<p>There is a strong trend in fabricating <i>miniaturized total analytical systems</i>, µTAS, for various biochemical and cell biology applications. These miniaturized systems could e.g. gain better separation performances, be faster, consume less expensive reagents and be used for studies that are difficult to access in the macro world. Disposable µTAS eliminate the risk of carry-over and can be fabricated to a low cost.</p><p>This work focused on the development of µTAS modules with the intentional use for miniaturized diagnostics. Modules for blood separation, desalting, enrichment, separation and ESI-MS detection were successfully fabricated. Surface coatings were additionally developed and evaluated for applications in µTAS with complex biological samples. The first heparin coating could be easily immobilized in a one-step-process, whereas the second heparin coating was aimed to form a hydrophilic surface that was able to draw blood or plasma samples into a microfluidic system by capillary forces. </p><p>The last mentioned heparin surface was further utilized when developing a chip-based sensor for performing CD4-count in human blood, an important marker to determine the stage of an HIV-infection.</p><p>All devices in this work were fabricated in PDMS, an elastomeric polymer with the advantage of rapid and less expensive prototyping of the microfabricated master. It was shown that PDMS could be considered as the material of choice for future commercial µTAS. The devices were intentionally produced using a low grade of fabrication complexity. It was however demonstrated that even with low complexity, it is possible to integrate several functional chip modules into a single microfluidic device.</p>

There is a strong trend in fabricating miniaturized total analytical systems, µTAS, for various biochemical and cell biology applications. These miniaturized systems could e.g. gain better separation performances, be faster, consume less expensive reagents and be used for studies that are difficult to access in the macro world. Disposable µTAS eliminate the risk of carry-over and can be fabricated to a low cost. This work focused on the development of µTAS modules with the intentional use for miniaturized diagnostics. Modules for blood separation, desalting, enrichment, separation and ESI-MS detection were successfully fabricated. Surface coatings were additionally developed and evaluated for applications in µTAS with complex biological samples. The first heparin coating could be easily immobilized in a one-step-process, whereas the second heparin coating was aimed to form a hydrophilic surface that was able to draw blood or plasma samples into a microfluidic system by capillary forces. The last mentioned heparin surface was further utilized when developing a chip-based sensor for performing CD4-count in human blood, an important marker to determine the stage of an HIV-infection. All devices in this work were fabricated in PDMS, an elastomeric polymer with the advantage of rapid and less expensive prototyping of the microfabricated master. It was shown that PDMS could be considered as the material of choice for future commercial µTAS. The devices were intentionally produced using a low grade of fabrication complexity. It was however demonstrated that even with low complexity, it is possible to integrate several functional chip modules into a single microfluidic device.

The field of micro total analysis systems (μTAS) aims at developments toward miniaturized and fully integrated lab-on-a-chip systems for applications, such as drug screening, drug delivery, cellular assays, protein analysis, genomic analysis and handheld point-of-care diagnostics. Such systems offer to dramatically reduce liquid sample and reagent quantities, increase sensitivity as well as speed of analysis and facilitate portable systems via the integration of components such as pumps, valves, mixers, separation units, reactors and detectors. Precise microfluidic control for such systems has long been considered one of the most difficult technical barriers due to integration of on-chip fluidic handling components and complicated off-chip liquid control as well as fluidic interconnections. Actuation principles and materials with the advantages of low cost, easy fabrication, easy integration, high reliability, and compact size are required to promote the development of such systems. Within this thesis, liquid displacement in microfluidic applications, by means of expandable microspheres, is presented as an innovative approach addressing some of the previously mentioned issues. Furthermore, these expandable microspheres are embedded into a PDMS matrix, which composes a novel thermally responsive silicone elastomer composite actuator for liquid handling. Due to the merits of PDMS and expandable microspheres, the composite actuator's main characteristic to expand irreversibly upon generated heat makes it