Nucleic acids are fundamental molecules of living organisms functioning essentially as the molecular information carriers of life. From how an organism is built to how it responds to external conditions, all of it, can be found in the form of nucleic acid sequences inside every single cell of every life form on earth. Therefore, accessing these sequences provides key information regarding the molecular identity and functional state of any living organism, this is very useful for areas like biomedicine, where accessing and understanding these molecular signatures is the key to develop strategies to understand, treat and diagnose diseases. Decades of research and technological advancements have led to the development of a number of molecular tools and engineering technologies that allow accessing the information contained in the nucleic acids. This thesis provides a general overview of the tools and technologies available for nucleic acid analysis, and proposes an illustrative concept on how molecular tools and emergent technologies can be combined in a modular fashion to design methods for addressing different biomedical questions. The studies included in this thesis, are focused on the particular use of the molecular tools named: padlock and selector probes, rolling circle amplification, and fluorescence detection of single molecules in combination with microfluidics and portable microscopy. By using this combination, it became possible to design and demonstrate novel approaches for integrated nucleic acid analysis, inexpensive digital quantification, mobile-phone based diagnostics and the description of viral infections. These studies represent a step forward towards the adoption of the selected group of tools and technologies, for the design and building of methods that can be used as powerful alternatives to conventional tools used in molecular diagnostics and virology. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 1: Manuscript.</p>

The rise of superbugs, including antibiotic-resistant bacteria, and virus outbreaks, such as the recent coronavirus scare, illustrate the need for rapid detection of disease pathogens. Widespread availability of rapid disease identification would facilitate outbreak prevention and specific treatment. The ARROW biosensor microchip can directly detect single molecules through fluorescence-based optofluidic interrogation. The nature of the microfluidic channels found on optofluidic sensor platforms sets some of the ultimate sensitivity and accuracy limits and can result in false negative test results. Yet higher sensitivity and specificity is desired through hydrodynamic focusing. Novel 3D hydrodynamic focusing designs were developed and implemented on the ARROW platform, an optofluidic lab-on-a-chip single-molecule detector device. Microchannels with cross-section dimensions smaller than 10 μm were formed using sacrificial etching of photoresist layers covered with plasma-enhanced chemical-vapor-deposited silicon dioxide on a silicon wafer. Buffer fluid carried to the focusing junction enveloped an intersecting sample fluid, resulting in 3D focusing of the sample stream. The designs which operate across a wide range of fluid velocities through pressure-driven flow were integrated with optical waveguides in order to interrogate fluorescing particles and confirm 3D focusing, characterize diffusion, and quantify optofluidic detection enhancement of single viruses on chip.

Le méthotrexate (MTX), un agent anti-cancéreux fréquemment utilisé en chimiothérapie, requiert généralement un suivi thérapeutique de la médication (Therapeutic Drug Monitoring, TDM) pour surveiller son niveau sanguin chez le patient afin de maximiser son efficacité tout en limitant ses effets secondaires. Malgré la fenêtre thérapeutique étroite entre l’efficacité et la toxicité, le MTX reste, à ce jour, un des agents anti-cancéreux les plus utilisés au monde. Les techniques analytiques existantes pour le TDM du MTX sont coûteuses, requièrent temps et efforts, sans nécessairement fournir promptement les résultats dans le délai requis. Afin d’accélérer le processus de dosage du MTX en TDM, une stratégie a été proposée basée sur un essai compétitif caractérisé principalement par le couplage plasmonique d’une surface métallique et de nanoparticules d’or. Plus précisément, l’essai quantitatif exploite la réaction de compétition entre le MTX et une nanoparticule d’or fonctionnalisée avec l’acide folique (FA-AuNP) ayant une affinité pour un récepteur moléculaire, la réductase humaine de dihydrofolate (hDHFR), une enzyme associée aux maladies prolifératives. Le MTX libre mixé avec les FA-AuNP, entre en compétition pour les sites de liaison de hDHFR immobilisés sur une surface active en SPR ou libres en solution. Par la suite, les FA-AuNP liées au hDHFR fournissent une amplification du signal qui est inversement proportionnelle à la concentration de MTX. La résonance des plasmons de surface (SPR) est généralement utilisée comme une technique spectroscopique pour l’interrogation des interactions biomoléculaires. Les instruments SPR commerciaux sont généralement retrouvés dans les grands laboratoires d’analyse. Ils sont également encombrants, coûteux et manquent de sélectivité dans les analyses en matrice complexe. De plus, ceux-ci n’ont pas encore démontré de l’adaptabilité en milieu clinique. Par ailleurs, les analyses SPR des petites molécules comme les médicaments n’ont pas été explorés de manière intensive dû au défi posé par le manque de la sensibilité de la