Common use of End User Agreement Clause in Contracts

End User Agreement. This publication is distributed under the terms of Article 25fa of the Dutch Copyright Act (Auteurswet) with explicit consent by the author. Dutch law entitles the maker of a short scientific work funded either wholly or partially by Dutch public funds to make that work publicly available for no consideration following a reasonable period of time after the work was first published, provided that clear reference is made to the source of the first publication of the work. This publication is distributed under The Association of Universities in the Netherlands (VSNU) ‘Article 25fa implementation’ pilot project. In this pilot research outputs of researchers employed by Dutch Universities that comply with the legal requirements of Article 25fa of the Dutch Copyright Act are distributed online and free of cost or other barriers in institutional repositories. Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication. You are permitted to download and use the publication for personal purposes. All rights remain with the author(s) and/or copyrights owner(s) of this work. Any use of the publication other than authorised under this licence or copyright law is prohibited. If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please contact the Library through email: ▇▇▇▇▇▇▇▇▇@▇▇▇.▇▇.▇▇, or send a letter to: University Library Radboud University Copyright Information Point PO Box 9100 6500 HA Nijmegen You will be contacted as soon as possible. Ultrarapid Generation of Femtoliter Microfluidic Droplets for Single- Molecule-Counting Immunoassays Jung-uk Shim,†,§,* ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇,† ▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇▇,‡ ▇▇▇▇▇▇▇ ▇. ▇. ▇▇▇▇,†,^ ▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇,† and ▇▇▇▇▇ ▇▇▇▇▇† †Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, U.K., CB2 1EW and ‡Department of Biochemistry, University of Cambridge, Cambridge, U.K., CB2 1GA. §Present address: Biomedical Engineering, University of Glasgow, Glasgow, UK, G12 8LT. ^Present address: Radboud University Nijmegen, Institute for Molecules and ▇▇▇▇▇▇▇▇▇, ▇▇▇▇▇▇▇▇▇▇▇▇▇▇ ▇▇▇, ▇▇▇▇ ▇▇ ▇▇▇▇▇▇▇▇, ▇▇▇ ▇▇▇▇▇▇▇▇▇▇▇. ARTICLE ABSTRACT We report a microfluidic droplet-based approach enabling the measurement of chemical reactions of individual enzyme molecules and its application to a single-molecule-counting immunoassay. A microfluidic 6 device is used to generate and manipulate <10 fL droplets at rates of up to 1.3 × 10 per second, about 2 orders of magnitude faster than has previously been reported. The femtodroplets produced with this device can be used to encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables the fluorescent product of a single enzyme molecule to be detected within 10 min of on-chip incubation. Our prototype system is validated by detection of a biomarker for prostate cancer in buffer, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte molecules.

End User Agreement. This publication is distributed under the terms of Article 25fa of the Dutch Copyright Act (Auteurswet) with explicit consent by the author. Dutch law entitles the maker of a short scientific work funded either wholly or partially by Dutch public funds to make that work publicly available for no consideration following a reasonable period of time after the work was first published, provided that clear reference is made to the source of the first publication of the work. This publication is distributed under The Association of Universities in the Netherlands (VSNU) ‘Article 25fa implementation’ pilot project. In this pilot research outputs of researchers employed by Dutch Universities that comply with the legal requirements of Article 25fa of the Dutch Copyright Act are distributed online and free of cost or other barriers in institutional repositories. Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication. You are permitted to download and use the publication for personal purposes. All rights remain with the author(s) and/or copyrights owner(s) of this work. Any use of the publication other than authorised under this licence or copyright law is prohibited. If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please contact the Library through email: ▇▇▇▇▇▇▇▇▇@▇▇▇.▇▇.▇▇, or send a letter to: University Library Radboud University Copyright Information Point PO Box 9100 6500 HA Nijmegen You will be contacted as soon as possible. Ultrarapid Generation View Article Online / Journal Homepage / Table of Femtoliter Microfluidic Droplets Contents for Single- Molecule-Counting Immunoassays Jung-uk Shim,†,§,* this issue ChemComm Dynamic Article Links Cite this: Chem. Commun., 2011, 47, 8740–8749 ▇▇▇.▇▇ ▇. ▇.▇▇▇/▇▇▇▇▇▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇,† ▇FEATURE ARTICLE Triazole: a unique building block for the construction of functional materials ▇▇▇▇▇▇ ▇▇▇▇´▇ˇ▇▇▇▇▇▇,‡ ▇▇▇, ▇▇▇▇ ▇. ▇. ▇▇▇▇,†,^ ▇▇ and ▇▇▇▇ ▇. ▇▇▇▇▇▇▇▇,† and * Downloaded by Radboud Universiteit Nijmegen on 06 December 2012 Published on 09 May 2011 on ▇▇▇▇▇ ://▇▇▇▇▇† †Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, U.K., CB2 1EW and ‡Department of Biochemistry, University of Cambridge, Cambridge, U.K., CB2 1GA. §Present address: Biomedical Engineering, University of Glasgow, Glasgow, UK, G12 8LT. ^Present address: Radboud University Nijmegen, Institute for Molecules and .▇▇▇▇▇▇▇▇▇, ▇▇▇▇▇▇▇▇▇▇▇.▇▇▇ ▇▇▇| doi:10.1039/C1CC10685F DOI: 10.1039/c1cc10685f Over the past 50 years, ▇▇▇▇ ▇▇ ▇▇▇▇▇▇▇▇, ▇▇▇ ▇▇▇▇▇▇▇▇▇▇▇. ARTICLE ABSTRACT We report a microfluidic dropletnumerous roads towards carbon-based approach enabling materials have been explored, all of them being paved using mainly one functional group as the measurement of chemical reactions of individual enzyme molecules and its application to a single-molecule-counting immunoassay. A microfluidic 6 device is used to generate and manipulate <10 fL droplets at rates of up to 1.3 × 10 per second, about 2 orders of magnitude faster than has previously been reportedbrick: acetylene. The femtodroplets produced with this device acetylene group, or the carbon–carbon triple bond, is one of the oldest and simplest functional groups in chemistry, and although not present in any of the naturally occurring carbon allotropes, it is an essential tool to access their synthetic carbon-rich family. In general, two strategies towards the synthesis of p-conjugated carbon-rich structures can be used employed: (a) either the acetylene group serves as a building block to encapsulate single biomolecular complexes tagged access acetylene-derived structures or (b) it serves as a synthetic tool to provide other, usually benzenoid, structures. The recently discovered copper-catalysed azide–alkyne cycloaddition (CuAAC) reaction, however, represents a new powerful alternative: it transforms the acetylene group into a five-membered heteroaromatic 1H-1,2,3-triazole (triazole) ring and this gives rise to new opportunities. Compared with all-carbon aromatic non-functional rings, the triazole ring possesses three nitrogen atoms and, thus, can serve as a reporter enzyme; their small volume enables ligand to coordinate metals, or as a hydrogen bond acceptor and donor. This Feature Article summarises examples of using the fluorescent product of triazole ring to construct conjugation- and/or function-related heteroaromatic materials, such as tuneable multichromophoric covalent ensembles, macrocyclic receptors or responsive foldamers. These recent examples, which open a single enzyme molecule new sub-field within organic materials, started to be detected within 10 min of onappear only few years ago and represent ‘‘a few more bricks’’ on the road to carbon-chip incubation. Our prototype system is validated by detection of a biomarker for prostate cancer in buffer, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte moleculesrich functional materials.

