Microfabrication of Organically Modified Ceramics for Bio-MEMS

A Bio-Micro-Electro-Mechanical-System (Bio-MEMS) is a miniaturized device that has mechanical, optical and/or electrical components for biomedical operations. High sensitivity, rapid response and integration capabilities are the main reasons for their attraction to researchers and adaptation of Bio-MEMS technology for many applications. Although the recent progress in microfabrication techniques has enabled a high degree of Bio-MEMS integration, many challenges remain. For example, extending the conventional cell monolayer cultures into 3D in vitro organ models often demands fabrication of round-cross sectional microstructures (microchannels and microwells) and integration of embedded metal-sensing elements. Owing to their low cost and the ease of the fabrication process, polymers have gained much attention in terms of biological microfluidic applications. Organically Modified Ceramics (ORMOCER) are hybrid inorganic-organic polymers, a new class of negative tone photoresist. Among polymers, ORMOCERs exhibit great potential with a view to biological microfluidic applications based on their inherent biocompatibility, transparency and mechanical stability. In this thesis, ORMOCER microfabrication methods were developed for implementation of optical, electrical and structural elements that are crucial for biological applications. A novel method, relying on controlled over-exposure of Ormocomp (a commercial formulation of ORMOCERs) was introduced for fabrication of tunable round cross-sectional microstructures, including microchannels (subprojects I-III) and microwells (subproject IV). Moreover, ORMOCER metallization was examined from the perspective of integration of embedded sensing elements (micromirrors and electrodes) into ORMOCER microfluidic channels to facilitate on-chip fluorescence (subprojects I and II) and electrochemical (subproject III) detection as well as electrical impedance spectroscopy (subproject IV). Metal adhesion, step coverage and bonding of embedded metal elements were addressed and new processes developed for various thin-film metals (subprojects III and IV). The round cross-sectional shape of the microchannel was exploited for implementation of thin-film reflective metal elements as concave micromirrors for optical detection of single cells, whereas the round shape of the microwells was applied to microfluidic three-dimensional (3D; spheroid) cell cultures. In addition to topography, the inherent surface properties of ORMOCERs were modified to allow for regulation of cell adhesion. As a result, cell monolayers (2D) and spheroids (3D) could be cultured side-by-side in a single microfluidic channel with non-invasive online impedance-based (monolayer) and optical monitoring (spheroids) of cell proliferation.Mikrovalmistustekniikat mahdollistavat sähkömekaanisten laitteiden miniatyrisoinnin biologisia ja lääketieteellisiä sovelluksia varten. Näistä laitteista käytetään yleisesti nimeä Bio-MEMS (engl. Bio-Micro-Electro-Mechanical-Systems). Bio-MEMS-laite koostuu mekaanisista, sähköjohtavista ja/tai optisista komponenteista, jotka mahdollistavat esimerkiksi soluviljelyn, lääkeaineiden kontrolloidun annostelun soluviljelmiin ja tutkittavien aineiden pitoisuuksien mittaamisen kemiallisesti mikrofluidistiikan avulla. Vaikka Bio-MEMS-laitteet ovat viime vuosina kehittyneet valtavin harppauksin, on mikrovalmistustekniikoissa ja materiaaleissa vielä paljon kehitettävää. Polymeeripohjaiset materiaalit ovat verrattain edullisia ja niiden valmistusprosessit suoraviivaisia, minkä vuoksi polymeereja käytetään paljon biologisissa mikrofluidistiikan sovelluksissa. Monet sovellukset, kuten 3D-solumallien kasvatus, edellyttävät pyöreäpohjaisia rakenteita ja mitta-antureiden yhdistämistä. Erityisesti pyöreäpohjaisten mikrorakenteiden valmistaminen on usein hidasta ja vaatii useita eri työvaiheita. Myös polymeerimateriaalien metallointi (anturien yhdistäminen) vaatii räätälöityjä mikrovalmistusmenetelmiä. Tässä työssä kehitettiin uusia mikrovalmistusmenetelmiä kaupalliselle ORMOCER-polymeerille (engl. organically modified ceramics), joka on luonnostaan bioyhteensopiva, läpinäkyvä ja mekaanisesti kestävä epäorgaaninen-orgaaninen hybridimateriaali. Työn ensimmäisessä osassa kehitettiin uusi yksivaiheinen litografinen menetelmä poikkileikkaukseltaan pyöreiden mikrorakenteiden, kuten mikrokanavien ja -kuoppien, valmistamiseen. Työn toisessa osassa kehitettiin ORMOCER-polymeerin metallointimenetelmiä, jotka mahdollistavat muun muassa mikropeilien ja sähköisten elektrodien yhdistämisen ORMOCER-polymeeristä valmistettuihin mikrokanaviin. Mikropeilien avulla on mahdollista parantaa optisen detektion herkkyyttä esimerkiksi yhden solun analytiikassa (osajulkaisu I) tai pienmolekyylien elektroforeettisessa erotuksessa (osajulkaisu II). Vastaavasti sähköisten elektrodien avulla voidaan mitata esimerkiksi pienmolekyylien pitoisuuksia amperometrisesti (osajulkaisu III) tai solujen jakautumista impedanssispektrosopiaan perustutuen (osajulkaisu IV). Lisäksi havaittiin, että ORMOCER-polymeerin pintaominaisuuksia muokkaamalla on mahdollista kontrolloida solujen polymeeripinnalle, mikä mahdollisti solujen kasvattamisen vierekkäin sekä perinteisenä viljelmänä (2D, soluyhteensopiva ja tasainen pinta) että sferoideina (3D, soluja hylkivä, pyöreäpohjainen pinta) samassa mikrofluidistisessa kanavassa