technique pour cette classe de molécules. Les développements récents en science des matériaux et chimie de surfaces exploitant l’intégration des nanoparticules d’or pour l’amplification de la réponse SPR et la chimie de surface peptidique ont démontré le potentiel de franchir les limites posées par le manque de sensibilité et l’adsorption non-spécifique pour les analyses directes dans les milieux biologiques. Ces nouveaux concepts de la technologie SPR seront incorporés à un système SPR miniaturisé et compact pour exécuter des analyses rapides, fiables et sensibles pour le suivi du niveau du MTX dans le sérum de patients durant les traitements de chimiothérapie. L’objectif de cette thèse est d’explorer différentes stratégies pour améliorer l’analyse des médicaments dans les milieux complexes par les biocapteurs SPR et de mettre en perspective le potentiel des biocapteurs SPR comme un outil utile pour le TDM dans le laboratoire clinique ou au chevet du patient. Pour atteindre ces objectifs, un essai compétitif colorimétrique basé sur la résonance des plasmons de surface localisée (LSPR) pour le MTX fut établi avec des nanoparticules d’or marquées avec du FA. Ensuite, cet essai compétitif colorimétrique en solution fut adapté à une plateforme SPR. Pour les deux essais développés, la sensibilité, sélectivité, limite de détection, l’optimisation de la gamme dynamique et l’analyse du MTX dans les milieux complexes ont été inspectés. De plus, le prototype de la plateforme SPR miniaturisée fut validé par sa performance équivalente aux systèmes SPR existants ainsi que son utilité pour analyser les échantillons cliniques des patients sous chimiothérapie du MTX. Les concentrations de MTX obtenues par le prototype furent comparées avec des techniques standards, soit un essai immunologique basé sur la polarisation en fluorescence (FPIA) et la chromatographie liquide couplée avec de la spectrométrie de masse en tandem (LC-MS/MS) pour valider l’utilité du prototype comme un outil clinique pour les tests rapides de quantification du MTX. En dernier lieu, le déploiement du prototype à un laboratoire de biochimie dans un hôpital démontre l’énorme potentiel des biocapteurs SPR pour utilisation en milieux clinique. / Methotrexate (MTX) cancer therapy requires therapeutic drug monitoring (TDM) for following its levels in a patient during the course of treatment in order to maximize efficacy while minimizing side effects. Despite its narrow therapeutic window, MTX remains until this date, one of the most employed chemotherapy agents. Existing TDM analytical techniques for MTX are costly, time-consuming and labor intensive which are not suitable to promptly generate results within the therapy timeframe. To provide rapid MTX quantification for TDM, a strategy is proposed based on a competitive assay featuring gold nanoparticles and surface plasmonic coupling. More specifically, the inhibition of MTX with its molecular receptor, human dihydrofolate reductase (hDHFR), an enzyme associated with proliferative diseases, is explored. Free MTX mixed with folic acid-functionalized gold nanoparticles (FA-AuNP) are in competition for hDHFR binding sites immobilized on a SPR active surface or free in solution. FA-AuNP binding to hDHFR provides signal amplification which is inversely proportional to the concentration of MTX. Surface plasmon resonance (SPR) is commonly used as a spectroscopic technique for the interrogation of biomolecular interactions. Current commercial SPR instruments are laboratory-based, bulky, expensive, lack sensitivity in complex matrix and have not shown adaptability in clinical settings. In addition, SPR analysis of small molecules such as drugs has not been extensively explored due to lack of sensitivity. The recent advances in materials science and surface chemistry exploiting gold nanoparticle integration for SPR response enhancement and peptide surface chemistry have shown potential in overcoming the poor sensitivity and surface-fouling limitations for crude biofluids analysis. These novel concepts of SPR technology are incorporated with a miniaturized fully integrated SPR prototype to conduct fast, reliable and sensitive analysis to monitor MTX levels of a patient undergoing chemotherapy. The objective of the thesis is to explore different strategies in improving drug analysis in a complex matrix using SPR biosensors and to put in perspective of the potential of SPR biosensors as a useful TDM tool in clinical laboratories or at a point-of-care situation. To achieve these objectives, a colorimetric solution-based MTX competitive assay is first established with FA-AuNP. Then, the solution-based MTX competitive assay is translated onto a SPR platform. For both developed assays, sensitivity, selectivity, detection limit, dynamic range optimization as well as analysis of methotrexate in complex matrix are inspected. Furthermore, the SPR prototype is validated by its equivalent performance to existing SPR systems and by its utility in executing MTX analysis in actual serum samples from patients undergoing chemotherapy. The concentrations of MTX obtained by SPR biosensing are compared to standard techniques: fluorescence polarization immunoassay (FPIA) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in order to confirm the feasibility of SPR biosensors as a useful clinical tool for performing rapid MTX concentration evaluation. Finally, the successful deployment of the prototype to a hospital laboratory demonstrates enormous prospective of SPR biosensors in clinical use.