End User Agreement. This publication is distributed under the terms of Article 25fa of the Dutch Copyright Act (Auteurswet) with explicit consent by the author. Dutch law entitles the maker of a short scientific work funded either wholly or partially by Dutch public funds to make that work publicly available for no consideration following a reasonable period of time after the work was first published, provided that clear reference is made to the source of the first publication of the work. This publication is distributed under The Association of Universities in the Netherlands (VSNU) ‘Article 25fa implementation’ pilot project. In this pilot research outputs of researchers employed by Dutch Universities that comply with the legal requirements of Article 25fa of the Dutch Copyright Act are distributed online and free of cost or other barriers in institutional repositories. Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication. You are permitted to download and use the publication for personal purposes. All rights remain with the author(s) and/or copyrights owner(s) of this work. Any use of the publication other than authorised under this licence or copyright law is prohibited. If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please contact the Library through email: ▇▇▇▇▇▇▇▇▇@▇▇▇.▇▇.▇▇, or send a letter to: University Library Radboud University Copyright Information Point PO Box 9100 6500 HA Nijmegen You will be contacted as soon as possible. Ultrarapid Generation of Femtoliter Microfluidic Droplets for Single- MoleculeVanaf het kabinet-▇▇▇ ▇ (vanaf 1994) vormen begrippen als ‘verantwoorde‐ lijkheid’, ‘meedoen’ en ‘zelfbeschikking’ ijkpunten in reflecties over burgerschap in Nederland (▇▇ ▇▇▇▇▇▇▇▇▇, 2014, 57-Counting Immunoassays Jung-uk Shim,†,§,* 59). In lijn hiermee nemen opeenvolgende kabinetten maatregelen waarbij verantwoordelijkheden en taken deels ▇▇▇▇▇▇ verplaatst van overheid naar burgers. Aan deze verplaatsing liggen doelmatig‐ heidsmotieven ten grondslag, maar ook een emancipatorisch burgerschapsideaal (Suvarierol, 2015; ▇▇▇▇▇▇▇▇▇ ▇.▇., 2013; Sociaal Cultureel Planbureau, 2012). Ook buiten Nederland zijn de afgelopen decennia normen van burgerschap geagen‐ deerd waarin noties als zelfredzaamheid, activiteit en verantwoordelijkheid een centrale positie innemen. In ▇▇▇▇▇▇ als Australië, de Verenigde Staten, Canada, Groot-Brittannië en andere Europese ▇▇▇▇▇▇ is het denken over de verdeling van verantwoordelijkheden tussen individuen, instanties en private organisaties ver‐ anderd. Onderzoekers zien veelal neoliberale agenda’s als aanjagers van het nieuwe burgerschapsmodel, dat is gebaseerd op marktprikkels en op autonome, kiezende individuen (Lister, 2015; Ilcan, 2009; Suvarierol, 2015; ▇▇▇▇▇▇, 2006; ▇▇▇▇▇▇▇, 2006; Bevir, 2011; Garthwaite, 2017). Tegelijk is ▇▇ ▇▇▇▇▇▇ van het zelf‐ redzaamheidsideaal juist dat het zich, als een ‘overlappende consensus,’ laat ver‐ enigen met uiteenlopende politieke agenda’s, zoals communitarisme (rol van de gemeenschap), liberalisme (terugtredende overheid), republikeinse opvattingen (vrijheid en morele plicht) en sociaaldemocratie (emancipatie, derde weg) (Sociaal Cultureel Planbureau, 2012; ▇▇ ▇▇▇▇▇▇▇▇▇, 2014; Kampen & Duyvendak, 2016; ▇▇▇▇▇▇ & ▇▇▇▇▇▇, 1997). Analyses over goed burgerschap beginnen vaak bij de constatering dat burgers te veel op de overheid zijn gaan leunen; ze stellen zich op als klant, slachtoffer of rechthebbende. Als ‘verzekeraar’ tegen risico’s en ongemakken zou de overheid haar burgers initiatief en verantwoordelijkheidsgevoel hebben ontnomen (▇▇ ▇▇▇▇▇▇▇▇▇, 2014, 60). Als reactie hierop ontwikkelde zich een burgerschapsideaal dat de nadruk legde op keuze, verantwoordelijkheid en participatie (▇▇▇▇▇▇ & * Dit onderzoek is onderdeel van het PhD-project van drs. ▇. ▇▇▇ ▇▇▇▇▇, die tevens werkzaam is in de organisatie waarin de casestudy is uitgevoerd. Basisdata, codeer- en analyseschema’s zijn opvraagbaar bij hem. ** Drs. ▇▇▇▇▇▇ ▇▇▇ ▇▇▇▇▇ is PhD-kandidaat (buitenpromovendus) bij het Institute for Science in Society (ISiS), Radboud University, en coördinerend adviseur corporate communicatie bij het ministerie van Financiën. ▇▇. ▇▇▇▇▇▇ ▇▇▇ Wessel is universitair docent, leerstoel Strategische Communicatie aan de Wageningen University & Research. Prof. ▇▇. ▇▇▇▇▇ is professor Socio- Ecological Interactions aan het Institute for Science in Society (ISiS), Radboud University. ▇▇▇▇▇▇ ▇▇▇ ▇▇▇▇▇, ▇▇▇▇▇▇ ▇▇▇ ▇▇▇▇▇▇ & ▇▇▇▇▇▇ ▇▇▇▇▇ Tonkens, 2011, 9). In Groot-Brittannië krijgt het ideaal van active citizenship ver‐ der betekenis in relatie tot het concept van de Big Society. De actieve burger heeft betaald werk, zet zich vrijwillig in voor de samenleving en is niet afhankelijk van de overheid (Garthwaite, 2017, 285.) Burgers zijn bovendien in veel gevallen, individueel of samen, in staat om tot betere oplossingen te ▇▇▇▇▇ ▇▇▇ de over‐ ▇▇▇▇ (▇▇▇▇▇ andere ▇▇▇▇▇▇, 2004, 33; ▇▇▇▇▇▇▇▇▇▇,† , 2015, 3). Volgens verschillende auteurs is er een trend waarneembaar waarbij burgers die niet aan het ideaal vol‐ doen, ▇▇▇▇▇▇ ▇. ▇▇▇▇▇,† ▇▇▇▇▇ ▇. voorgesteld als onvolledige burgers die niet helemaal klaar zijn voor hun vrijheid (Suvarierol, 2015) of die afkeuring verdienen (Garthwaithe, 2017; ▇▇▇▇▇▇▇,† ▇▇ & ▇▇▇▇▇▇▇, 2013). In weerwil van ▇▇ ▇▇▇▇▇ omarming van het begrip door politici, professionals en opiniemakers lijkt ▇▇ ▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇▇,‡ ▇▇ overeenstemming over wat zelfredzaamheid precies is. Het begrip refereert aan een breed spectrum van maatschappelijke waarden die beleidsmakers en professionals in staat stellen om uiteenlopende praktijken te legitimeren. Zelfredzaamheid is vooral een onbepaald, negatief geformuleerd ideaal, in de zin dat betekenissen zich aanvankelijk beperken tot wat burgers geacht ▇▇▇▇▇▇ ▇vooral níet te doen: op de overheid leunen, overlast veroorzaken, zich negatief gedragen. ▇. Pas later ▇▇▇▇,†,^ ▇▇▇▇ ook positieve noties als fat‐ soen en verantwoordelijkheid toegevoegd (Tonkens, 2006, 8-9). Verschillende op Nederland gerichte studies thematiseren in dit verband een spanningsveld waarin de overheid tegelijk wil loslaten én afdwingen (▇▇ ▇▇▇▇▇▇▇▇▇,† and , 2014; Van Alke‐ made, 2015; Raad voor Maatschappelijke Ontwikkeling, 2013, 2014; Sociaal Cul‐ tureel Planbureau, 2012). In internationale publicaties komen vergelijkbare analyses naar voren over de meervoudige en soms tegengestelde motieven die onder burgerschapsnoties als actief burgerschap en zelfredzaamheid werkzaam zijn (vergelijk Lister, 2015, 357-358). Door alle observaties heen verschijnt een burgerschapsideaal dat breed en amorf is, en waarmee soms tegengestelde praktijken ▇▇▇▇▇▇ gelegitimeerd. Het krijgt uiteenlopende toepassingen en betekenissen