Human induced pluripotent stem cell (iPSCs)-derived brain organoids have potential to recapitulate the earliest stages of brain development, serving as an effective in vitro model for studying both normal brain development and disorders. In this study, we demonstrate a straightforward approach of generating multiple cerebral organoids from iPSCs on a pillar plate platform, eliminating the need for labor-intensive, multiple transfer and encapsulation steps to ensure the reproducible generation of cerebral organoids. We formed embryoid bodies (EBs) in an ultra-low attachment (ULA) 384-well plate and subsequently transferred them to the pillar plate containing Matrigel, using a straightforward sandwiching and inverting method. Each pillar on the pillar plate contains a single spheroid, and the success rate of spheroid transfer was in a range of 95 - 100%. Using this approach, we robustly generated cerebral organoids on the pillar plate and demonstrated an intra-batch coefficient of variation (CV) below 9 – 19% based on ATP-based cell viability and compound treatment. Notably, our spheroid transfer method in combination with the pillar plate allows miniaturized culture of cerebral organoids, alleviates the issue of organoid variability, and has potential to significantly enhance assay throughput by allowing in situ organoid assessment as compared to conventional organoid culture in 6-/24-well plates, petri dishes, and spinner flasks.

The persistence of biofilms in hospital settings are associated with Healthcare Associated Infections (HCAI), causing increased morbidity, mortality and healthcare costs. The resistance of biofilms against commonly used hospital disinfectants has been well reported. Metal and metal oxide nanoparticles (NP) such as silver (Ag), copper (Cu), zinc oxide (ZnO) and copper oxide (CuO) exhibit antimicrobial properties against various pathogens. Methods: Biofilm formation of Pseudomonas aeruginosa and Staphylococcus aureus in a Centre for Disease Control (CDC) biofilm reactor and a 96 well plate was compared. A three stage approach including Minimum Biofilm Reduction Concentration (MBRC), R2 values and log(10) reductions was used to assess the efficacy of Ag and ZnO NPs both alone and in combination against P. aeruginosa and S. aureus biofilms. Atomic Absorption Spectroscopy (AAS), Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM) was used to further assess the antimicrobial ability of the metal and metal oxide NPs. The prevention of P. aeruginosa and S. aureus adherence on Ag and ZnO thin film coating on silicon (Si) surfaces was also investigated, as well as icaC, ebpS and fnbB gene expression in S. aureus biofilms. Results: The CDC biofilm reactor demonstrated to be the most effective method for P. aeruginosa and S. aureus biofilm production in comparison to 96 well plates, with lower standard errors of the mean (SE) and higher replicability. Individual MBRC of ZnO and Ag NPs in suspension were 256 and 50 µg/ml for P. aeruginosa and 16 and 50 µg/ml for S. aureus respectively. The concentrations in combination were reduced by at least a half, with concentrations of 32/25 µg/ml of ZnO/Ag NPs in suspension resulting in a significant (p ≤0.05) reduction of 3.77 log(10) against P. aeruginosa biofilms and 8/12 µg/ml of ZnO/Ag NPs in suspension resulted in a 3.91 log(10) (p ≤0.05) against S. aureus biofilms. Both combinations showed an additive effect. Time point analysis confirmed that a 24 hour treatment is vital for any significant (p ≤0.05) antimicrobial activity. AAS data suggested that the Ag+ ions quenched Zn2+ ions, therefore the antimicrobial efficacy of the combination is mainly due to Ag+ ions. Damage of the biofilms from Ag and ZnO NPs was observed in the SEM imaging and energy dispersive X-ray (EDX) analysis confirmed the adherence of Zn and Ag within the biofilms. CLSM imaging showed dead (red) cells of P. aeruginosa and S. aureus biofilms throughout the depth of the biofilm. P. aeruginosa formation was reduced by 1.41 log(10) and 1.43 log(10) on Ag and ZnO thin film coatings respectively. For S. aureus, a reduction of 1.82 log(10) and 1.65 log(10) was obtained for Ag and ZnO coating respectively. Only low levels of ribonucleic acid (RNA) were achieved so no further gene analysis could occur. Conclusion: Reductions of ≥3 log(10) were observed for P. aeruginosa and S. aureus biofilm treatment with ZnO/Ag NP suspensions. It can be concluded that the ZnO/Ag NP suspensions had greater antimicrobial activity than Ag and ZnO coated surfaces owing to large concentrations of Ag+ and Zn2+ ions acting upon the biofilms. The slower release of ions from coated surfaces suggest an inadequate concentration of ions in the media, which are therefore unable to prevent biofilm formation as rapidly as NP suspensions, however provide a sustained release of ions over time. The results from this investigation propose that Ag and ZnO NPs in suspension could be a potential alternative to disinfectants for use in nosocomial environments against P. aeruginosa and S. aureus biofilms.

