Fish-Liver-on-Chip: A Microfluidic Model to Assess Bioaccumulation of Environmental Drug Residues In Vitro

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

Immobilization of proteolytic enzymes on replica-molded thiol-ene micropillar reactors via thiol-gold interaction

Julkaistaan OA-artikkelina.We introduce rapid replica molding of ordered, high-aspect-ratio, thiol-ene micropillar arrays for implementation of microfluidic immobilized enzyme reactors (IMERs). By exploiting the abundance of free surface thiols of off-stoichiometric thiol-ene compositions, we were able to functionalize the native thiol-ene micropillars with gold nanoparticles (GNPs) and these with proteolytic alpha-chymotrypsin (CHT) via thiol-gold interaction. The micropillar arrays were replicated via PDMS soft lithography, which facilitated thiol-ene curing without the photoinitiators, and thus straightforward bonding and good control over the surface chemistry (number of free surface thiols). The specificity of thiol-gold interaction was demonstrated over allyl-rich thiol-ene surfaces and the robustness of the CHT-IMERs at different flow rates and reaction temperatures using bradykinin hydrolysis as the model reaction. The product conversion rate was shown to increase as a function of decreasing flow rate (increasing residence time) and upon heating of the IMER to physiological temperature. Owing to the effective enzyme immobilization onto the micropillar array by GNPs, no further purification of the reaction solution was required prior to mass spectrometric detection of the bradykinin hydrolysis products and no clogging problems, commonly associated with conventional capillary packings, were observed. The activity of the IMER remained stable for at least 1.5 h (continuous use), suggesting that the developed protocol may provide a robust, new approach to implementation of IMER technology for proteomics research.Peer reviewe

Exploitation of block copolymers as coating material in capillary electromigration techniques

This Master's thesis deals with the use of block copolymers in capillary electromigration techniques (literature part) and both in material chemistry and capillary electrophoresis (experimental part). Amphiphilic block copolymers are an interesting research topic due to their specific molecular structure, which consists of at least two parts with different chemical natures. The great potential of block copolymers arises from their tunability of size, shape and composition. In recent years, numerous copolymer architectures have been developed and the demand to find new materials for biomolecule separations remains high. The literature part introduces rarely used coating materials, block copolymers, in capillary electromigration techniques. The two main electromigration techniques where block copolymers have been tested are capillary electrophoresis and capillary gel electrophoresis. Block copolymers have been attached to capillary inner surface permanently and dynamically. In capillary gel electrophoresis the micellization ability of block copolymers has been already well-known for many decades, and specific studies of copolymer phases have been published. In the experimental part of this M.Sc. thesis, double-hydrophilic poly(N-methyl-2-vinylpyridinium iodide- block –ethylene oxide) diblock copolymer was used in two very different applications to emphasize the potential of block copolymers in various fields. In both studies, the hydrophilicity of ethylene oxide block and polycationic nature of vinylpyridinium block were utilized. First poly(N-methyl-2-vinylpyridinium iodide- block –ethylene oxide) was used to mediate the self-assembly of ferritin protein cages. The aim of this research was to explore complexation of double-hydrophilic diblock copolymers with protein cages and to study the molecular morphology of the formed nanoparticle/copolymer assemblies. Complexation process was studied in aqueous solvent medium and formation of complexes was investigated with dynamic light