possible to locally alter its surface topography. The composite actuator concept, along with a novel adhesive PDMS bonding technique, is used to design and fabricate liquid handling components such as pumps and valves, which operate at work-ranges from nanoliters to microliters. The integration of several such microfluidic components promotes the development of disposable lab-on-a-chip platforms for precise sample volume control addressing, e.g. active dosing, transportation, merging and mixing of nanoliter liquid volumes. Moreover, microfluidic pumps based on the composite actuator have been incorporated with sharp and hollow microneedles to realize a microneedle-based transdermal patch which exhibits on-board liquid storage and active dispensing functionality. Such a system represents a first step toward painless, minimally invasive and transdermal administration of macromolecular drugs such as insulin or vaccines. The presented on-chip liquid handling concept does not require external actuators for pumping and valving, uses low-cost materials and wafer-level processes only, is highly integrable and potentially enables controlled and cost-effective transdermal microfluidic applications, as well as large-scale integrated fluidic networks for point-of care diagnostics, disposable biochips or lab-on-a-chip applications. This thesis discusses several design concepts for a large variety of microfluidic components, which are promoted by the use of the novel composite actuator. Results on the successful fabrication and evaluation of prototype devices are reported herein along with comprehensive process parameters on a novel full-wafer adhesive bonding technique for the fabrication of PDMS based microfluidic devices. / QC 20100817

The analysis of low abundant biological molecules is often challenging due to their chemical properties, low concentration and limited sample volumes. Neuropeptides are one group of molecules that fits these criteria. Neuropeptides also play an important role in biological functions, which makes them extra interesting to analyze. A classic chemical analysis involves sampling, sample preparation, separation and detection. In this thesis, an enhanced solid supported microdialysis method was developed and used as a combined sampling- and preparation technique. In general, significantly increased extraction efficiency was obtained for all studied peptides. To be able to control the small sample volumes and to minimize the loss of neuropeptides because of unwanted adsorption onto surfaces, the subsequent analysis steps were miniaturized to a micro total analysis system (µ-TAS), which allowed sample pre-treatment, injection, separation, manipulation and detection. In order to incorporate these analysis functions to a microchip, a novel microfabrication protocol was developed. This method facilitated three-dimensional structures to be fabricated without the need of clean room facilities. The sample pre-treatment step was carried out by solid phase extraction from beads packed in the microchip. Femtomole levels of neuropeptides were detected from samples possessing the same properties as microdialysates. The developed injection system made it possible to conduct injections from a liquid chromatographic separation into a capillary electrophoresis channel, which facilitated for advanced multidimensional separations. An electrochemical sample manipulation system was also developed. In the last part, different electrospray emitter tip designs made directly from the edge of the microchip substrate were developed and evaluated. The emitters were proven to be comparable with conventional, capillary based emitters in stability, durability and dynamic flow range. Although additional developments remain, the analysis steps described in this thesis open a door to an integrated, on-line µ-TAS for neuropeptides analysis in complex biological samples.

Submitted by Jaqueline Silva (jtas29@gmail.com) on 2016-10-03T14:17:36Z No. of bitstreams: 2 Dissertação - Eunício de Oliveira Lobo Junior - 2016.pdf: 5628245 bytes, checksum: cb5cfdfdb21a0a9bed0292decf6c094f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2016-10-03T14:18:22Z (GMT) No. of bitstreams: 2 Dissertação - Eunício de Oliveira Lobo Junior - 2016.pdf: 5628245 bytes, checksum: cb5cfdfdb21a0a9bed0292decf6c094f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-10-03T14:18:22Z (GMT). No. of bitstreams: 2 Dissertação - Eunício de Oliveira Lobo Junior - 2016.pdf: 5628245 bytes, checksum: cb5cfdfdb21a0a9bed0292decf6c094f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-03-10 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / This report describes the development of two alternative platforms for electrophoretic runs in microsystems. Firstly, the development of a hybrid capillary system that dispenses microfabrication steps is presented using fused silica capillaries interconnected by a commercial crossed