Cette thèse porte sur l’étude des propriétés plasmoniques de réseaux nanostructurés dans le but de développer des applications de bioanalyse. L'intérêt de travailler avec ces structures est dû à leur grande sensibilité de surface, leur facilité de fabrication et leur simplicité d'analyse par spectrophotométrie en transmission. L'objectif était de fabriquer un dispositif capable d'effectuer du criblage à haut débit pour des fins biomédicales. Le premier objectif de la thèse porte sur l’étude des propriétés plasmoniques des réseaux de nanotrous. Une compréhension approfondie de ces structures a permis d’exploiter efficacement leur performance pour des applications de bioanalyse plasmonique. Une solution analytique fut établie pour étudier les modes de diffractions des polaritons de plasmons de surface d’onde de Bloch (BW-SPP). Cette équation a permis de corroborer les observations expérimentales avec des calculs théoriques par rapport au couplage plasmonique des réseaux de nanotrous. De plus, la variation de l'angle d'incidence a permis de déplacer la fréquence à laquelle les modes plasmoniques sont excités. Il était donc possible d'ajuster la position des BWSPP de façon à maximiser un couplage à une longueur d'onde désirée. Cet effet a été exploité avec la technique d'amplification de surface de diffusion Raman exaltée (SERS). Finalement, la sensibilité en surface de réseaux de nanotrous a été amplifiée selon l’angle d’excitation en transmission. Ce gain en sensibilité permet la détection de protéines d’IgG humain pour des basses concentrations de l’ordre du nanomolaire (nM). Le second objectif de la thèse traite du développement d’un lecteur multipuits couplé avec la technologie des réseaux de nanotrous afin de créer une plateforme de détection plasmonique pour du criblage à haut débit. Cet instrument offre une analyse en transmission d’échantillons nanostructurés à l’aide d’une plaque 96-puits pour des angles d’incidence allant jusqu’à 50°. Une nouvelle méthode de microfabrication de réseaux de nanotrous par photolithographie fut établie. Cette technique a permis de fabriquer des réseaux de nanotrous sur de grandes surfaces avec uniformité. L’efficacité du système fut démontrée pour la détection de protéines d’IgG humain, du méthotrexate (MTX) et le criblage d’anticorps de l’antigène prostatique spécifique (PSA). Le dernier volet de la thèse discute de l’étude des propriétés plasmoniques de réseaux de nanodisques recouverts d’un film d’or pour amplifier plus fortement la sensibilité des capteurs plasmoniques. Cette section de la thèse a démontré la performance des réseaux de nanodisques en tant que capteur plasmonique. En effet, les réseaux de nanodisques ont l’avantage d’exciter un mode de Bragg (BM, Bragg modes) en transmission directe générant une bande plasmonique fine ayant un facteur de mérite (FOM, figure of merit) élevé (sensiblité/réponse plasmonique). L’excitation de ces structures en transmission directe a simplifié énormément l’utilisation du robot multipuits par l’excitation à incidence normale tout en offrant une FOM supérieure aux réseaux de nanotrous. Pour continuer, des simulations 3D et une image Raman du signal SERS des structures ont démontré que le champ plasmonique des BM est grandement confiné autour des nanodisques. Ce confinement du champ plasmonique des réseaux de nanodisques à générer un facteur d’amplification SERS de l’ordre de 107. En somme, cette thèse démontre une étude des propriétés plasmoniques de réseaux nanostructurés pour des applications de bioanalyse par criblage à haut débit. Les études rapportées dans cette thèse ont