Characterization of dry-film photolithography for the study of B. burgdorferi motility

Kuivafilmilitografian karakterisointi Borrelia burgdorferi:n liikkuvuuden tutkimiselle. Borrelia burgdorferi on puutiaisten kantama spirokeettabakteeri, joka voi tarttua useisiin eläinlajeihin. Ihmisillä B. burgdorferi aiheuttaa borrelioosia. Tauti on laajalle levinnyt monissa pohjois- ja länsi...

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Päätekijä: Pudas, Arttur
Muut tekijät: Matemaattis-luonnontieteellinen tiedekunta, Faculty of Sciences, Bio- ja ympäristötieteiden laitos, Department of Biological and Environmental Science, University of Jyväskylä, Jyväskylän yliopisto
Aineistotyyppi: Pro gradu
Kieli:eng
Julkaistu: 2017
Aiheet:
Linkit: https://jyx.jyu.fi/handle/123456789/56026
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author Pudas, Arttur
author2 Matemaattis-luonnontieteellinen tiedekunta Faculty of Sciences Bio- ja ympäristötieteiden laitos Department of Biological and Environmental Science University of Jyväskylä Jyväskylän yliopisto
author_facet Pudas, Arttur Matemaattis-luonnontieteellinen tiedekunta Faculty of Sciences Bio- ja ympäristötieteiden laitos Department of Biological and Environmental Science University of Jyväskylä Jyväskylän yliopisto Pudas, Arttur Matemaattis-luonnontieteellinen tiedekunta Faculty of Sciences Bio- ja ympäristötieteiden laitos Department of Biological and Environmental Science University of Jyväskylä Jyväskylän yliopisto
author_sort Pudas, Arttur
datasource_str_mv jyx
description Kuivafilmilitografian karakterisointi Borrelia burgdorferi:n liikkuvuuden tutkimiselle. Borrelia burgdorferi on puutiaisten kantama spirokeettabakteeri, joka voi tarttua useisiin eläinlajeihin. Ihmisillä B. burgdorferi aiheuttaa borrelioosia. Tauti on laajalle levinnyt monissa pohjois- ja länsimaissa ja leviää entisestään ilmastonmuutoksen laajentaessa punkkien elinaluetta. Monet tekijät vaikeuttavat borrelioosin hoitoa. B. burgdorferin kyky välttää ihmisen luontaista immuunijärjestelmää ovat tässä suhteessa merkittävä. Bakteerien liikkuvuus on olennainen tämän kyvyn kannalta, mutta sen tarkka rooli bakteerien kyvyssä liikkua verisuonistossa on osittain tuntematon. Mikrofluidistiikka hyödyntää tiettyjä nesteiden dynamiikkaa ohjaavia voimia, jotka aiheuttavat nesteitä ja niiden kantamia partikkeleita käyttäytymään eri tavoin mikroskooppisessa ympäristössä makroskooppisiin ympäristöihin verrattuna. Valmistustekniikoiden edistyminen on mahdollistanut mikrofluidiikan etujen hyväksikäytön lisääntymisen ja edesauttanut sen kasvua voimakkaana biologisten tieteiden välineenä. Kuivakalvo-litografia tarjoaa helppokäyttöisen ja edullisen vaihtoehdon perinteisille mikrofluidisille valmistusmenetelmille, jotka edellyttävät puhdastiloja. Mikrofluidisten sirujen valmistukseen voidaan käyttää polydimetyylisiloksaani-polymeeria (PDMS), joka on joustava,  bioyhteensopiva ja yksinkertaistaa valmistusprosessia. Mikrofluidistiikka soveltuu erinomaisesti verisuonien, imusuonijärjestelmän ja muiden in vivo -järjestelmien, simulointiin. Tämän tutkimuksen tarkoituksena oli optimoida kuivakalvolitografiamenetelmä biologisissa kokeissa käytettäväksi ja suorittaa mikrofluidinen virtauskoe käyttäen eläviä B. burgdorferi soluja. Mikrofluidinen valmistusmenetelmä perustui mustesuihkutulostuksella tehtyihin peittomaskeihin. Kuparilevyt, joissa oli DuPont Riston 200 -sarjan negatiivinen fotoresistipäällystys, liitettiin maskeihin ja altistettiin UV:lle läheisyyslitografialla. Lopulliset mikofluidiset sirut valmistettiin PDMS:stä ja liitettiin lasipinnalle aktivoimalla sekä lasi että PDMS-pinnat plasmalla. Valmistusmenetelmä karakterisoitiin pyyhkäisyelektroni- ja stereomikroskopialla. Vihreää fluoresoivaa proteiinia tuottava B. burgdorferi GCB726 kanta syötettiin mikrokanavaan eri virtausnopeuksilla, jotka olivat korkeintaan 0,1 ml/min. Bakteerien liikkuvuus videoitiin fluoresenssimikroskopialla. Kuivakalvolitografian avulla mikrokanavat voitiin valmistaa luotettavasti 75 µm läpimittaan asti alle 5 tunnin kuluessa peittomaskin valmistuksesta. Vaikka kanavissa havaittiin pieniä vikoja, niiden vaikutus laminaariseen virtaukseen oli vähäpätöinen. Kuvataajuuden rajoitteiden vuoksi B. burgdorferi videokuva-aineistosta käytettiin vain 0,0073 cm/s:n virtausnopeudella tallennettuja videoita. B. burgdorferin liikkuvuus ilmeni kolmella tavalla: "Tanssimisessa", voltteina ja vuorovaikutuksena kanavan seinämän kanssa. Suurin osa mikrofluidisten sirujen valmistusprosesseissa havaituista vioista johtui läheisyyslitografiamenetelmän ja mustesuihkutulostimen rajoista. Tuloksia voitaisiin parantaa litografiaa optimoimalla ja parempi laatuisella maskitulostimella. Mikrosiruvalmistusprosessi oli tyydyttävä B. burgdorferin