Le méthotrexate (MTX), un agent anti-cancéreux fréquemment utilisé en chimiothérapie, requiert généralement un suivi thérapeutique de la médication (Therapeutic Drug Monitoring, TDM) pour surveiller son niveau sanguin chez le patient afin de maximiser son efficacité tout en limitant ses effets secondaires. Malgré la fenêtre thérapeutique étroite entre l’efficacité et la toxicité, le MTX reste, à ce jour, un des agents anti-cancéreux les plus utilisés au monde. Les techniques analytiques existantes pour le TDM du MTX sont coûteuses, requièrent temps et efforts, sans nécessairement fournir promptement les résultats dans le délai requis. Afin d’accélérer le processus de dosage du MTX en TDM, une stratégie a été proposée basée sur un essai compétitif caractérisé principalement par le couplage plasmonique d’une surface métallique et de nanoparticules d’or. Plus précisément, l’essai quantitatif exploite la réaction de compétition entre le MTX et une nanoparticule d’or fonctionnalisée avec l’acide folique (FA-AuNP) ayant une affinité pour un récepteur moléculaire, la réductase humaine de dihydrofolate (hDHFR), une enzyme associée aux maladies prolifératives. Le MTX libre mixé avec les FA-AuNP, entre en compétition pour les sites de liaison de hDHFR immobilisés sur une surface active en SPR ou libres en solution. Par la suite, les FA-AuNP liées au hDHFR fournissent une amplification du signal qui est inversement proportionnelle à la concentration de MTX. La résonance des plasmons de surface (SPR) est généralement utilisée comme une technique spectroscopique pour l’interrogation des interactions biomoléculaires. Les instruments SPR commerciaux sont généralement retrouvés dans les grands laboratoires d’analyse. Ils sont également encombrants, coûteux et manquent de sélectivité dans les analyses en matrice complexe. De plus, ceux-ci n’ont pas encore démontré de l’adaptabilité en milieu clinique. Par ailleurs, les analyses SPR des petites molécules comme les médicaments n’ont pas été explorés de manière intensive dû au défi posé par le manque de la sensibilité de la technique pour cette classe de molécules. Les développements récents en science des matériaux et chimie de surfaces exploitant l’intégration des nanoparticules d’or pour l’amplification de la réponse SPR et la chimie de surface peptidique ont démontré le potentiel de franchir les limites posées par le manque de sensibilité et l’adsorption non-spécifique pour les analyses directes dans les milieux biologiques. Ces nouveaux concepts de la technologie SPR seront incorporés à un système SPR miniaturisé et compact pour exécuter des analyses rapides, fiables et sensibles pour le suivi du niveau du MTX dans le sérum de patients durant les traitements de chimiothérapie. L’objectif de cette thèse est d’explorer différentes stratégies pour améliorer l’analyse des médicaments dans les milieux complexes par les biocapteurs SPR et de mettre en perspective le potentiel des biocapteurs SPR comme un outil utile pour le TDM dans le laboratoire clinique ou au chevet du patient. Pour atteindre ces objectifs, un essai compétitif colorimétrique basé sur la résonance des plasmons de surface localisée (LSPR) pour le MTX fut établi avec des nanoparticules d’or marquées avec du FA. Ensuite, cet essai compétitif colorimétrique en solution fut adapté à une plateforme SPR. Pour les deux essais développés, la sensibilité, sélectivité, limite de détection, l’optimisation de la gamme dynamique et l’analyse du MTX dans les milieux complexes ont été inspectés. De plus, le prototype de la plateforme SPR miniaturisée fut validé par sa performance équivalente aux systèmes SPR existants ainsi que son utilité pour analyser les échantillons cliniques des patients sous chimiothérapie du MTX. Les concentrations de MTX obtenues par le prototype furent