scattering. Transmission electron microscopy and small-angle x-ray scattering technique were used to characterize the size and shape of the particles. In the second approach the double-hydrophilic block copolymer was used as capillary coating material in two different capillary electromigration techniques. The possibility to alter the electro-osmotic flow and to gain a new tool for biomolecule studies was explored. Our results indicated that poly(N-methyl-2-vinylpyridinium iodide- block- ethylene oxide) binds efficiently with oppositely charged objects and surfaces via electrostatic interactions, and the polyethylene oxide block gives good stability in aqueous medium. Nanoparticle co-assembly studies showed that the poly(N-methyl-2-vinylpyridinium iodide- block- ethylene oxide) complexes were approximately 200-400 nm in diameter. For capillary coating studies, the polymer suppressed electro-osmotic flow efficiently and showed good run-to-run stability with RSD values from 1.4 to 7.9 %. Coating was observed to be very stable at pH range from 4.5 to 8.5 with ultra-low mobilities. The results achieved prove the potential of double-hydrophilic block copolymers in different various fields in the future.Tämän pro gradu–tutkielman kirjallinen osa käsittelee lohkopolymeerejä ja niiden hyödyntämistä kapillaarielektromigraatiotekniikoissa. Tutkielman kokeellisessa osassa lohkopolymeeria hyödynnetään kahdessa eri sovelluksessa. Amfifiiliset lohkopolymeerit ovat mielenkiintoinen tutkimuskohde niiden erityisen rakenteen vuoksi. Ne koostuvat hydrofiilisesta ja hydrofobisesta osasta, joka mahdollistaa hyvin erilaisten pintojen ja funktionaalisten ryhmien liittämisen toisiinsa. Viime vuosina on tutkittu paljon erilaisia kopolymeerirakenteita ja niiden käyttömahdollisuuksia biomolekyylitutkimuksissa. Kirjallisuusosassa esitellään tutkimuksia, joissa kopolymeereihin kuuluvia lohkopolymeerejä käytetään silikakapillaarien päällystysmateriaalina kapillaarielektromigraatiotekniikoista erityisesti kapillaarivyöhyke- ja kapillaarigeelielektroforeesissa. Päällystyksissä lohkopolymeerit on kiinnitetty joko kovalenttisesti tai elektrosaattisin vuorovaikutuksin silikakapillaarin pintaan. Kapillaarigeelielektroforeesissa lohkopolymeerien misellinmuodostusta ja erilaisia kopolymeerirakenteita on hyödynnetty ansiokkaasti. Pro gradu-tutkielman kokeellisessa osassa selvitettiin saman lohkopolymeerin soveltuvuutta kahteen eri käyttötarkoitukseen. Molemmissa sovelluksissa amfifiilinen lohkopolymeeri muokattiin ensin kemiallisesti- hydrofiiliseksi lohkopolymeeriksi, jonka jälkeen sovelluksissa hyödynnettiin lohkopolymeerin PEO-lohkon hydrofiilisyyttä ja kvaternarisoidun P2QVP-lohkon polyelektrolyyttiluonnetta. Ensimmäisessä sovelluksessa poly(N-metyyli-2-vinyylipyridinium jodidi – lohko - etyleenioksidi)-rakennetta käytettiin avustamaan ferritiiniproteiinihäkkien itsejärjestäytymistä. Tämän tutkimuksen tarkoituksena oli selvittää lohkopolymerien ja proteiinihäkkien kompleksoitumista ja tutkia muodostuvan nanopartikkeli/kopolymeerin morfologiaa. Kompleksointitutkimuksissa hyödynnettiin dynaamista valonsirontaa sekä transmissioelektronimikroskopiaa ja pienkulmaröntgensirontaa. Toisessa sovelluksessa hydrofiilisella lohkopolymeerilla päällystettiin silikapillaarin sisäpinta ilman kovalenttisia sidoksia ja selvitettiin päällysteen soveltuvuutta kapillaarivyöhyke-elektroforeesiin. Tutkimus keskittyi päällysteen ominaisuuksien tutkimiseen ja etenkin sen vaikutukseen elektro-osmoottiseen virtaukseen sekä biomolekyylien erotuksiin. Saadut tulokset osoittivat, että käytetty lohkopolymeeri sitoutuu vahvasti elektrostaattisin vuorovaikutuksin vastakkaismerkkisten funktionaalisten ryhmien ja pintojen kanssa. Muodostuneet nanopartikkelikompleksit olivat kooltaan 200-400 nm ja sauvamaisia. Kapillaarin päällystystutkimukset osoittivat, että käytetyn lohkopolymeeripäällystyksen ansiosta elekro-osmoottisesta virtauksesta päästiin lähes kokonaan eroon ja että päällyste muodosti stabiilin ja neutraalin pinnan laajalla pH-alueella