in relatie tot onder andere zorg, vei‐ ligheid, welzijn, sociale zekerheid en financiën (onder andere Helsloot & Van ’t Padje, 2010; Movisie, 2013; Nibud, 2017). Wie vraagt naar betekenissen van zelf‐ redzaamheid, zal daarom de plaatsen moeten opzoeken waar mensen het ideaal concretiseren en materialiseren (▇▇▇▇▇▇ & Tonkens, 2011, 180-200). In dit artikel gaan we op zoek naar de toepassing van zelfredzaamheidsopvattin‐ gen in een context van beleidsuitvoering. We ▇▇▇▇▇▇ weten wat er gebeurt als amb‐ tenaren een burgerschapsideaal, op maatschappelijk niveau gevormd, in hun eigen praktijk van betekenissen voorzien. De volgende vraag staat centraal: hoe geven ambtenaren het maatschappelijke ideaal van zelfredzaamheid betekenis in hun werkpraktijk en op ▇▇▇▇▇ ▇▇▇▇▇† †Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, U.K., CB2 1EW and ‡Department of Biochemistry, University of Cambridge, Cambridge, U.K., CB2 1GA▇ speelt het een rol bij het vormgeven en legi‐ timeren van beleidskeuzes? We combineren in dit onderzoek een macroperspectief met een meso- en micro‐ perspectief. §Present address: Biomedical Engineering, University of Glasgow, Glasgow, UK, G12 8LT. ^Present address: Radboud University Nijmegen, Institute for Molecules and Met de term ‘micro’ verwijzen we naar het niveau van tweegesprek‐ ▇▇▇ en ▇▇▇▇▇▇ groepen, ‘meso’ verwijst naar het organisatieniveau, en ‘macro’ naar het niveau van maatschappij of beroepsveld. Een burgerschapsopvatting die func‐ tioneert als een ‘ideologie’ op maatschappelijk niveau wordt binnen een organisa‐ tie verwerkt en omgewerkt. Dat professionals ideeën naar specifieke contexten vertalen en aanpassen is in heel wat studies beschreven (bijvoorbeeld ▇▇▇▇▇▇▇ & Nielsen, 2016; ▇▇▇▇▇▇▇ & ▇▇▇, 2017; ▇▇▇▇▇▇▇▇▇▇▇ & ▇▇▇▇▇▇▇▇, 2012). Gedetail‐ leerde beschrijvingen van de interacties die hieraan ten grondslag liggen zijn schaarser. Bovendien richten deze studies zich vaak op de vertaling van manage‐ ment- en organisatieconcepten. Er is nog weinig onderzoek beschikbaar over ▇▇ ▇▇▇▇▇▇ waarop ambtenaren maatschappelijke ideeën over burgerschap toepassen – maar ook aanpassen – in de praktijk van een uitvoeringsorganisatie. Meer in het algemeen is er een beperkte basis van theorievorming over de wisselwerking tussen interacties op een micro- en macroniveau. De structuratietheorie (Gid‐ dens, 1995) leert dat niveaus elkaar wederzijds constitueren, maar ▇▇ ▇▇▇▇▇▇ waarop dat via (micro)interacties gebeurt – hoe bijvoorbeeld ‘bottom-up’ nieuwe interpretaties ontstaan die leiden tot aanpassing van macroframes – is tot nu toe in weinig studies beschreven (▇▇▇▇, ▇▇▇▇▇ & ▇▇▇▇▇▇, 2015; ▇▇▇▇▇▇, 2008; ▇▇▇▇▇ & ▇▇▇▇, 2009). Met dit casusonderzoek ▇▇▇▇▇▇ we bijdragen aan theorievorming over ▇▇ ▇▇▇▇▇▇ waarop ambtenaren (stille of luidere) maatschappelijke ideolo‐ gieën over burgerschap in een organisatiecontext toepassen en betekenis geven. We laten zien hoe ambtenaren noties over zelfredzame burgers hanteren in een organisatiecontext waarin altijd sprake is van schaarste en dilemma’s. Daarbij zul‐ len we argumenteren dat typeringen van burgers nauw verbonden zijn met zowel ideologische opvattingen over burgerschap als met organisatiekenmerken. Dit artikel is als volgt opgebouwd. Eerst volgt een bespreking van het theoretisch kader. Daarna ▇▇▇▇▇▇▇ we de casus en de methode van onderzoek toe. Vervolgens presenteren we de belangrijkste bevindingen van het veldwerk, dat is uitgevoerd bij de Belastingdienst. Ten slotte reflecteren we op de resultaten en verbinden we er conclusies aan. Om te bestuderen wat er gebeurt als ambtenaren praten over zelfredzame burgers combineren we drie theoretische invalshoeken. Studies naar interactional framing laten zien hoe mensen via taal betekenis geven aan wat er gebeurt. Het concept van organisaties als zelfreferentiële systemen stelt ons in staat om vanuit een orga‐ nisatieperspectief naar deze framingprocessen te kijken. Omdat we ▇▇▇▇▇▇ weten hoe tijdens deze processen (ongearticuleerde) ▇▇▇▇▇▇ ▇▇▇▇▇▇ gevormd, aange‐ past en aangewend, maken we bovendien gebruik van de ▇▇▇▇▇ ▇▇▇ ▇▇▇▇▇▇ ideologie in organisaties. Het concept framing helpt ons ▇▇ ▇▇ ‘▇▇▇▇▇▇’ te begrijpen waarmee we de wereld om ons heen betekenis geven. Frames kunnen enerzijds ▇▇▇▇▇▇ opgevat als cog‐ nitieve of mentale schema’s, perspectieven op de werkelijkheid die als het ware zijn opgeslagen in het geheugen. Frames filteren de waarneming op zodanige wijze dat deze aansluiten bij bestaande manieren om verschijnselen of gebeurte‐ ▇▇▇▇▇▇ ▇▇ begrijpen (Aarts & Van Woerkum, 2006; ▇▇▇▇▇ & ▇▇▇▇▇▇▇, 2012). ▇▇▇▇▇▇ ▇▇▇ ▇▇▇▇▇, ▇▇▇▇▇▇ ▇▇▇ ▇▇▇▇▇▇ & ▇▇▇▇▇▇ ▇▇▇▇▇ Anderzijds wordt framing opgevat als doelgeoriënteerde betekenisproductie. Mensen hanteren frames als instrumenten waarmee ze, meer of minder doelbe‐ wust, anderen proberen te overtuigen van een specifieke interpretatie. Door informatie te selecteren, elementen uit ▇▇ ▇▇▇▇▇▇▇, voorop te plaatsen, uit te ver‐ groten of weg te laten belichten we onderwerpen op een ▇▇▇▇▇▇ die bevordert dat we medewerking genereren (▇▇▇▇▇▇▇, 1974; Aarts & ▇▇▇ ▇▇▇▇▇▇▇, 2006; ▇▇▇ ▇▇▇▇▇▇▇ & ▇▇▇▇▇, 2013; ▇▇▇▇▇▇▇ & ▇▇▇▇, 2000; ▇▇▇▇▇▇, 1993; ▇▇ ▇▇▇▇▇▇, ▇▇▇ ▇▇▇▇▇▇ & ▇▇▇▇▇▇▇, 2012). ARTICLE ABSTRACT Effecten van framing zijn divers. Krachtige frames kun‐ nen ervoor zorgen dat mensen informatie beter onthouden, dat standpunten moeilijk te weerleggen zijn, dat onderwerpen moeilijk of juist makkelijk bespreek‐ baar zijn, of dat waarden makkelijk ▇▇▇▇▇▇ geactiveerd (De Bruijn e.a., 2012). Het herhalen en circuleren van frames draagt bovendien bij aan het ontstaan van dominante discoursen op een macroniveau (▇▇▇▇ ▇.▇., 2015). Framing is ondertussen geen individuele of geïsoleerde bezigheid. Studies laten zien hoe mensen in voortdurende interactie met elkaar betekenissen vormen en veranderen (Aarts & Van Woerkum, 2006; ▇▇▇ ▇▇▇▇▇▇▇ & Aarts, 2013). Indivi‐ duen en groepen geven hun ervaringen betekenis in een proces van coproductie terwijl ze al ▇▇▇ niet in fysieke nabijheid met elkaar aan het praten, schrijven, onderhandelen en onderzoeken zijn. Hierbij ontstaan geïnstitutionaliseerde fra‐ mes die als het ware de ruimte comprimeren waarbinnen interactanten bepalen wat de situatie is en hoe te handelen. Een benadering waarbij interactionele framing wordt opgevat als betekenisconstructie vestigt aandacht op de dynamiek van processen waarbij ▇▇▇▇▇▇ ▇▇▇▇▇▇ geïnstitutionaliseerd. Mensen passen bestaande normen en machtsverhoudingen toe op nieuwe situaties op een ▇▇▇▇▇▇ die leidt tot bestendiging of aanpassing van bestaande frames (▇▇▇▇ ▇.