virtauksessa käyttäytymisen tarkkailemiseksi. B. burgdorferin liikkuvuus vaikutti aktiivisesti bakteerisolujen orientaatioon laminaarisessa virtauksessa, mikä johti hypoteesiin, että tämä liikkuvuus voisi antaa soluille mahdollisuuden uida verisuonijärjestelmässä. Lisätutkimuksia tarvitaan varmistamaan, onko tämä kardiovaskulaarinen liikkuvuus kehittynyt nimenomaan ratkaisevaksi ektravasaatiossa vai pelkkä välituote solujen kyvystä liikkua pehmytkudoksissa. Nämä tulokset osoittavat kuitenkin, että B. burgdorferin liikkuvuus saattaa auttaa bakteeria löytämään verisuonten seinä ekstravasaation aikana. Borrelia burgdorferi is a species of sphirochete bacteria infecting multiple different species of animals via a tick vector. In humans B. burgdorferi causes lyme disease. In many northern and western countries the disease has advanced to an endemic state, and is spreading further due to climate change affecting the spread of its carrier vector. Many factors contribute to the difficulty controlling B. burgdorferi infections and Lyme disease. One of the main factors are the methods through which the bacteria are able to evade the immune system. The bacterial mobility of B. burgdorferi is a large contributing factor to this ability, but the extent to which this mobility is crucial in the extravasation step in its pathogenic life cycle remains partially unknown. Microfluidics takes advantage of the different scale dependencies of certain forces governing fluid dynamics that causes the fluids and particles within to behave differently in a microscale environment compared to the traditional macroscale. Advances in fabrication techniques have made utilizing the advantages of microfluidic techniques more widespread and enabled it to potentially grow into a powerful tool for biological sciences. Dry-film photolithography offers ease of use and a cheap alternative to traditional microfluidic fabrication methods which require access to clean room facilities. These microfluidic chips can be made with flexible polydimethylsiloxane (PDMS) polymer, which is biocompatible, and makes the fabrication process relatively simple. Microfluidics is ideally suited for simulating cardiovascular environments, and other in vivo systems, such as the lymphatic system. The aim of this study was to characterize and optimize a dry-film photolithography method for use in biological experiments, and to carry out a flow experiment using living B. burgdorferi cells. The microfluidic fabrication technique relied on masks made with inkjet printing. Pre laminated copper plates with DuPont Riston 200 series negative photoresist were then coupled with the masks and exposed under UV with a proximity lithography method. The final chips were manufactured in PDMS, and coupled with a glass substrate by activating both the glass and PDMS surfaces with air plasma. The fabrication method was characterized with scanning electron microscopy and stereo microscopy. A green fluorescent protein expressig strain of B. burgdorferi GCB726 flown in a microchannel at flow rates of up to 0.1 ml/min. Video footage of these experiments were captured with fluorescent microscopy technique. With the dry-film photolithography technique microchannel structures could be reliably fabricated down to 75 µm resolution in less than 5 h from the mask fabrication to the final product. Though small defects were observed in the channel their effect on the laminar flow was negligible. Due to framerate constreaints only footage of B. burgdorferi in 0.0073 cm/s flow speed used. B. burgdorferi mobility in flow was observed in three distinct patterns: ‘Dancing’, flipping, and interacting with the channel wall. Most of the defects observed in the microfluidic chip fabrication processes were due to intrinsic to our proximity photolithography set-up and limits of the ink-jet printer. However, the results could be improved by further optimizing the photolithography set-up and with higher quality mask printing. The microchip fabrication process was satisfactory for the fast and cheap production of microfluidic experiment set-ups to observe B. burgdorferi in flow. The mobility of B. burgdorferi actively changed the orientation of the bacterial cells in laminar flow, which lead to the hypothesis that their mobility could confer the cells an ability to swim in the cardiovascular system. Additional studies are required to confirm whether or not this cardiovascular mobility is crucial for the pathogenesis or simply a byproduct of the cell’s ability to move within tissues and therefore inconsequential to whether or not they find the target tissues to infect. However, these results indicate that the mobility of B. burgdorferi contribute to its ability to find the vascular wall during extravasation.
