single stranded DNA probes) is immobilized as a continuous density gradient of probes along the length of a microfluidic channel instead of a standard array format commonly used in microarray technologies or DNA based biosensors. The development of continuous density gradients of immobilized probe was achieved by electrokinetically subjecting probes that were terminated with an appropriate functional group for a surface coupling reaction to increasing convective velocity along the length of the microfluidic channel. This gradient format was able to discriminate between a fully complementary target and one containing 3 BPM based on the spatial pattern of hybridization for picomole quantities of DNA targets. Temperature mediated destabilization of DNA hybrids demonstrated that the density of immobilized probes plays an important role in the thermodynamic stability of DNA hybrids. In addition, it was found that efficiency, selectivity and melt temperature of DNA hybrids for surface based hybridization is dependent on the density of the probe molecules.

High-resolution lattice periodicity images of a variety of well-defined surfaces, including graphite, mica, and Au(111), validated the good stability of our atomic force microscope (AFM) system. Combining self-assembled monolayer (SAM) and AFM technology, we demonstrated the capabilities of pattern fabrication as well as modification of surface functionality. AFM-based nanolithography operating conditions, such as scan rate, deflection setpoint, and number of scan were studied to obtain the optimized quality of the fabricated patterns. Thiolated-DNA probe molecules could be patterned at a nanometer scale on a gold substrate. However, we found that the surface coverage began to drop notably with the probe length (number of DNA bases). Therefore, the displaced DNA molecules during nanoshaving were reversibly adsorbed, and patterning became unreliable. We were unsuccessful in detecting the subsequent hybridization reactions at these nanopatterns from AFM measurements. To realize the DNA hybridization, further studies on the incubation temperature, probe length and even DNA sequences are required to demonstrate that this AFM-based gene diagnostic method is truly operational.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 122-137).

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2015-12-14T21:26:35Z No. of bitstreams: 1 GeorgeAlexandreDeBarrosLucena_DISSERT.pdf: 3317276 bytes, checksum: 22082dfd410296351adf3deb96db86ff (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2015-12-15T21:24:23Z (GMT) No. of bitstreams: 1 GeorgeAlexandreDeBarrosLucena_DISSERT.pdf: 3317276 bytes, checksum: 22082dfd410296351adf3deb96db86ff (MD5) / Made available in DSpace on 2015-12-15T21:24:23Z (GMT). No. of bitstreams: 1 GeorgeAlexandreDeBarrosLucena_DISSERT.pdf: 3317276 bytes, checksum: 22082dfd410296351adf3deb96db86ff (MD5) Previous issue date: 2014-06-04 / Introduction: Infiltration of organic fluids and microorganisms at the abutment/implant interface may result in bacterial infection of peri-implant tissues. Internal colonization of periodontal pathogens may be caused by bacteria trapped during installation or penetration of abutment/implant leakage. The aim of this study was to detect periodontal pathogens in the internal area of dental implants before loading. Materials and Methods: Seventy-eight implants in 32 partially edentulous subjects were selected for this evaluation. A bacterial biofilm sample of the internal surface of each implant was taken and analyzed for the presence of 40 microorganisms by checkerboard DNA-DNA hybridization, prior to installation of healing or any other prosthetic abutment. Discussion: Bacteria were detected in 20 patients (62.5%), distributed in 41 implants (52.6%). Forty-seven percent of implants showed no bacterial detection. Spontaneous early implant exposure to oral cavity during the healing period was not significant (P >0.05) to increase bacterial prevalence, but implants placed at mandible had higher bacterial prevalence than maxillary ones. Conclusion: The internal surface of dental implants can serve as a reservoir of periodontal pathogens for future implant/abutment interface.