▇., 2015; Poole, ▇▇▇▇▇▇▇ & ▇▇▇▇▇▇, 1985; ▇▇▇▇▇▇, 2008, 164; ▇▇▇▇▇▇▇, 1974). Ook een organisatie is geen geïsoleerde omgeving. Interactanten in organisaties maken gebruik van frames uit de bredere context van cultuur, samenleving en werkveld die dienen als basis voor gezamenlijke interpretaties en interactieregels. Deze kunnen tijdens interacties op microniveau ▇▇▇▇▇▇ bevestigd en gerituali‐ seerd, maar ook uitgedaagd, bevraagd of vervangen. Zodoende liggen macrofra‐ mes (zoals maatschappelijke en culturele normen) aan de basis van gedeelde interpretaties via de interacties van een organisatie, maar omgekeerd kunnen micro-interacties ook leiden tot aanpassing en omvorming van deze macroframes (▇▇▇▇ ▇.▇., 2015, 11-12; ▇▇▇▇▇▇▇, 2004, 20). Een framingperspectief biedt de mogelijkheid om betekenis niet als statische enti‐ teit te onderzoeken, maar als een proces van voortdurend aanpassen, omvormen, vernieuwen en leren, waarbij institutionalisering van betekenissen plaatsvindt in voortdurende top-down- en bottom-upbewegingen, waarbij ‘micro’ en ‘macro’ elkaar wederzijds constitueren (▇▇▇▇▇, ▇▇▇▇▇▇ & Gray, 2017, 18). Analyse van (talige) frames kan informatie opleveren die niet vanzelfsprekend naar voren komt bij methoden van onderzoek waarin de inhoudelijke zelfrapportages van respondenten centraal staan. Frameanalyses richten zich op wat mensen zeggen, maar ook op wat ze doen als ze iets zeggen. Ze leveren inzicht in (deels) onbewuste overwegingen, dilemma’s en strategieën die door gespreksdeelnemers niet expli‐ ciet onder woorden ▇▇▇▇▇▇ gebracht, maar die onderdeel uitmaken van repertoi‐ res om doelen te bereiken. Framingtheorie maakt dus duidelijk hoe mensen betekenis geven aan wat er gebeurt op een ▇▇▇▇▇▇ die de kans op het bereiken van doelen vergroot, of dat nu officiële organisatiedoelen zijn of meer persoonlijke en sociale doelen. Daartoe zullen verbindingen moeten ▇▇▇▇▇▇ gelegd met bestaande betekenissen van het organisatiesysteem. ▇▇▇▇ hierbij bijvoorbeeld aan beleids- en organisatiedoelen, maar ook aan alle praktijken, opvattingen, zienswijzen en gebruiken die de orga‐ nisatie kenmerken. We report a microfluidic droplet-based approach enabling the measurement of chemical reactions of individual enzyme molecules and its application to a single-molecule-counting immunoassaygaan er daarom van uit dat bepalende elementen van het organisatiesysteem tijdens processen van framing een belangrijke rol spelen. A microfluidic 6 device Deze aanname kunnen we verder onderbouwen met theorievorming over organisaties als ‘zelfreferentiële’ systemen. Volgens ▇▇▇▇▇▇▇ (1995, 24) is used to generate and manipulate <10 fL droplets at rates of up to 1.3 × 10 per secondbetekenisgeving voor een organisatiesysteem een ▇▇▇▇▇▇ ▇▇ via selectie met complexiteit om te gaan. Omdat de complexe werkelijkheid altijd veel meer mogelijkheden omvat ▇▇▇ waaraan een organisatie kan beantwoorden, about 2 orders of magnitude faster than has previously been reportedmaakt de organisatie deze com‐ plexiteit hanteerbaar en benaderbaar door te reduceren en te selecteren. The femtodroplets produced with this device can be used to encapsulate single biomolecular complexes tagged with a reporter enzymeVolgens ▇▇▇▇▇▇▇ kiezen organisaties uit hun omgeving die elementen die helpen om de interne werkelijkheid te definiëren en te reproduceren (▇▇▇▇▇▇▇, 1995; their small volume enables the fluorescent product of a single enzyme molecule to be detected within 10 min of on-chip incubationVan Her‐ zele & Aarts, 2013; Hernes & ▇▇▇▇▇▇, 2003). Our prototype system is validated by detection of a biomarker for prostate cancer in bufferOrganisaties zijn dus ‘zelfreferenti‐ eel’. Ze zijn geneigd en genoodzaakt om de omgeving terug te brengen naar de termen van het eigen systeem (▇▇▇▇▇▇▇, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte molecules.1990, 1995; Wagemans, 1998, 2002). Dat betekent niet dat organisaties geen oog hebben voor hun omgeving, wel dat vragen over wat relevant en betekenisvol is, ▇▇▇▇▇▇ ▇▇(her)formuleerd vanuit de

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Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication. You are permitted to download and use the publication for personal purposes. All rights remain with the author(s) and/or copyrights owner(s) of this work. Any use of the publication other than authorised under this licence or copyright law is prohibited. If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please contact the Library through email: ▇▇▇▇▇▇▇▇▇@▇▇▇.▇▇.▇▇, or send a letter to: University Library Radboud University Copyright Information Point PO Box 9100 6500 HA Nijmegen You will be contacted as soon as possible. Ultrarapid Generation of Femtoliter Microfluidic Droplets for Single- MoleculeMacromolecules 2010, 43, 10157–10161 10157 DOI: 10.1021/ma1024889 Convenient Route To Initiate Kumada Catalyst-Counting Immunoassays Jung-uk Shim,†,§,* Transfer Polycondensation Using Ni(dppe)Cl2 or Ni(dppp)Cl2 and Sterically Hindered Grignard Compounds ▇▇▇▇▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇▇▇▇,† *,†,§ ▇▇▇▇▇▇▇ ▇. ▇▇▇▇▇,† *,‡,§ ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇,† ▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇▇,‡ † ▇▇▇▇▇▇▇ ▇. ▇. ▇▇▇▇,†,^ ▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇,† ‡ and ▇▇▇▇▇ ▇▇▇▇▇*,† †Department of Chemistry†Leibniz-Institut fu€r Polymerforschung Dresden e.V., University of Cambridge, Lensfield Road, Cambridge, U.K., CB2 1EW and ‡Department of Biochemistry, University of Cambridge, Cambridge, U.K., CB2 1GA. §Present address: Biomedical Engineering, University of Glasgow, Glasgow, UK, G12 8LT. ^Present address: Radboud University Nijmegen, Institute for Molecules and ▇▇▇▇ ▇▇▇▇▇▇▇ ▇, ▇▇▇▇▇ ▇▇▇▇▇▇▇, ▇▇▇▇▇▇▇, and ‡Melville Laboratory for Polymer Synthesis, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K. §V.S. and M.S. contributed equally to the work Conjugated polymers (CPs) attract considerable attention as promising materials for solar cells, field effect transistors, light- emitting diodes, etc.1 However, the properties of existing CPs that are prepared predominantly by conventional step-growth poly- condensations are still far from optimal. For industrial-scale applications, CPs with controllable molecular weight (MW), MW distribution, chain-end functionality, minimum amounts of defects, and as a result, controlled and reproducible optoelec- tronic properties are required. In addition, new CP architectures are needed that predictably self-assemble into desirable nano- morphologies, thus solving a longstanding problem with improp- er morphologies of active layers in optoelectronic devices.1 Nowadays, chain-growth Kumada catalyst transfer polyconden- sations (KCTP), also referred to as Grignard metathesis poly- merization (GRIM),2 has become a powerful tool for the synthe- sis of well-defined polymers,3 