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Ihmisill\u00e4 B. burgdorferi aiheuttaa borrelioosia. Tauti on laajalle levinnyt monissa pohjois- ja l\u00e4nsimaissa ja levi\u00e4\u00e4 entisest\u00e4\u00e4n ilmastonmuutoksen laajentaessa punkkien elinaluetta. Monet tekij\u00e4t vaikeuttavat borrelioosin hoitoa. B. burgdorferin kyky v\u00e4ltt\u00e4\u00e4 ihmisen luontaista immuunij\u00e4rjestelm\u00e4\u00e4 ovat t\u00e4ss\u00e4 suhteessa merkitt\u00e4v\u00e4. Bakteerien liikkuvuus on olennainen t\u00e4m\u00e4n kyvyn kannalta, mutta sen tarkka rooli bakteerien kyvyss\u00e4 liikkua verisuonistossa on osittain tuntematon.\r\nMikrofluidistiikka hy\u00f6dynt\u00e4\u00e4 tiettyj\u00e4 nesteiden dynamiikkaa ohjaavia voimia, jotka aiheuttavat nesteit\u00e4 ja niiden kantamia partikkeleita k\u00e4ytt\u00e4ytym\u00e4\u00e4n eri tavoin mikroskooppisessa ymp\u00e4rist\u00f6ss\u00e4 makroskooppisiin ymp\u00e4rist\u00f6ihin verrattuna. Valmistustekniikoiden edistyminen on mahdollistanut mikrofluidiikan etujen hyv\u00e4ksik\u00e4yt\u00f6n lis\u00e4\u00e4ntymisen ja edesauttanut sen kasvua voimakkaana biologisten tieteiden v\u00e4lineen\u00e4. Kuivakalvo-litografia tarjoaa helppok\u00e4ytt\u00f6isen ja edullisen vaihtoehdon perinteisille mikrofluidisille valmistusmenetelmille, jotka edellytt\u00e4v\u00e4t puhdastiloja. Mikrofluidisten sirujen valmistukseen voidaan k\u00e4ytt\u00e4\u00e4 polydimetyylisiloksaani-polymeeria (PDMS), joka on joustava, \u00a0bioyhteensopiva ja yksinkertaistaa valmistusprosessia. Mikrofluidistiikka soveltuu erinomaisesti verisuonien, imusuonij\u00e4rjestelm\u00e4n ja muiden in vivo -j\u00e4rjestelmien, simulointiin.\r\nT\u00e4m\u00e4n tutkimuksen tarkoituksena oli optimoida kuivakalvolitografiamenetelm\u00e4 biologisissa kokeissa k\u00e4ytett\u00e4v\u00e4ksi ja suorittaa mikrofluidinen virtauskoe k\u00e4ytt\u00e4en el\u00e4vi\u00e4 B. burgdorferi soluja. Mikrofluidinen valmistusmenetelm\u00e4 perustui mustesuihkutulostuksella tehtyihin peittomaskeihin. Kuparilevyt, joissa oli DuPont Riston 200 -sarjan negatiivinen fotoresistip\u00e4\u00e4llystys, liitettiin maskeihin ja altistettiin UV:lle l\u00e4heisyyslitografialla. Lopulliset mikofluidiset sirut valmistettiin PDMS:st\u00e4 ja liitettiin lasipinnalle aktivoimalla sek\u00e4 lasi ett\u00e4 PDMS-pinnat plasmalla. Valmistusmenetelm\u00e4 karakterisoitiin pyyhk\u00e4isyelektroni- ja stereomikroskopialla. Vihre\u00e4\u00e4 fluoresoivaa proteiinia tuottava B. burgdorferi GCB726 kanta sy\u00f6tettiin mikrokanavaan eri virtausnopeuksilla, jotka olivat korkeintaan 0,1 ml/min. Bakteerien liikkuvuus videoitiin fluoresenssimikroskopialla.\r\nKuivakalvolitografian avulla mikrokanavat voitiin valmistaa luotettavasti 75 \u00b5m l\u00e4pimittaan asti alle 5 tunnin kuluessa peittomaskin valmistuksesta. Vaikka kanavissa havaittiin pieni\u00e4 vikoja, niiden vaikutus laminaariseen virtaukseen oli v\u00e4h\u00e4p\u00e4t\u00f6inen. Kuvataajuuden rajoitteiden vuoksi B. burgdorferi videokuva-aineistosta k\u00e4ytettiin vain 0,0073 cm/s:n virtausnopeudella tallennettuja videoita. B. burgdorferin liikkuvuus ilmeni kolmella tavalla: \"Tanssimisessa\", voltteina ja vuorovaikutuksena kanavan sein\u00e4m\u00e4n kanssa.