Simultaneous Culturing of Cell Monolayers and Spheroids on a Single Microfluidic Device for Bridging the Gap between 2D and 3D Cell Assays in Drug Research

Two‐dimensional (2D) cell cultures have been the primary screening tools to predict drug impacts in vitro for decades. However, owing to the lack of tissue‐specific architecture of 2D cultures, secondary screening using three‐dimensional (3D) cell culture models is often necessary. A microfluidic approach that facilitates side‐by‐side 2D and 3D cell culturing in a single microchannel and thus combines the benefits of both set‐ups in drug screening; that is, the uniform spatiotemporal distributions of oxygen, nutrients, and metabolic wastes in 2D, and the tissue‐like architecture, cell–cell, and cell–extracellular matrix interactions only achieved in 3D. The microfluidic platform is made from an organically modified ceramic material, which is inherently biocompatible and supports cell adhesion (2D culture) and metal adhesion (for integration of impedance electrodes to monitor cell proliferation). To induce 3D spheroid formation on another area, a single‐step lithography process is used to fabricate concave microwells, which are made cell‐repellant by nanofunctionalization (i.e., plasma porosification and hydrophobic coating). Thanks to the concave shape of the microwells, the spheroids produced on‐chip can also be released, with the help of microfluidic flow, for further off‐chip characterization after culturing. In this study, the methodology is evaluated for drug cytotoxicity assessment on human hepatocytes.Peer reviewe

Aqueous and non-aqueous microchip electrophoresis with on-chip electrospray ionization mass spectrometry on replica-molded thiol-ene microfluidic devices

This work describes aqueous and non-aqueous capillary electrophoresis on thiol-ene-based microfluidic separation devices that feature fully integrated and sharp electrospray ionization (ESI) emitters. The chip fabrication is based on simple and low-cost replica-molding of thiol-ene polymers under standard laboratory conditions. The mechanical rigidity and the stability of the materials against organic solvents, acids and bases could be tuned by adjusting the respective stoichiometric ratio of the thiol and allyl ("ene") monomers, which allowed us to carry out electrophoresis separation in both aqueous and non-aqueous (methanol- and ethanol-based) background electrolytes. The stability of the ESI signal was generallyPeer reviewe

Metallization of Organically Modified Ceramics for Microfluidic Electrochemical Assays

Organically modified ceramic polymers (ORMOCERs) have attracted substantial interest in biomicrofluidic applications owing to their inherent biocompatibility and high optical transparency even in the near-ultraviolet (UV) range. However, the processes for metallization of ORMOCERs as well as for sealing of metallized surfaces have not been fully developed. In this study, we developed metallization processes for a commercial ORMOCER formulation, Ormocomp, covering several commonly used metals, including aluminum, silver, gold, and platinum. The obtained metallizations were systematically characterized with respect to adhesion (with and without adhesion layers), resistivity, and stability during use (in electrochemical assays). In addition to metal adhesion, the possibility for Ormocomp bonding over each metal as well as sufficient step coverage to guarantee conductivity over topographical features (e.g., over microchannel edges) was addressed with a view to the implementation of not only planar, but also three-dimensional on-chip sensing elements. The feasibility of the developed metallization for implementation of microfluidic electrochemical assays was demonstrated by fabricating an electrophoresis separation chip, compatible with a commercial bipotentiostat, and incorporating integrated working, reference, and auxiliary electrodes for amperometric detection of an electrochemically active pharmaceutical, acetaminophen.Peer reviewe

Side-by-side 2D and 3D Cell Culturing Microdevices for Drug Toxicity Screening

Peer reviewe

Aqueous and non-aqueous microchip electrophoresis with on-chip electrospray ionization mass spectrometry on replica-molded thiol-ene microfluidic devices

This work describes aqueous and non-aqueous capillary electrophoresis on thiol-ene-based microfluidic separation devices that feature fully integrated and sharp electrospray ionization (ESI) emitters. The chip fabrication is based on simple and low-cost replica-molding of thiol-ene polymers under standard laboratory conditions. The mechanical rigidity and the stability of the materials against organic solvents, acids and bases could be tuned by adjusting the respective stoichiometric ratio of the thiol and allyl (“ene”) monomers, which allowed us to carry out electrophoresis separation in both aqueous and non-aqueous (methanol- and ethanol-based) background electrolytes. The stability of the ESI signal was generally ≤10% RSD for all emitters. The respective migration time repeatabilities in aqueous and non-aqueous background electrolytes were below 3 and 14% RSD (n = 4-6, with internal standard). The analytical performance of the developed thiol-ene microdevices was shown in mass spectrometry (MS) based analysis of peptides, proteins, and small molecules. The theoretical plate numbers were the highest (1.2–2.4 × 104 m−1) in ethanol-based background electrolytes. The ionization efficiency also increased under non-aqueous conditions compared to aqueous background electrolytes. The results show that replica-molding of thiol-enes is a feasible approach for producing ESI microdevices that perform in a stable manner in both aqueous and non-aqueous electrophoresis.Peer reviewe