shape interface. This hybrid system was coupled with contactless conductivity detector (C4D) to allow the determination of inorganic cations in biological samples. Electrokinetic sample injection was performed through gated mode approach for the first time in this arrangement. Operational parameters such as: (i) wave frequency and amplitude applied in C4D system, (ii) electrical potential applied in injection, (iii) injection time, (iv) detection point, (v) effect of capillary conditioning as well as and (vi) recovery percentage were extensively investigated and optimized. Better separations of cationic mixture containing NH4+, K+, Na+, Ca2+ and Mg2+ were achieved using a buffer system composed of 50 mM Lactic Acid, 20 mM Histidine and 3 mM 18-crown-6 on a capillary with effective length of 14.5 cm. . Addition of internal standard was used to ensure analytical reproducibility and allow the recording of merit figures. Linear behaviors were observed in concentration ranges between 10 and 100 M for NH4+, K+, Ca2+ e Mg2+, and 20-200 M for Na+. The limit of detection values calculated were 3.75 μM (NH4+), 3.70 μM (K+), 7.50 μM (Na+), 5.00 μM (Ca2+) and 5.35 μM (Mg2+). The concentration levels achieved for cations in biological samples ranged from 4,1 μM to 200 μM. Besides the hybrid system, this report also describes the development of an alternative methodology for the fabrication of high-relief masters for soft-lithography in poly(dimethylsiloxane) (PDMS) substrate. One of the innovative features makes reference to the use of low cost commercial photoresist from textile industry - poly(vinyl acetate) (PVAc) - which exhibits low toxicity. PVAc films were deposited on printed cirtuitry boards through the use of a homemade spincoater developed by desktop cooler, with rotation time control. This methodology allowed the production of high relief masters and PDMS channels with width and depth of 50 μm and 40 μm, respectively. Channels and masters profiles They were characterized with the following techniques: scan electron microscopy, perfilometry, optical and electrical. PDMS electrophoresis devices were successfully used for the separation of major inorganic cations. / Esta dissertação descreve o desenvolvimento de duas plataformas alternativas para a realização de eletroforese em microssistemas. Inicialmente é descrita um sistema eletroforético híbrido que dispensa etapas de microfabricação utilizando capilares de sílica fundida, conectados por uma interface comercial com formato em cruz. Este sistema capilar híbrido foi acoplado com detecção condutométrica sem contato (C4D) e aplicado na determinação de cátions inorgânicos (NH4+, K+, Na+, Ca2+, Mg2+) em amostras biológicas. A injeção de amostras foi realizada eletrocineticamente no modo gated, sendo o primeiro estudo em capilares utilizando esta modalidade de injeção. Foram avaliados os parâmetros operacionais de funcionamento do sistema incluindo (i) frequência e amplitude da onda senoidal aplicada ao sistema de detecção, (ii) potencial elétrico aplicado na injeção, (iii) tempo de injeção, (iv) composição do tampão, (v) ponto de detecção, (vi) efeito do condicionamento do capilar e (vii) recuperação. As melhores separações para uma mistura contendo os cátions inorgânicos foram obtidas usando-se um sistema tamponante composto de ácido lático 50 mM, histidina 20 mM e éter coroa (18-crown-6) 3 mM em um capilar com comprimento efetivo de 14,5 cm. As figuras de mérito analítico foram obtidas a partir da adição do íon Li+ como padrão interno, o qual assegurou confiabilidade nas análises quantitativas. A partir da otimização dos parâmetros analíticos, as curvas analíticas para os íons NH4+, K+, Ca2+ e Mg2+ exibiram comportamento linear (R2>0,99) na faixa de 10-100 M enquanto a curva analítica para o íon Na+ proporcionou resposta linear na faixa de 20-200 M. Os limites de detecção encontrados para os cinco cátions foram entre 3,75 μM (NH4+), 3,75 μM (K+), 7,50 μM (Na+), 5,00 μM (Ca2+) e 5,35 μM (Mg2+). O sistema desenvolvido foi explorado para a determinação dos cátions inorgânicos em amostras de urina, saliva e lágrimas. As concentrações encontradas nas amostras biológicas variaram de 4,1 μM a 200 μM. Além do sistema híbrido, a dissertação também apresenta uma metodologia de baixo custo para produção de moldes em alto relevo para litografia suave em poli(dimetilsiloxano) (PDMS). A principal inovação é o uso de fotoresiste de baixo custo, que se trata de uma emulsão fotossensível de poli(acetato de vinila) (PVAc) utilizada na indústria têxtil e que apresenta baixa toxicidade. Outra inovação é o controle da altura dos moldes utilizando um spincoater de produção própria, com controle de tempo de rotação. Com esta metodologia foram produzidos moldes em alto relevo, e microchips em PDMS com 50 μm de largura e 40 μm de altura. Foram realizadas separações eletroforéticas dos cátions NH4+,K+,Na+,Ca2+,Mg2+e Li+. As eficiências de separação variaram entre 73.000 e 120.000 pratos/m. O que comprova que a metodologia alternativa apresenta aplicabilidade microfluídica