\r\nSuurin osa mikrofluidisten sirujen valmistusprosesseissa havaituista vioista johtui l\u00e4heisyyslitografiamenetelm\u00e4n ja mustesuihkutulostimen rajoista. Tuloksia voitaisiin parantaa litografiaa optimoimalla ja parempi laatuisella maskitulostimella. Mikrosiruvalmistusprosessi oli tyydytt\u00e4v\u00e4 B. burgdorferin virtauksessa k\u00e4ytt\u00e4ytymisen tarkkailemiseksi. B. burgdorferin liikkuvuus vaikutti aktiivisesti bakteerisolujen orientaatioon laminaarisessa virtauksessa, mik\u00e4 johti hypoteesiin, ett\u00e4 t\u00e4m\u00e4 liikkuvuus voisi antaa soluille mahdollisuuden uida verisuonij\u00e4rjestelm\u00e4ss\u00e4. Lis\u00e4tutkimuksia tarvitaan varmistamaan, onko t\u00e4m\u00e4 kardiovaskulaarinen liikkuvuus kehittynyt nimenomaan ratkaisevaksi ektravasaatiossa vai pelkk\u00e4 v\u00e4lituote solujen kyvyst\u00e4 liikkua pehmytkudoksissa. N\u00e4m\u00e4 tulokset osoittavat kuitenkin, ett\u00e4 B. burgdorferin liikkuvuus saattaa auttaa bakteeria l\u00f6yt\u00e4m\u00e4\u00e4n verisuonten sein\u00e4 ekstravasaation aikana.", "language": "fi", "element": "description", "qualifier": "abstract", "schema": "dc"}, {"key": "dc.description.abstract", "value": "Borrelia burgdorferi is a species of sphirochete bacteria infecting multiple different species of animals via a tick vector. In humans B. burgdorferi causes lyme disease. In many northern and western countries the disease has advanced to an endemic state, and is spreading further due to climate change affecting the spread of its carrier vector. Many factors contribute to the difficulty controlling B. burgdorferi infections and Lyme disease. One of the main factors are the methods through which the bacteria are able to evade the immune system. The bacterial mobility of B. burgdorferi is a large contributing factor to this ability, but the extent to which this mobility is crucial in the extravasation step in its pathogenic life cycle remains partially unknown.\r\n\tMicrofluidics takes advantage of the different scale dependencies of certain forces governing fluid dynamics that causes the fluids and particles within to behave differently in a microscale environment compared to the traditional macroscale. Advances in fabrication techniques have made utilizing the advantages of microfluidic techniques more widespread and enabled it to potentially grow into a powerful tool for biological sciences. Dry-film photolithography offers ease of use and a cheap alternative to traditional microfluidic fabrication methods which require access to clean room facilities. These microfluidic chips can be made with flexible polydimethylsiloxane (PDMS) polymer, which is biocompatible, and makes the fabrication process relatively simple. Microfluidics is ideally suited for simulating cardiovascular environments, and other in vivo systems, such as the lymphatic system. \r\n\tThe aim of this study was to characterize and optimize a dry-film photolithography method for use in biological experiments, and to carry out a flow experiment using living B. burgdorferi cells. The microfluidic fabrication technique relied on masks made with inkjet printing. Pre laminated copper plates with DuPont Riston 200 series negative photoresist were then coupled with the masks and exposed under UV with a proximity lithography method. The final chips were manufactured in PDMS, and coupled with a glass substrate by activating both the glass and PDMS surfaces with air plasma. The fabrication method was characterized with scanning electron microscopy and stereo microscopy. A green fluorescent protein expressig strain of B. burgdorferi GCB726 flown in a microchannel at flow rates of up to 0.1 ml/min. Video footage of these experiments were captured with fluorescent microscopy technique.