comparées avec des techniques standards, soit un essai immunologique basé sur la polarisation en fluorescence (FPIA) et la chromatographie liquide couplée avec de la spectrométrie de masse en tandem (LC-MS/MS) pour valider l’utilité du prototype comme un outil clinique pour les tests rapides de quantification du MTX. En dernier lieu, le déploiement du prototype à un laboratoire de biochimie dans un hôpital démontre l’énorme potentiel des biocapteurs SPR pour utilisation en milieux clinique. / Methotrexate (MTX) cancer therapy requires therapeutic drug monitoring (TDM) for following its levels in a patient during the course of treatment in order to maximize efficacy while minimizing side effects. Despite its narrow therapeutic window, MTX remains until this date, one of the most employed chemotherapy agents. Existing TDM analytical techniques for MTX are costly, time-consuming and labor intensive which are not suitable to promptly generate results within the therapy timeframe. To provide rapid MTX quantification for TDM, a strategy is proposed based on a competitive assay featuring gold nanoparticles and surface plasmonic coupling. More specifically, the inhibition of MTX with its molecular receptor, human dihydrofolate reductase (hDHFR), an enzyme associated with proliferative diseases, is explored. Free MTX mixed with folic acid-functionalized gold nanoparticles (FA-AuNP) are in competition for hDHFR binding sites immobilized on a SPR active surface or free in solution. FA-AuNP binding to hDHFR provides signal amplification which is inversely proportional to the concentration of MTX. Surface plasmon resonance (SPR) is commonly used as a spectroscopic technique for the interrogation of biomolecular interactions. Current commercial SPR instruments are laboratory-based, bulky, expensive, lack sensitivity in complex matrix and have not shown adaptability in clinical settings. In addition, SPR analysis of small molecules such as drugs has not been extensively explored due to lack of sensitivity. The recent advances in materials science and surface chemistry exploiting gold nanoparticle integration for SPR response enhancement and peptide surface chemistry have shown potential in overcoming the poor sensitivity and surface-fouling limitations for crude biofluids analysis. These novel concepts of SPR technology are incorporated with a miniaturized fully integrated SPR prototype to conduct fast, reliable and sensitive analysis to monitor MTX levels of a patient undergoing chemotherapy. The objective of the thesis is to explore different strategies in improving drug analysis in a complex matrix using SPR biosensors and to put in perspective of the potential of SPR biosensors as a useful TDM tool in clinical laboratories or at a point-of-care situation. To achieve these objectives, a colorimetric solution-based MTX competitive assay is first established with FA-AuNP. Then, the solution-based MTX competitive assay is translated onto a SPR platform. For both developed assays, sensitivity, selectivity, detection limit, dynamic range optimization as well as analysis of methotrexate in complex matrix are inspected. Furthermore, the SPR prototype is validated by its equivalent performance to existing SPR systems and by its utility in executing MTX analysis in actual serum samples from patients undergoing chemotherapy. The concentrations of MTX obtained by SPR biosensing are compared to standard techniques: fluorescence polarization immunoassay (FPIA) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in order to confirm the feasibility of SPR biosensors as a useful clinical tool for performing rapid MTX concentration evaluation. Finally, the successful deployment of the prototype to a hospital laboratory demonstrates enormous prospective of SPR biosensors in clinical use.