all-conjugated block copolymers4 and polymer brushes.8-10 However, despite impressive progress, several important challenges still remain. Although a number of model thiophene-based conjugated block copolymers were already synthesized via the sequential polymerization of different monomers,4a-e examples of all-conjugated block copolymers com- posed of two substantially different blocks remain scarce.4f-i While some effort has been made in synthesizing various types of donor-acceptor block copolymers,5 all-conjugated block copo- lymers composed of state-of-the-art electron-donor and electron- acceptor conjugated blocks have not been synthesized to date. Such architectures are potentially promising materials for inter- face engineering in organic solar cells, especially when consider- ing that power conversion efficiencies of all-polymer blend photovoltaics are currently lagging behind polymer/fullerene derivative-based devices.6 Difficulties in preparing such block copolymers originate from the synthetic requirements that the two different blocks should be formed with the aid of the same catalyst, under approximately the same polymerization condi- tions and without a sacrifice in the chain-growth polymerization performance.7 One way to solve this problem is to optimize the polymerization conditions and catalysts that are suitable to grow both blocks.4f-i An alternative strategy implies that two blocks are polymerized in two steps under conditions optimal for polymerization of each block.8 Here, the most challenging step is to selectively prepare functional Ni-initiators in high yield (frequently called as externally added initiators) that are properly attached to molecules or objects, from which polymerization of + *Corresponding author. Telephone: ▇▇-▇▇▇-▇▇▇▇▇▇▇ -▇▇▇, ▇▇▇▇ ▇▇ ▇▇▇▇▇▇▇▇, ▇▇▇ E-mail: (V.S.) ▇▇▇▇▇▇▇▇▇▇@▇. ARTICLE ABSTRACT We report a microfluidic droplet-based approach enabling the measurement of chemical reactions of individual enzyme molecules and its application to a single-molecule-counting immunoassay. A microfluidic 6 device is used to generate and manipulate <10 fL droplets at rates of up to 1.3 × 10 per second, about 2 orders of magnitude faster than has previously been reported. The femtodroplets produced with this device can be used to encapsulate single biomolecular complexes tagged with a reporter enzyme▇▇▇▇.▇▇; their small volume enables the fluorescent product of a single enzyme molecule to be detected within 10 min of on-chip incubation. Our prototype system is validated by detection of a biomarker for prostate cancer in buffer, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte molecules(A.K.) ▇▇▇▇▇@▇▇▇▇▇.▇▇; (M.S.) ▇▇▇▇▇@▇▇▇.▇▇.▇▇.

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Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication. You are permitted to download and use the publication for personal purposes. All rights remain with the author(s) and/or copyrights owner(s) of this work. Any use of the publication other than authorised under this licence or copyright law is prohibited. If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please contact the Library through email: ▇▇▇▇▇▇▇▇▇@▇▇▇.▇▇.▇▇, or send a letter to: University Library Radboud University Copyright Information Point PO Box 9100 6500 HA Nijmegen You will be contacted as soon as possible. Ultrarapid Generation View Article Online / Journal Homepage / Table of Femtoliter Microfluidic Droplets Contents for Single- Molecule-Counting Immunoassays Jung-uk Shim,†,§this issue PAPER ▇▇▇.▇▇▇.▇▇▇/▇▇▇▇▇▇▇▇▇ | Journal of Materials Chemistry Synthesis and characterization of low bandgap conjugated donor–acceptor polymers for polymer:PCBM solar cells† x ▇▇▇▇▇ ▇▇,* ‡ a ▇▇▇▇▇▇▇ ▇▇▇▇▇▇,‡b ▇▇▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇▇▇▇,† ‡b Ximin He,a ▇▇▇▇▇ ▇. ▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇,† ▇▇▇b ▇▇▇▇ ▇. ▇▇▇▇▇▇▇▇▇▇,‡ ▇ and ▇▇▇▇▇▇▇ ▇. ▇. ▇▇▇▇,†,^ ▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇,† and ▇▇▇▇▇ ▇▇▇▇▇† †Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, U.K., CB2 1EW and ‡Department of Biochemistry, University of Cambridge, Cambridge, U.K., CB2 1GA*a Published on 20 September 2010. §Present address: Biomedical Engineering, University of Glasgow, Glasgow, UK, G12 8LT. ^Present address: Downloaded by Radboud University Nijmegen, Institute for Molecules and ▇▇▇▇▇▇▇▇▇, ▇▇▇▇▇▇▇▇▇▇▇▇▇▇ ▇▇▇, ▇▇▇▇ ▇▇ ▇▇▇▇▇▇▇▇, ▇▇▇ ▇▇▇▇▇▇▇▇▇▇▇Nijmegen on 9/8/2022 1:31:43 PM. ARTICLE ABSTRACT DOI: 10.1039/c0jm01641a We report on the synthesis, characterization and photovoltaic performance of three novel semiconducting polymers based on poly[bis-N,N0-(4-octylphenyl)-bis-N,N0-phenyl-1,4- phenylenediamine-alt-5,50-40,70,-di-2-thienyl-20,10,30-benzothiadiazole]. They differ only in the presence and position of hexyl side-chains on the thienyl groups. T8TBT-0 has no such side-chains, they face towards the benzothiadiazole in T8TBT-in and away in T8TBT-out. Based on electron-donating triarylamine and electron-accepting dithienyl-benzothiadiazole groups, the new polymers exhibit low bandgaps and enhanced absorption in the red part of the visible spectrum. Despite their identical backbone they differ in their synthesis and photophysics: T8TBT-0 and T8TBT-in can be synthesized by direct Suzuki coupling but a microfluidic dropletnew synthesis procedure is necessary for T8TBT-based approach enabling out. In absorption and luminescence a blue shift is induced by the measurement of chemical reactions of individual enzyme molecules inward facing, and its application to a singlelesser extent by the outward-moleculefacing side-counting immunoassaychains. A microfluidic 6 From comparison of the photophysics in solutions and films, we conclude that the addition of side-chains reduces formation of aggregates in films and that this effect is stronger for inward-facing side-chains. By blending the three polymers with PCBM in a standard photovoltaic device is used to generate and manipulate <10 fL droplets at rates of up to 1.3 × 10 per secondarchitecture, about 2 orders of magnitude faster than has previously been reported. The femtodroplets produced with this device can be used to encapsulate single biomolecular complexes tagged T8TBT-0 performs best with a reporter enzyme; their small volume enables the fluorescent product power conversion efficiency (PCE) of a single enzyme molecule 1.0% (under AM1.5G illumination at 100 mW cm—2) compared to be detected within 10 min of on0.17% and 0.27% for T8TBT-chip incubation. Our prototype system is validated by detection of a biomarker for prostate cancer in bufferout and T8TBT-in, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte moleculesrespectively.