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Die Einbrennsilikonisierung wird schon lange verwendet, um spezielle Eigenschaften bei pharmazeutischen Glasbehältnissen zu erzielen. Üblicherweise werden Silikonöle verwendet um Gleitfilme auf den Oberflächen vorgefüllter Glasspritzen zu erzeugen oder die Glaswand zu hydrophobieren damit wäßrige Inhalte sauber ablaufen können. Weiterhin kann durch die hydrophobe Deaktivierung der Glaswände eine Reduktion der Adsorption von Wirk- und Hilfsstoffen sowie eine Erhöhung der hydrolytischen Resistenz erreicht werden. Wegen des parenteralen Verwendungszweckes werden meistens Gläser der Glasart 1 entsprechend dem europäischen Arzneibuch eingesetzt. Die Silikonölfilme (Trimethylsiloxy-endgeblockte Polydimethylsiloxane, PDMS) werden aus verdünnter wäßriger Emulsion auf der inneren Behältnisoberfläche gespreitet und anschließend bei Temperaturen oberhalb von 300°C für ca. 10-30 min hitzebehandelt. Neben der Sterilisation und Entpyrogenisierung wird der Silikonfilm durch das Einbrennen verändert. In der Literatur wurde dieser Prozeß bisher als Hitzefixierung oder -härtung bezeichnet in der Annahme, daß das Silikonöl durch Glasbindung oder Quervernetzung immobilisiert wird. Es wurden weitere Effekte wie Verdampfung oder Zersetzungsreaktionen als Folge der Hitzebehandlung angenommen. Die Dissertation hatte daher zum Ziel diese Vorstellungen analytisch zu belegen. Die Eigenschaften einbrennsilikonisierter Gläser lassen sich durch einfache Experimente beschreiben. Durch organische Extraktion der Gläser (z.B. Toluol, Dichlormethan) läßt sich der überwiegende Silikonanteil (ca. 80%) wiederfinden. Nach erschöpfender Extraktion zeigt sich eine beständige Hydrophobie z.B. durch die Ausbildung hoher Randwinkel von Wasser oder das veränderte Ablaufverhalten von Flüssigkeiten in diesen Behältnissen festgestellt werden. Basierend auf diesen Beobachtungen wurde die Anwesenheit einer gebundenen neben einer löslichen Silikonölfraktion nach dem Einbrennen angenommen. Alle analytischen Studien wurden an dieser zwei Lagen Theorie orientiert und durchgeführt. Dazu wurden einige oberflächensensitive Techniken wie z.B. die kombinierte Röntgenphotoelektronen- und Augerelektronenspektroskopie (XPS/AES) und die Flugzeit-Sekundärionenmassenspektroskopie (TOF-SIMS) eingesetzt. Durch chemische Zustandsdiagramme und massenspektroskopische Strukturanalyse konnte die Ausbildung von Bindungen zwischen Glas und Silikon detektiert werden. Kontaktwinkelmessungen zeigten, daß das gebundene Silikon bis ca. 400°C thermostabil ist und oberhalb von 450°C pyrolysiert wird. Die extrahierbare Silikonfraktion wurde ebenfalls mit einigen analytischen Techniken im Vergleich mit den jeweiligen unbehandelten Ausgangsmaterialien untersucht. Chromatographische Studien mittels Größenausschluß- und Hochtemperatur-Gaschromatographie (SEC,GC) zeigten signifikante Unterschiede in der polymeren Zusammensetzung des PDMS. Die Molekulargewichtsmittel stiegen als Folge von Verlusten von niedermolekularen Siloxanen einer Größe zwischen 0-100 Siloxaneinheiten (SU) an. Diese Defizite können auf Verdampfungs- (0-50 SU) und Zersetzungsreaktionen (50-100 SU) zurückgeführt werden. Die Untersuchung des extrahierbaren eingebrannten Silikonöls mit der Fourier Transform Infrarot Spektroskopie (FTIR) zeigte keine strukturelle Veränderung der PDMS Moleküle, z.B. durch Hydrolyse, Oxidation oder Verzweigung an. Die Emulgatoren werden durch das Einbrennen zerstört. Dies wurde durch die 1H-Kernresonanz Spektroskopie (1H-NMR) nachgewiesen. Die Thermostabilität des PDMS wird durch katalytische Effekte der Emulsionshilfsstoffe und durch die Anwesenheit von Sauerstoff erniedrigt, wie aus thermogravimetrische Analysen (TGA) mit kommerziellen Silikonölen und Ölemulsionen modellhaft abgeleitet wurde. Um die Migration von Silikonölspuren in Arzneimittel in silikonisierten Glasbehältnissen zu bewerten wurde eine Atom Absorptions spektroskopische (AAS) Methode entwickelt und entsprechend den ICH-Richtlinien validiert (Bestimmungsgrenze in Liquida 10 mg/L). Die Schlußfolgerung aus den Analysen ist, daß nach der Einbrennsilikonisierung zwei differenzierbare Silikonfraktionen an der Glasoberfläche anwesend sind. Die kovalent gebundene Dünnschicht ist unlöslich und verliert ihren Ölcharakter, ist aber für die Hydrophobisierung ausreichend. In der löslichen Fraktion, werden die mittleren Molekulargewichte durch den Verlust von niedermolekularen Anteilen erhöht. Der Anteil dieser Schicht hängt von der eingesetzten Ausgangsmenge an Silikonöl ab. Durch die Entfernung der niedermolekularen Anteile und die Zerstörung der Emulgatoren können toxikologische Bedenken zurückgestellt werden. / Heat Curing Siliconization of Pharmaceutical Glass Containers The heat curing siliconization has long since