O presente trabalho teve por objetivo investigar a microbiota de canais radiculares relacionadas ao insucesso do tratamento endodôntico, buscando a identificação e a quantificação destes micro-organismos. Foram selecionados 36 dentes com infecção endodôntica persistente. O material obturador foi removido do canal radicular e amostras microbiológicas foram coletadas dos canais com o auxílio de limas tipo Hedströen e cones de papel absorvente estéril. A técnica do Checkerboard DNA-DNA hybridization foi utilizada para detecção de até 79 espécies bacterianas em cada amostra, utilizando sondas de DNA específicas. Os dados microbiológicos foram expressos em percentagem média (prevalência), proporção e nível médio de cada espécie em cada amostra. Os testes t independente e de correlação de Pearson foram usados para correlacionar a contagem das bactérias testadas com os dados clínicos (p&#8804; 0,05). Foi encontrada uma média de 11 espécies por amostra. E. faecium (36%), S. epidermidis (36%), E. saburreum (28%), P. micra (28%), S. sanguis (28%), C. sputigena (28%), L. buccalis (28%), E. faecalis (28%) e S. warneri (28%) foram as espécies mais prevalentes, e as espécies encontradas em níveis médios mais altos foram E. faecium, D. pneumosintes, S. epidermidis, H. pylori e C. sputigena. T. socranskii (3%), F. periodonticum (3%), C. gingivalis (3%), S. ixodetis (3%) apresentaram prevalências mais baixas. E. faecium e S. epidermidis apresentaram os maiores valores de prevalência, níveis médios e proporção. Não houve correlação entre a microbiota detectada nas amostras com os sinais e sintomas clínicos apresentados pelos pacientes, porém nas lesões periapicais de maior área foi detectada contagem significativamente maior de bacilos e espécies Gram-negativas (p<0,05). Baseado nos resultados obtidos é possível concluir que a microbiota presente em dentes com periodontite apical persistente possui perfil misto e complexo, e que uma maior área de lesão perirradicular pode estar associada a contagem elevada de bacilos e de espécies Gram-negativas. / The present study investigated the composition of the root canal microbiota in endodontic failures, aiming to identify and quantify these microorganisms. Thirty six teeth with persistent endodontic infection were selected. The root-filling materials were removed and microbiological samples were taken from the root canals with a Hedströen-type file and sterile paper points. The Checkerboard DNA-DNA hybridization technique was used for the detection of 79 bacterial species in each sample, using specific DNA probes. Microbiological data were express in mean prevalence, proportions and levels of each species in each sample. t independent test and Pearson correlation test were use to correlate bacterial counts and clinical conditions (p&#8804; 0,05). There were found a mean of 11 different species per sample. E. faecium (36%), S. epidermidis (36%), E. saburreum (28%), P. micra (28%), S. sanguis (28%), C. sputigena (28%), L. buccalis (28%), E. faecalis (28%) and S. warneri (28%) were the most prevalent species, and the species found in highest mean levels were E. faecium, D. pneumosintes, S. epidermidis, H. pylori and C. sputigena. T. socranskii (3%), F. periodonticum (3%), C. gingivalis (3%) and S. ixodetis (3%) were found in low prevalence. E. faecium and S. epidermidis presented the highest values of prevalence, means levels and proportions. No correlation was found between the detected microbiota and clinical findings; however in periapical lesions with highest areas, higher levels of rods and Gram-negative species were detected (p<0.05). Based on these results it may be concluded that the microbiota in teeth with persistent apical periodontitis presents a mixed and complex profile, and periapical lesions with larger area might be high associated with higher counts of rods and Gram-negative species.

O presente trabalho teve por objetivo investigar a microbiota de canais radiculares relacionadas ao insucesso do tratamento endodôntico, buscando a identificação e a quantificação destes micro-organismos. Foram selecionados 36 dentes com infecção endodôntica persistente. O material obturador foi removido do canal radicular e amostras microbiológicas foram coletadas dos canais com o auxílio de limas tipo Hedströen e cones de papel absorvente estéril. A técnica do Checkerboard DNA-DNA hybridization foi utilizada para detecção de até 79 espécies bacterianas em cada amostra, utilizando sondas de DNA específicas. Os dados microbiológicos foram expressos em percentagem média (prevalência), proporção e nível médio de cada espécie em cada amostra. Os testes t independente e de correlação de Pearson foram usados para correlacionar a contagem das bactérias testadas com os dados clínicos (p&#8804; 0,05). Foi encontrada uma média de 11 espécies por amostra. E. faecium (36%), S. epidermidis (36%), E. saburreum (28%), P. micra (28%), S. sanguis (28%), C. sputigena (28%), L. buccalis (28%), E. faecalis (28%) e S. warneri (28%) foram as espécies mais prevalentes, e as espécies encontradas em níveis médios mais altos foram E. faecium, D. pneumosintes, S. epidermidis, H. pylori e C. sputigena. T. socranskii (3%), F. periodonticum (3%), C. gingivalis (3%), S. ixodetis (3%) apresentaram prevalências mais baixas. E. faecium e S. epidermidis apresentaram os maiores valores de prevalência, níveis médios e proporção. Não houve correlação entre a microbiota detectada nas amostras com os sinais e sintomas clínicos apresentados pelos pacientes, porém nas lesões periapicais de maior área foi detectada contagem significativamente maior de bacilos e espécies Gram-negativas (p<0,05). Baseado nos resultados obtidos é possível concluir que a microbiota presente em dentes com periodontite apical persistente possui perfil misto e complexo, e que uma maior área de lesão perirradicular pode estar associada a contagem elevada de bacilos e de espécies Gram-negativas. / The present study investigated the composition of the root canal microbiota in endodontic failures, aiming to identify and quantify these microorganisms. Thirty six teeth with persistent endodontic infection were selected. The root-filling materials were removed and microbiological samples were taken from the root canals with a Hedströen-type file and sterile paper points. The Checkerboard DNA-DNA hybridization technique was used for the detection of 79 bacterial species in each sample, using specific DNA probes. Microbiological data were express in mean prevalence, proportions and levels of each species in each sample. t independent test and Pearson correlation test were use to correlate bacterial counts and clinical conditions (p&#8804; 0,05). There were found a mean of 11 different species per sample. E. faecium (36%), S. epidermidis (36%), E. saburreum (28%), P. micra (28%), S. sanguis (28%), C. sputigena (28%), L. buccalis (28%), E. faecalis (28%) and S. warneri (28%) were the most prevalent species, and the species found in highest mean levels were E. faecium, D. pneumosintes, S. epidermidis, H. pylori and C. sputigena. T. socranskii (3%), F. periodonticum (3%), C. gingivalis (3%) and S. ixodetis (3%) were found in low prevalence. E. faecium and S. epidermidis presented the highest values of prevalence, means levels and proportions. No correlation was found between the detected microbiota and clinical findings; however in periapical lesions with highest areas, higher levels of rods and Gram-negative species were detected (p<0.05). Based on these results it may be concluded that the microbiota in teeth with persistent apical periodontitis presents a mixed and complex profile, and periapical lesions with larger area might be high associated with higher counts of rods and Gram-negative species.