End User Agreement. This publication is distributed under the terms of Article 25fa of the Dutch Copyright Act (Auteurswet) with explicit consent by the author. Dutch law entitles the maker of a short scientific work funded either wholly or partially by Dutch public funds to make that work publicly available for no consideration following a reasonable period of time after the work was first published, provided that clear reference is made to the source of the first publication of the work. This publication is distributed under The Association of Universities in the Netherlands (VSNU) ‘Article 25fa implementation’ pilot project. In this pilot research outputs of researchers employed by Dutch Universities that comply with the legal requirements of Article 25fa of the Dutch Copyright Act are distributed online and free of cost or other barriers in institutional repositories. Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication. You are permitted to download and use the publication for personal purposes. All rights remain with the author(s) and/or copyrights owner(s) of this work. Any use of the publication other than authorised under this licence or copyright law is prohibited. If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please contact the Library through email: ▇▇▇▇▇▇▇▇▇@▇▇▇.▇▇.▇▇, or send a letter to: University Library Radboud University Copyright Information Point PO Box 9100 6500 HA Nijmegen You will be contacted as soon as possible. Ultrarapid Generation of Femtoliter Microfluidic Droplets for Single- Molecule-Counting Immunoassays Jung-uk Shim,†,§,* ▇▇▇▇▇▇▇ ▇. ▇ ▇▇▇▇, MD ▇▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇,† ▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇-▇▇,‡ ▇▇▇▇, MD, PhD ▇▇▇▇▇▇ ▇. ▇. ▇▇▇▇,†,^ ▇▇, MD, PhD ▇▇▇ ▇. ▇▇ ▇▇▇▇, MD, PhD ▇▇▇▇ ▇▇▇ ▇▇▇▇▇▇▇▇, PhD ▇▇▇ ▇. ▇▇▇ ▇▇▇▇▇▇▇▇, MD, PhD ▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇,† and ▇▇▇▇▇ ▇▇▇▇▇† †Department , MD, PhD Screening for Lung Cancer with Digital Chest Radiography: ORIGINAL RESEARCH THORACIC IMAGING ■ Purpose: To estimate the performance of Chemistry, University of Cambridge, Lensfield Road, Cambridge, U.K., CB2 1EW and ‡Department of Biochemistry, University of Cambridge, Cambridge, U.K., CB2 1GA. §Present address: Biomedical Engineering, University of Glasgow, Glasgow, UK, G12 8LT. ^Present address: Radboud University Nijmegen, Institute digital chest radiography for Molecules and ▇▇▇▇▇▇▇▇▇, ▇▇▇▇▇▇▇▇▇▇▇▇▇▇ ▇▇▇, ▇▇▇▇ ▇▇ ▇▇▇▇▇▇▇▇, ▇▇▇ ▇▇▇▇▇▇▇▇▇▇▇. ARTICLE ABSTRACT We report a microfluidic droplet-based approach enabling the measurement of chemical reactions of individual enzyme molecules and its application to a single-molecule-counting immunoassay. A microfluidic 6 device is used to generate and manipulate <10 fL droplets at rates of up to 1.3 × 10 per second, about 2 orders of magnitude faster than has previously been reported. The femtodroplets produced with this device can be used to encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables the fluorescent product of a single enzyme molecule to be detected within 10 min of on-chip incubation. Our prototype system is validated by detection of a biomarker for prostate cancer in buffer, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte moleculeslung cancer.

End User Agreement. This publication is distributed under the terms of Article 25fa of the Dutch Copyright Act (Auteurswet) with explicit consent by the author. Dutch law entitles the maker of a short scientific work funded either wholly or partially by Dutch public funds to make that work publicly available for no consideration following a reasonable period of time after the work was first published, provided that clear reference is made to the source of the first publication of the work. This publication is distributed under The Association of Universities in the Netherlands (VSNU) ‘Article 25fa implementation’ pilot project. In this pilot research outputs of researchers employed by Dutch Universities that comply with the legal requirements of Article 25fa of the Dutch Copyright Act are distributed online and free of cost or other barriers in institutional repositories. Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication. You are permitted to download and use the publication for personal purposes. All rights remain with the author(s) and/or copyrights owner(s) of this work. Any use of the publication other than authorised under this licence or copyright law is prohibited. If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please contact the Library through email: ▇▇▇▇▇▇▇▇▇@▇▇▇.▇▇.▇▇, or send a letter to: University Library Radboud University Copyright Information Point PO Box 9100 6500 HA Nijmegen You will be contacted as soon as possible. Ultrarapid Generation JOURNAL OF MAGNETIC RESONANCE IMAGING 36:1104–1112 (2012) Original Research Computer Aided Analysis of Femtoliter Microfluidic Droplets for Single- Molecule-Counting Immunoassays Jung-uk Shim,†,§,* ▇Breast MRI Enhancement Kinetics Using Mean Shift C Lustering and Multifeature Iterative Region of Interest Selection ▇▇▇▇ ▇. ▇▇▇▇▇▇▇▇▇▇,† ▇▇, MD, PhD,1* ▇▇▇▇▇▇▇ ▇. ▇▇▇, MSc,2 ▇▇▇▇▇ ▇▇▇▇▇,† ▇, PhD,2,3 ▇▇▇▇ ▇▇▇▇▇▇▇▇▇▇▇▇, PhD,2 ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇,† ▇, MD, PhD,2 and ▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇▇,‡ ▇▇▇▇▇▇▇ ▇, PhD2 1Ikazia Hospital Radiology, Rotterdam, The Netherlands. ▇2Radboud University Medical Centre, Nijmegen, The Netherlands. ▇▇▇▇,†,^ 3Maastricht University Medical Center, Maastricht, The Netherlands. ▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇,† and ▇▇▇▇▇ ▇▇▇▇▇† †Department , MD, PhD, recently passed away. *Address reprint requests to: M.J.S., Ikazia Hospital, department of ChemistryRadiology, University of Cambridge, Lensfield Road, Cambridge, U.K., CB2 1EW and ‡Department of Biochemistry, University of Cambridge, Cambridge, U.K., CB2 1GA. §Present address: Biomedical Engineering, University of Glasgow, Glasgow, UK, G12 8LT. ^Present address: Radboud University Nijmegen, Institute for Molecules and ▇▇▇▇▇▇▇▇▇, ▇▇▇▇▇▇▇▇▇▇▇▇▇▇ ▇▇▇, ▇▇▇▇ ▇▇ ▇▇▇▇▇▇▇▇, ▇▇▇ ▇▇▇▇▇▇▇▇▇▇▇. ARTICLE ABSTRACT We report a microfluidic dropletE-based approach enabling the measurement mail: ▇. ▇▇▇▇▇▇▇▇▇▇▇▇@▇▇▇▇▇▇.▇▇ Received June 16, 2011; Accepted June 1, 2012. DOI 10.1002/jmri.23746 View this article online at ▇▇▇▇▇▇▇▇▇▇▇▇▇▇▇▇▇▇.