been used to introduce special properties to pharmaceutical glass containers. Silicone oils are usually applied to the containers to form lubricating films on the inner surface of prefilled syringes or to gain hydrophobic container walls for clear draining aqueous solutions, e.g. in injection vials. Furthermore the deactivation of the glass wall reduces adsorption of hydrophilic drug compounds and increases the hydrolytic stability of the glass surface. Due to the parenteral targeting the glass containers are made from glass type 1 according to the European Pharmacopoeia. The silicone oil films (= trimethylsiloxy endcapped polydimethylsiloxane, PDMS) are spread from diluted aqueous silicone oil emulsions on the inner surface and successively heat treated above 300°C for 10-30 minutes. Apart from sterilization and depyrogenation, the silicone layer is changed by the burning-in treatment. In literature the process has previously been designated as heat fixing or heat curing assuming that silicone oil is immobilized by bonding to the glass or branching. Further effects such as vaporization or degradation were suggested along with the heating. Proving these suggestions analytically was therefore the aim of the dissertation. The properties of siliconized glass containers can be characterized by simple experiments. Extraction of the glasses with suitable organic solvents (e.g. toluene, dichloromethane) recovers most of the silicone (approximately 80%) after a typical heat curing process. Still, after exhaustive solvent extraction there are persisting hydrophobic properties of the glasses evidenced by high contact angles of water and changed draining behaviour of fluids in the containers. From this observations it was assumed that a bound and a soluble silicone species is present after the heat curing siliconization. All analytical studies were based on the two-layer hypothesis and thus divided in two parts. Several surface analysis techniques were employed to detect and characterize the fixed silicone layer among them the combined X-ray photoelectron and augerelectron spectroscopy (XPS/AES) and time-of-flight secondary ion mass spectroscopy (TOF-SIMS). The formation of new bonds between silicone and glass was evidenced by chemical state plots and mass spectroscopic structural analysis. Contact angle measurements showed that the bound silicone was thermostable up to 400°C and pyrolyzed at temperatures above 450°C. The extractable silicone was also studied with several analytical techniques in comparison with the respective uncured starting materials. Chromatographic studies using size exclusion (SEC) and high temperature gas chromatography (GC) revealed significant changes in the composition of the silicone polymer. The molecular weight averages increase because low molecular weight siloxanes (LMWS) with 0-100 dimethylsiloxane units are removed through the heat curing process. These deficits can be related to vaporization (molecules from 0-50 units) and degradation (molecules from 0-100 units) effects. A Fourier transform infrared spectroscopic (FTIR) investigation of the molecular structure of the extractable PDMS after heat treatment showed no chemical changes e.g. by hydrolysis, branching or oxidation. The emulsifiers are destroyed in the heating process which was proved by a proton nuclear magnetic resonance (1H-NMR) study. The thermostability of the silicones is decreased by katalytic effects of auxiliary substances in the emulsions and the presence of oxygen. This was concluded from model thermogravimetric studies (TGA) with commercial medical grade silicone oils and emulsions. To evaluate migration of silicone traces to drug formulations in siliconized glass containers a trace analytical method by graphite furnace atomic absorption spectrometry (GF-AAS) was developed and validated according to the ICH guidelines. In liquid formulations levels of 10µg/L can be properly quantified. It was concluded that two main species of silicone can be differentiated on the glass surface after heat curing siliconization. The bonded thin layer is insoluble and sufficient for hydrophobic deactivation. The oil properties are lost. The extractable fraction is altered by removal of LMWS which increases molecular weight and viscosity and determines the lubricating properties. Its amount depends on the used silicone mass. No toxicological concern has to be made about LMWS and emulsifiers in the drug product due to their removal in the curing step.

A spin-cast silicone membrane has been successfully bonded between two injection-molded microstructured plastic discs. This sandwich structure creates a useful platform for mass production of microfluidic systems, provided that the bonds are leakproof. The bonds were achieved by a silicon dioxide coating deposited on the plastic discs by evaporation. This investigation is concerned with the process and the result only, no theory is discussed.