prouvés que le champ plasmonique des réseaux de nanotrous peut être contrôlé afin d’amplifier leur sensibilité. De plus, la thèse rapporte la première plateforme de bioanalyse plasmonique utilisant un lecteur multipuits. Finalement, la fabrication de structures plasmoniques composés de nanodisques d’or a permis de mettre en évidence des propriétés optiques qui peuvent être mises à profit pour des mesures optiques ultras sensibles. / This thesis describes the plasmonic properties of nanostructured arrays towards development of biosensing applications. These structures exhibited several advantages such as high surface sensitivity, ease of microfabrication and simple excitation setup in transmission spectroscopy. The goal was to design a plasmonic device able to achieve high throughput analysis for biomedical purposes. The first section of the thesis covers a study of the plasmonic properties of nanohole arrays. An analytical solution was derived to assess plasmonic properties of the diffraction modes of Bloch-Wave surface plasmon polaritons (BW-SPP). Tuning of the excitation angle allowed for a precise control of the plasmonic signal’s position and an optimal coupling at a specific wavelength. This feature of nanohole arrays was demonstrated for applications in surface-enhanced Raman scattering (SERS). Finally, this section described the enhancement of the surface sensitivity of nanohole arrays through variation of the excitation angle in transmission. Such enhancement of the sensitivity allowed for detection of the concentration of human IgG proteins in the low nanomolar range. The second section of the thesis discusses the development of a multi-well plate reader coupled with the nanohole arrays technology. A custom-built plasmonic reader, designed at University of Montreal, allowed analysis of plasmonic structures in transmission with a 96-well plate for excitation where the incident angle is up to 50° relative to normal. A novel microfabrication technique of nanohole arrays, based on photolithography, is described. This technique allowed fabrication of nanohole arrays on a large scale with great surface uniformity. The performance of the plasmonic reader is demonstrated for sensing of human IgG proteins, methotrexate (MTX) and screening of prostate specific antigen (PSA) antibodies. The final section of the thesis describes studies on the plasmonic properties of nanodisk arrays coated with a gold film. This section described the performance of nanodisk arrays for plasmonic sensing. This structure benefited from the excitation of Bragg modes (BM) in direct transmission, which generated a sharp plasmonic band with a high figure of merit (FOM). The excitation of nanodisk arrays in direct transmission simplified the design of the plasmonic reader while providing a greater FOM than nanohole arrays. Furthermore, 3D simulations and a Raman image of the nanodisk arrays’ SERS intensity showed the confinement of the plasmonic field of the BM at the edges of the nanodisk. Such confinement of the plasmonic field of nanodisk arrays led to high SERS enhancements to a factor of 10^7. In summary, this thesis studied the plasmonic properties of nanostructured arrays towards development of applications for high throughput biosensing. These studies proved that the plasmonic field of nanohole arrays can be tuned to enhance their surface sensitivity. Furthermore, the thesis revealed the first plasmonic sensing platform using a multiwell plate reader. Finally, the thesis describes a novel plasmonic structure with outstanding optical properties; the gold coated nanodisk arrays.