▇▇▇. DYNAMIC CONTRAST ENHANCED magnetic reso- ▇▇▇▇▇ imaging has become an integral part of chemical reactions breast imaging. There are several well-established indica- tions for its use (1): exclusion of individual enzyme molecules and its application to a single-molecule-counting immunoassay. A microfluidic 6 device malignancy when conventional imaging is used to generate and manipulate <10 fL droplets at rates of up to 1.3 × 10 per secondinconclusive, about 2 orders of magnitude faster than has previously been reported. The femtodroplets produced with this device can be used to encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables the fluorescent product of a single enzyme molecule to be detected within 10 min of on-chip incubation. Our prototype system is validated by preoperative staging, detection of a biomarker possible primary breast tumor in cases with metastatic disease of unknown origin, evaluation of response to neoadjuvant therapy, follow- up after breast conserving therapy, screening for prostate cancer women at increased risk of breast cancer, and prosthe- sis imaging. Nearly all breast MRI examinations require differentiation between benign and malignant origin of enhancing tissue. For the evaluation of a detected lesion, both morphology and dynamic enhancement characteristics are valuable (2–6). The ACR BI-RADSTM lexicon (7) offers a useful guideline in bufferthe assessment of an enhancing lesion on breast MRI. Guidelines like this are somewhat limited by their dependence on proper description of a lesion by the human reader; we previously showed that there is considerable variability in this description (8), down a finding that extended to both morphological and kinetic features. Kinetic features must be evaluated in the most suspicious area of a concentration tu- mor (9–11), but our study showed that choosing the location and size of 46 fM. This work demonstrates this region of interest (ROI) was a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation major source of monodisperse femtoliter droplets for the counting variability. Purpose: To evaluate automatic characterization of individual analyte moleculesa breast MR ▇▇▇▇▇▇ by its spatially coherent region of interest (ROI).

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Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication. You are permitted to download and use the publication for personal purposes. All rights remain with the author(s) and/or copyrights owner(s) of this work. Any use of the publication other than authorised under this licence or copyright law is prohibited. If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please contact the Library through email: ▇▇▇▇▇▇▇▇▇@▇▇▇.▇▇.▇▇, or send a letter to: University Library Radboud University Copyright Information Point PO Box 9100 6500 HA Nijmegen You will be contacted as soon as possible. Ultrarapid Generation of Femtoliter Microfluidic Droplets for Single- Molecule-Counting Immunoassays Jung-uk Shim,†,§,* ▇▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇ , ▇▇▇▇▇ ▇▇▇▇▇▇▇ , ▇▇▇▇▇▇▇▇▇▇ ▇▇▇▇▇▇ , ▇▇▇▇▇ ▇▇▇▇▇▇▇▇ , and ▇▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇▇ Toxicologic Pathology 2021, Vol. 49(4) 714-719 ª The Author(s) 2021 Article reuse guidelines: ▇▇▇,† ▇▇▇▇.▇▇▇/▇▇▇▇▇▇▇▇-▇▇▇▇▇▇▇▇▇▇▇ ▇. DOI: 10.1177/0192623321990375 ▇▇▇▇▇,† ▇▇▇.▇▇▇▇▇▇▇.▇▇▇/▇▇▇▇/▇▇▇ The 2019 manuscript by the Special Interest Group on Digital Pathology and Image Analysis of the Society of Toxicologic pathology suggested that a synergism between artificial intelligence (AI) and machine learning (ML) technologies and digital toxicologic pathology would improve the daily workflow and future impact of toxicologic pathologists globally. Now 2 years later, the authors of this review consider whether, in their opinion, there is any evidence that supports that thesis. Specifically, we consider the opportunities and challenges for applying ML (the study of computer algorithms that are able to learn from example data and extrapolate the learned information to unseen data) algorithms in toxicologic pathology and how regulatory bodies are navigating this rapidly evolving field. Although we see similarities with the “Last Mile” metaphor, the weight of evidence suggests that tox- icologic pathologists should approach ML with an equal dose of skepticism and enthusiasm. There are increasing opportunities for impact in our field that leave the authors cautiously excited and optimistic. Toxicologic pathologists have the opportunity to critically evaluate ML applications with a “call-to-arms” mentality. Why should we be late adopters? There is ample evidence to encourage engagement, growth, and leadership in this field. artificial intelligence, machine learning, deep learning, neural networks, digital toxicologic pathology The “Last Mile” metaphor was first used by the early land-based telecommunications industry1 to describe the dif- ficulty of connecting end-user homes and businesses to the main telecommunication network. One of the main barriers was the cost of installing and maintaining this infrastructure, because it could only be amortized over 1 subscriber, com- pared to many customers in the main trunks of the network. Other challenges of “Last Mile” delivery included ensuring transparency with the customer; following guidelines from local, state, and federal regulatory agencies; increasing effi- ciency of the workflow; and improving infrastructure support- thoughts on early qualification and validation efforts and the challenges therein. In our opinion, and as pointed to in the 2019 Special Interest Group manuscript,3 a continued rapid growth in each of the 3 key ingredients of ML: (1) massive computer power, (2) big data and (3) inherent knowledge, has fueled accelerated use of artificial intelligence (AI) and ML in almost all areas of sci- ence. More and larger partnerships between AI scientists and medical specialties are being established, and the inference (causal and counterfactual) and probability (prediction) output ing the network. Interestingly, these 4 challenges overlap with common ones we currently face as toxicologic pathologists when confronted with the notion of applying machine learn- ing (ML)2 in the digital histopathology space. In this review, we highlight both published medical pathology examples and our personal experiences to date with ML applications in toxicologic pathology, which indicate that we are starting to overcome some of the “Last Mile” challenges. As most of us work in a highly regulated environment and are at least cau- tious about implementing new approaches or technology because of the perceived burden of qualification or Good Laboratory Practice (GLP) validation, we also share personal 1 Novartis, Novartis Institutes for BioMedical Research, Preclinical Safety, East Hanover, NJ, USA 2 Boehringer Ingelheim Pharmaceuticals Incorporated, Nonclinical Drug Safety, Ridgefield, CT, USA 3 ▇▇▇▇▇▇▇ River Laboratories, Pathology, Frederick, MD, USA 4 Diagnostic Image Analysis Group Radboud University Medical Center Nijmegen, the Netherlands 5 ▇▇▇▇▇▇▇ River Laboratories, Pathology, Ashland, OH, USA ▇▇▇▇▇▇ ▇. ▇▇▇▇▇▇, Novartis, Novartis Institutes for BioMedical Research, Preclinical Safety-Pathology, ▇,† ▇▇ ▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇▇,‡ ▇▇▇▇▇▇▇ ▇. ▇. ▇▇▇▇,†,^ ▇, ▇▇▇▇ ▇▇▇▇▇▇▇▇▇,† and ▇▇▇, ▇▇ ▇▇▇▇▇† †Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, U.K., CB2 1EW and ‡Department of Biochemistry, University of Cambridge, Cambridge, U.K., CB2 1GA. §Present address: Biomedical Engineering, University of Glasgow, Glasgow, UK, G12 8LT. ^Present address: Radboud University Nijmegen, Institute for Molecules and ▇▇▇▇▇▇▇▇▇, ▇▇▇. Email: ▇▇▇▇▇▇.▇▇▇▇▇▇@▇▇▇▇▇▇▇▇.