Thiol-ene based microfluidic devices for bioanalysis

Microfluidic devices have been noticed potential platforms for bioanalytical research. The small size enables rapid analyses, reduced sample and reagent consumption and microtechnology allow integration of multiple analytical units on-chip. Despite the progress made in the past years, the current materials for these devices have challenges with the cost and/or mass production possibilities, unit compatibility and reproducible performance. Thiol-ene polymers have been proposed as alternative materials for microsystems to address the above-mentioned challenges. The aim of this thesis was to study the feasibility of thiol-enes for out-of-cleanroom replica molding of analytical devices. Thiol-ene photopolymerization was studied in the absence of a photoinitiator. Without the initiator the bulk properties were noticed relatively similar compared with the initiated-thiol-enes, but the curing time was shown to have a notable impact on the surface properties in the absence of the initiator. This allowed control over the surface chemistry and bonding of even two alike thiol-ene layers. These obtained properties of thiol-enes were utilized in enzyme microreactor, in microchip electrophoresis (MCE), and in mass spectrometric (MS) applications. Proteolytic peptide digestion was obtained with immobilized enzymes on the microreactor. Straightforward enzyme immobilization was developed by the use of thiol-rich surfaces and gold nanoparticles via thiol-gold chemistry. In MCE, the controllability of the surface properties allowed production of devices with stable and high electroosmotic flow. Owing to the good solvent stability of thiol-enes, MCE separations could be conducted even in non-aqueous media with enhanced resolving power. For online MS applications, the microdevices were integrated with on-chip electrospray tip. With optimized polymer composition, stable electrospray and good chip-to-chip reproducibility was achieved. The tunable surface chemistry allowed development of inert surfaces by polyethylene glycol coating to fully eliminate nonspecific surface interactions. This thesis work increases our understanding of the material and surface properties of thiol-enes with a view to their use in mass spectrometry based bioanalysis as well as in other fields of microfluidics. The impact of the work increases the customization possibilities in low-cost microfabrication.Mikrofluidiset laitteet ovat yhä merkittävämmässä osassa nykypäivän bioanalytiikan tutkimusta. Laitteiden pieni koko mahdollistaa yhä nopeammat mittausanalyysit kuin myös kemiallisten jätteiden ja reagenssien määrän merkittävän vähenemisen. Mikroteknologian avulla voidaan lisäksi liittää kaikki kemiallisen analyysin vaiheet samalle laitteelle. Nykypäivän mikrolaitteilla on kuitenkin ongelmana mittaussysteemien toistettavuus sekä materiaalien epästabiilit ominaisuudet ja kallis tuotanto. Yhtenä ratkaisuna tähän on uusien materiaalien tutkimus. Eräs uusi varteen otettava materiaali on tioleenipolymeerit. Tässä väitöskirjatyössä tutkittiin näiden polymeerien soveltuvuutta analyyttisiksi mittalaitteiksi, ja tioleenipohjaiset mikrolaiteet valmistettiin edullisella replikointi menetelmällä. Lisäksi tämän väitöskirjatyön tavoitteena oli tutkia tioleenien materiaali- ja pintaominaisuuksia ja valmistusta ilman yleisesti käytettyä initiaattoria tai muita apuaineita. Tässä väitöskirjatyössä havaittiin, että ilman initiaattoria valmistettujen tioleenien materiaaliominaisuudet ovat samankaltaisia kuin initiaattorin kanssa. Pintaominaisuudet olivat taas selkeästi riippuvaiset valmistus olosuhteista, mikä mahdollisti pintakemian kontrolloinnin ja erityisesti kahden saman kemian omaavan pinnan liittämisen yhteen. Näitä havaittuja tioleenien ominaisuuksia hyödynnettiin näytteen esikäsittely, mikrosiruelektroforeesi (MCE) sekä massaspektrometria (MS) sovelluksissa. Näytteen esikäsittelyssä proteolyyttinen peptidien pilkkominen saavutettiin mikroreaktorisirulla immobilisoitujen entsyymien avulla. Mikrosiruelektroforeesissa pintaominaisuuksien kontrolloinnilla saavutettiin stabiili elektroosmoottinen virtaus. Tioleenien hyvä liuotin yhteensopivuus mahdollisti lisäksi selektiiviset bioanalyysit orgaanisissa olosuhteissa. Optimoiduilla materiaaliominaisuuksilla mikrosiruihin pystyttiin valmistamaan toistettavasti sähkösumutuskärki MS detektointia varten. Vaativimpiin biologisiin mittauksiin kehitettiin lisäksi inertti polymeeri pinnoitus. Tämä väitöskirjatutkimus laajentaa tietämystä tioleenipolymeerien yhteensopivuudesta bioanalyyseihin, mutta myös muihin analytiikan ja mikrofluidistiikan käyttötarkoituksiin. Tässä työssä kehitetyt mikrosirut mahdollistavat lisäksi niiden edullisen valmistuksen sekä räätälöinnin erilaisiin sovelluksiin

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

PeptiCHIP : A Microfluidic Platform for Tumor Antigen Landscape Identification

Publisher Copyright: © 2021 The Authors. Published by American Chemical Society.Identification of HLA class I ligands from the tumor surface (ligandome or immunopeptidome) is essential for designing T-cell mediated cancer therapeutic approaches. However, the sensitivity of the process for isolating MHC-I restricted tumor-specific peptides has been the major limiting factor for reliable tumor antigen characterization, making clear the need for technical improvement. Here, we describe our work from the fabrication and development of a microfluidic-based chip (PeptiCHIP) and its use to identify and characterize tumor-specific ligands on clinically relevant human samples. Specifically, we assessed the potential of immobilizing a pan-HLA antibody on solid surfaces via well-characterized streptavidin-biotin chemistry, overcoming the limitations of the cross-linking chemistry used to prepare the affinity matrix with the desired antibodies in the immunopeptidomics workflow. Furthermore, to address the restrictions related to the handling and the limited availability of tumor samples, we further developed the concept toward the implementation of a microfluidic through-flow system. Thus, the biotinylated pan-HLA antibody was immobilized on streptavidin-functionalized surfaces, and immune-affinity purification (IP) was carried out on customized microfluidic pillar arrays made of thiol-ene polymer. Compared to the standard methods reported in the field, our methodology reduces the amount of antibody and the time required for peptide isolation. In this work, we carefully examined the specificity and robustness of our customized technology for immunopeptidomics workflows. We tested this platform by immunopurifying HLA-I complexes from 1 × 106 cells both in a widely studied B-cell line and in patients-derived ex vivo cell cultures, instead of 5 × 108 cells as required in the current technology. After the final elution in mild acid, HLA-I-presented peptides were identified by tandem mass spectrometry and further investigated by in vitro methods. These results highlight the potential to exploit microfluidics-based strategies in immunopeptidomics platforms and in personalized immunopeptidome analysis from cells isolated from individual tumor biopsies to design tailored cancer therapeutic vaccines. Moreover, the possibility to integrate multiple identical units on a single chip further improves the throughput and multiplexing of these assays with a view to clinical needs.Peer reviewe