Abstract Small-scale cultivation methods are a necessity for the development of new biotechnological processes. The most common method for submerged microbial cultivation is a shake flask used with a batch operation protocol. Well plate cultivation formats have also increased their importance, due to the need to utilize high-throughput cultivations for efficient product development. However, batch cultivation is often not the optimal method for obtaining high cell densities and good product quality, due to unlimited microbial growth. The aim of this dissertation was to improve small-scale microbial cultivations for microbial growth and product formation. Hydrolytic enzymes were utilized to relieve nutrient limitation by hydrolysis of proteins in lactic acid bacteria cultures to improve lactic acid production from dairy side products. Hydrolytic enzymes were also utilized in the enzymatic release of glucose from starch to create a fed-batch-like cultivation system applicable on small scale. The wireless sensor system developed was applied in shake flask cultivations to monitor oxygen and pH levels. Enzymatic polymer processing was applicable for small-scale cultivations. Lactic acid production by Lactobacillus salivarius ssp. salicinius was enhanced four-fold when the proteins were hydrolyzed either by proteases or by proteolytic microbes. The fed-batch-mimicking controlled glucose feeding and growth control were obtained by means of the simultaneous enzymatic hydrolysis of starch-polymer during cultivation. Controlled growth, higher cell densities, decreased side product formation and increased amount of soluble protein product were obtained in Escherichia coli cultivations. When this method was applied to the cultivation and recombinant protein production of the methylotrophic yeast Pichia pastoris, higher cell densities and higher amounts of active protein were obtained. The glucose concentration remained low enough to avoid the substrate repression of the alcohol oxidase promoter. The fed-batch method is suitable for high-throughput cultivations since the method can be utilized in well plate formats without external feeding devices. The method can be utilized in the development of new biotechnological products, especially when the production system is sensitive to growth conditions, and growth control is preferred. / Tiivistelmä Pienen mittakaavan mikrobikasvatusmenetelmiä tarvitaan kehitettäessä uusia bioteknologisia prosesseja. Tavallisin menetelmä mikrobien liuoksessa tapahtuvaan kasvatukseen on panostyyppisesti tehtävä sekoituspullokasvatus. Kuoppalevykasvatukset ovat myös tulleet entistä tärkeämmiksi, koska tuotekehityksen tehostamiseksi on tarvetta käyttää high-throughput-menetelmiä. Tavoiteltaessa korkeita mikrobisolutiheyksiä ja tuotteen hyvää laatua, panostyyppinen kasvatus ei ole usein paras vaihtoehto, johtuen mikrobien rajoittamattomasta kasvusta. Tämän työn tarkoituksena oli parantaa mikrobien kasvua ja tuotteen muodostusta pienen mittakaavan kasvatuksissa. Meijeriteollisuuden sivutuotteiden proteiineja pilkottiin entsyymien avulla, jotta maitohappobakteerit pystyivät hyödyntämään proteiinit tehokkaammin ja tuottamaan enemmän maitohappoa. Hydrolyyttisiä entsyymejä hyödynnettiin myös glukoosin vapauttamiseen tärkkelyksestä, jolloin saatiin luotua pieneen mittakaavaan sopiva panossyöttötyyppinen kasvatusmenetelmä. Työn aikana kehitettyä langatonta mittausjärjestelmää hyödynnettiin sekoituspullokasvatuksissa happipitoisuuden ja pH:n seurantaan. Entsymaattinen polymeerien käsittely oli soveltuva menetelmä pienen mittakaavan kasvatuksiin. Maitohapon tuotto Lactobacillus salivarius ssp. salicinius -mikrobilla nelinkertaistui, kun ravinneproteiinit pilkottiin joko proteaasien tai proteolyyttisten mikrobien avulla. Panossyöttömenetelmää muistuttava hallittu glukoosin syöttö ja mikrobin kasvun hallinta saavutettiin pilkkomalla tärkkelystä glukoosiksi kasvatuksen aikana. Escherichia coli kasvatuksissa saavutettiin hallittu solumäärän kasvu, korkeammat solutiheydet, vähentynyt sivutuotteiden muodostus ja suurempi liukoisen tuoteproteiinin määrä. Tätä menetelmää sovellettiin myös vierasproteiinin tuottoon metylotrofisella Pichia pastoris -hiivalla, jolloin saavutettiin korkeammat solutiheydet ja suurempi aktiivisen tuoteproteiinin määrä. Glukoosin määrä kasvatusliuoksessa pysyi riittävän alhaisena, jotta se ei repressoinut hiivan alkoholioksidaasi-promoottoria. Panossyöttömenetelmä on sopiva high-throughput-mikrobikasvatuksiin, koska sitä voidaan käyttää kuoppalevyillä ilman syöttölaitteita. Menetelmää voidaan hyödyntää uusien bioteknisten tuotteiden kehittämisessä erityisesti silloin, kun tuottoisäntä on herkkä kasvuolosuhteiden suhteen ja mikrobin kasvua halutaan hallita.