\r\n\tWith the dry-film photolithography technique microchannel structures could be reliably fabricated down to 75 \u00b5m resolution in less than 5 h from the mask fabrication to the final product. Though small defects were observed in the channel their effect on the laminar flow was negligible. Due to framerate constreaints only footage of B. burgdorferi in 0.0073 cm/s flow speed used. B. burgdorferi mobility in flow was observed in three distinct patterns: \u2018Dancing\u2019, flipping, and interacting with the channel wall.\r\n\tMost of the defects observed in the microfluidic chip fabrication processes were due to intrinsic to our proximity photolithography set-up and limits of the ink-jet printer. However, the results could be improved by further optimizing the photolithography set-up and with higher quality mask printing. The microchip fabrication process was satisfactory for the fast and cheap production of microfluidic experiment set-ups to observe B. burgdorferi in flow. The mobility of B. burgdorferi actively changed the orientation of the bacterial cells in laminar flow, which lead to the hypothesis that their mobility could confer the cells an ability to swim in the cardiovascular system. Additional studies are required to confirm whether or not this cardiovascular mobility is crucial for the pathogenesis or simply a byproduct of the cell\u2019s ability to move within tissues and therefore inconsequential to whether or not they find the target tissues to infect. However, these results indicate that the mobility of B. burgdorferi contribute to its ability to find the vascular wall during extravasation.", "language": "en", "element": "description", "qualifier": "abstract", "schema": "dc"}, {"key": "dc.description.provenance", "value": "Submitted using Plone Publishing form by Arttur Pudas (arriripu) on 2017-11-28 07:38:32.952863. Form: Pro gradu -lomake (https://kirjasto.jyu.fi/julkaisut/julkaisulomakkeet/pro-gradu-lomake). 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spellingShingle Pudas, Arttur Characterization of dry-film photolithography for the study of B. burgdorferi motility mikrofluidiikka kuivafilmilitografia mikrofluidics Solu- ja molekyylibiologia Cell and molecular biology 4013 bakteerit liikkuvuus Borrelia burgdorferi Lymen borrelioosi borrelioosi
title Characterization of dry-film photolithography for the study of B. burgdorferi motility
title_full Characterization of dry-film photolithography for the study of B. burgdorferi motility
title_fullStr Characterization of dry-film photolithography for the study of B. burgdorferi motility Characterization of dry-film photolithography for the study of B. burgdorferi motility
title_full_unstemmed Characterization of dry-film photolithography for the study of B. burgdorferi motility Characterization of dry-film photolithography for the study of B. burgdorferi motility
title_short Characterization of dry-film photolithography for the study of B. burgdorferi motility
title_sort characterization of dry film photolithography for the study of b burgdorferi motility
title_txtP Characterization of dry-film photolithography for the study of B. burgdorferi motility
topic mikrofluidiikka kuivafilmilitografia mikrofluidics Solu- ja molekyylibiologia Cell and molecular biology 4013 bakteerit liikkuvuus Borrelia burgdorferi Lymen borrelioosi borrelioosi
topic_facet 4013 Borrelia burgdorferi Cell and molecular biology Lymen borrelioosi Solu- ja molekyylibiologia bakteerit borrelioosi kuivafilmilitografia liikkuvuus mikrofluidics mikrofluidiikka
url https://jyx.jyu.fi/handle/123456789/56026 http://www.urn.fi/URN:NBN:fi:jyu-201711284397
work_keys_str_mv AT pudasarttur characterizationofdryfilmphotolithographyforthestudyofbburgdorferimotility