A new method, probability distribution of diffusivities (time scaled square displacements between succeeding video frames), was developed to analyze single molecule tracking (SMT) experiments. This method was then applied to SMT experiments on ultrathin liquid tetrakis(2-ethylhexoxy)silane (TEHOS) films on Si wafer with 100 nm thermally grown oxide, and on thin semectic liquid crystal films. Spatial maps of diffusivities from SMT experiments on 220 nm thick semectic liquid crystal films reveal structure related dynamics. The SMT experiments on ultrathin TEHOS films were complemented by fluorescence correlation spectroscopy (FCS). The observed strongly heterogeneous single molecule dynamics within those films can be explained by a three-layer model consisting of (i) dye molecules adsorbed to the substrate, (ii) slowly diffusing molecules in the laterally heterogeneous near-surface region of 1 - 2 molecular diameters, and (iii) freely diffusing dye molecules in the upper region of the film. FCS and SMT experiments reveal a strong influence of substrate heterogeneity on SM dynamics. Thereby chemisorption to substrate surface silanols plays an important role. Vertical mean first passage times (mfpt) in those films are below 1 µs. This appears as fast component in FCS autocorrelation curves, which further contain a contribution from lateral diffusion and from adsorption events. Therefore, the FCS curves are approximated by a tri-component function, which contains an exponential term related to the mfpt, the correlation function for translational diffusion and a stretched exponential term for the broad distribution of adsorption events. Lateral diffusion coefficients obtained by FCS on 10 nm thick TEHOS films, thereby, are effective diffusion coefficients from dye transients in the focal area. They strongly depend on the substrate heterogeneity. Variation of the frame times for the acquisition of SMT experiments in steps of 20 ms from 20 ms to 200 ms revealed a strong dependence of the corresponding probability distributions of diffusivities on time, in particular in the range between 20 ms and 100 ms. This points to average dwell times of the dye molecules in at least one type of the heterogeneous regions (e.g. on and above silanol clusters) in the range of few tens of milliseconds. Furthermore, time series of SM spectra from Nile Red in 25 nm thick poly-n-alkyl-methacrylate (PnAMA) films were studied. In analogy to translational diffusion, spectral diffusion (shifts in energetic positions of SM spectra) can be studied by probability distributions of spectral diffusivities, i.e. time scaled square energetic displacements. Simulations were run and analyzed to study contributions from noise and fitting uncertainty to spectral diffusion. Furthermore the effect of spectral jumps during acquisition of a SM spectrum was investigated. Probability distributions of spectral diffusivites of Nile Red probing vitreous PnAMA films reveal a two-level system. In contrast, such probability distributions obtained from Nile Red within a 25 nm thick poly-n-butylmethacrylate film around glass transition and in the melt state, display larger spectral jumps. Moreover, for longer alkyl side chains a solvent shift to higher energies is observed, which supports the idea of nanophase separation within those polymers.

A new method, probability distribution of diffusivities (time scaled square displacements between succeeding video frames), was developed to analyze single molecule tracking (SMT) experiments. This method was then applied to SMT experiments on ultrathin liquid tetrakis(2-ethylhexoxy)silane (TEHOS) films on Si wafer with 100 nm thermally grown oxide, and on thin semectic liquid crystal films. Spatial maps of diffusivities from SMT experiments on 220 nm thick semectic liquid crystal films reveal structure related dynamics. The SMT experiments on ultrathin TEHOS films were complemented by fluorescence correlation spectroscopy (FCS). The observed strongly heterogeneous single molecule dynamics within those films can be explained by a three-layer model consisting of (i) dye molecules adsorbed to the substrate, (ii) slowly diffusing molecules in the laterally heterogeneous near-surface region of 1 - 2 molecular diameters, and (iii) freely diffusing dye molecules in the upper region of the film. FCS and SMT experiments reveal a strong influence of substrate heterogeneity on SM dynamics. Thereby chemisorption to substrate surface silanols plays an important role. Vertical mean first passage times (mfpt) in those films are below 1 µs. This appears as fast component in FCS autocorrelation curves, which further contain a contribution from lateral diffusion and from adsorption events. Therefore, the FCS curves are approximated by a tri-component function, which contains an exponential term related to the mfpt, the correlation function for translational diffusion and a stretched exponential term for the broad distribution of adsorption events. Lateral diffusion coefficients obtained by FCS on 10 nm thick TEHOS films, thereby, are effective diffusion coefficients from dye transients in the focal area. They strongly depend on the substrate heterogeneity. Variation of the frame times for the acquisition of SMT experiments in steps of 20 ms from 20 ms to 200 ms revealed a strong dependence of the corresponding probability distributions of diffusivities on time, in particular in the range between 20 ms and 100 ms. This points to average dwell times of the dye molecules in at least one type of the heterogeneous regions (e.g. on and above silanol clusters) in the range of few tens of milliseconds. Furthermore, time series of SM spectra from Nile Red in 25 nm thick poly-n-alkyl-methacrylate (PnAMA) films were studied. In analogy to translational diffusion, spectral diffusion (shifts in energetic positions of SM spectra) can be studied by probability distributions of spectral diffusivities, i.e. time scaled square energetic displacements. Simulations were run and analyzed to study contributions from noise and fitting uncertainty to spectral diffusion. Furthermore the effect of spectral jumps during acquisition of a SM spectrum was investigated. Probability distributions of spectral diffusivites of Nile Red probing vitreous PnAMA films reveal a two-level system. In contrast, such probability distributions obtained from Nile Red within a 25 nm thick poly-n-butylmethacrylate film around glass transition and in the melt state, display larger spectral jumps. Moreover, for longer alkyl side chains a solvent shift to higher energies is observed, which supports the idea of nanophase separation within those polymers.