▇▇▇ ▇▇▇of machines are married with the high-level decision-making and reasoning of humans at an increasing frequency. Yet we need to be cautious of the hype surrounding all things AI. In its June 2020 edition, ▇▇▇▇ the Technology Quarterly of the journal The Economist4 states that we should be taking a “Reality Check” and consider that there are very real limita- tions to AI, naive and fallible. Is AI harder to implement than expected? Is the AI promise still greater than the science? Are the costs and IT resources prohibitive to many? We have expe- rienced the so-called “▇▇ ▇▇▇▇▇▇▇▇” (a period of reduced fund- ing and interest in AI research), ▇▇▇ ▇▇▇▇▇▇▇▇▇▇▇The Economist asks is an “AI Autumn” now coming? We also acknowledge, as did the authors of the Special Interest Group publication in 2019, that strong/general AI aiming at mimicking human capabilities remains a distant but very important philosophical idea, and that in 2021, we are still creating artificial “idiot savants”— narrow AI applications that can excel at well-bounded tasks, but make serious mistakes if faced with unexpected input. ARTICLE ABSTRACT We report a microfluidic dropletComputers do not exhibit true intelligence since they are incap- able of thought; however, they can “learn” from data and improve their performance through training on relevant exam- ples provided by experts, such as toxicologic pathologists. Based on the authors’ experiences of developing and apply- ing ML solutions to digital histopathology data, as was the case for ML-based approach enabling image analysis and stereology solutions, we feel that general adoption of ML is achievable and the measurement potential return on investment favorable. However, ML is not a panacea and, like all scientific methods, should be applied when its use adds clear scientific or operational value. We currently see several application areas and opportunities for ML growth in digital toxicologic pathology: (1) abnormality detection, (2) decision support, (3) tissue lesion screening, (4) diagnostic scoring simplification, (5) counting automation, and (6) object quantification. Examples of chemical reactions of individual enzyme molecules these are shown in Figure 1 and its application to a single-molecule-counting immunoassay. A microfluidic 6 device is used to generate and manipulate <10 fL droplets at rates of up to 1.3 × 10 per second, about 2 orders of magnitude faster than has previously been reported. The femtodroplets produced with this device can be used to encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables described in the fluorescent product of a single enzyme molecule to be detected within 10 min of on-chip incubation. Our prototype system is validated by detection of a biomarker for prostate cancer in buffer, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte moleculesfollowing paragraphs.

End User Agreement. This publication is distributed under the terms of Article 25fa of the Dutch Copyright Act (Auteurswet) with explicit consent by the author. Dutch law entitles the maker of a short scientific work funded either wholly or partially by Dutch public funds to make that work publicly available for no consideration following a reasonable period of time after the work was first published, provided that clear reference is made to the source of the first publication of the work. This publication is distributed under The Association of Universities in the Netherlands (VSNU) ‘Article 25fa implementation’ pilot project. In this pilot research outputs of researchers employed by Dutch Universities that comply with the legal requirements of Article 25fa of the Dutch Copyright Act are distributed online and free of cost or other barriers in institutional repositories. Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication. You are permitted to download and use the publication for personal purposes. All rights remain with the author(s) and/or copyrights owner(s) of this work. Any use of the publication other than authorised under this licence or copyright law is prohibited. If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please contact the Library through email: ▇▇▇▇▇▇▇▇▇@▇▇▇.▇▇.▇▇, or send a letter to: University Library Radboud University Copyright Information Point PO Box 9100 6500 HA Nijmegen You will be contacted as soon as possible. Ultrarapid Generation Impaired epithelial differentiation of Femtoliter Microfluidic Droplets for Single- Moleculeinduced pluripotent stem cells from ectodermal dysplasia-Counting Immunoassays Jungrelated patients is rescued by the small compound APR-246/PRIMA-1MET ▇▇▇▇ ▇▇▇▇▇▇-uk Shim,†,§▇▇▇▇▇▇▇▇▇▇▇,b,1, ▇▇▇▇▇ ▇▇▇▇▇▇▇,* 1, ▇▇▇▇▇ ▇▇▇▇▇▇▇▇,b, ▇▇▇▇▇-▇▇▇▇▇ ▇▇▇▇▇▇▇, ▇▇▇▇ ▇▇▇ ▇▇▇▇▇▇▇▇▇, ▇▇▇▇ ▇. ▇▇▇▇▇▇, ▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇,† ▇, ▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇,b,1,2, and ▇▇,‡ ▇▇▇▇▇▇▇ ▇. ▇. ▇▇▇▇,†,^ b,1 aInstitut National de la Santé et de la Recherche Médicale U898, University of Nice, 06107 Nice, France; bStem Cell Research Center, ▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇ Faculty of Medicine, INSERTECH, Technion, Haifa 31096, Israel; cZentrum für Integrative Psychiatrie, 24105 Kiel, Germany; dDepartment of Human Genetics, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centrum, 6500 HB, Nijmegen, The Netherlands; and eDepartment of Oncology- Pathology, Karolinska Institutet, Cancer Center Karolinska, SE-171 76 Stockholm, Sweden Edited by ▇,† ▇▇ ▇. ▇▇▇, The ▇▇▇▇▇▇▇▇ Family Institute for Breast Cancer Research, Ontario Cancer Institute at Princess ▇▇▇▇▇▇▇▇ Hospital, University Health Network, Toronto, ON, Canada, and approved May 8, 2012 (received for review February 7, 2012) Ectodermal dysplasia is a group of congenital syndromes affecting a variety of ectodermal derivatives. Among them, ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome is caused by single point mutations in the p63 gene, which controls epidermal development and homeostasis. Phenotypic defects of the EEC syn- drome include skin defects and limbal stem-cell deficiency. In this study, we designed a unique cellular model that recapitulated major embryonic defects related to EEC. Fibroblasts from healthy donors and EEC patients carrying two different point mutations in the DNA binding domain of p63 were reprogrammed into induced pluripo- tent stem cell (iPSC) lines. EEC-iPSC from both patients showed early ectodermal commitment into K18+ cells but failed to further differ- entiate into K14+ cells (epidermis/limbus) or K3/K12+ cells (corneal epithelium). APR-246 (PRIMA-1MET), a small compound that restores functionality of mutant p53 in human tumor cells, could revert cor- ▇▇▇▇ epithelial lineage commitment and reinstate a normal p63-re- lated signaling pathway. This study illustrates the relevance of iPSC for p63 related disorders and paves the way for future therapy of EEC. TRP63 | rare disease | cornea E ctodermal dysplasia are rare syndromes characterized by ab- normal development of the skin and ectodermal derivatives, like teeth, hair, cornea, and nails. Among these syndromes, some are related to mutations on the transcription factor p63 (TP63) and represent a group of autosomal dominant ectodermal dys- plasia associated with orofacial clefting and limb abnormalities. The severe phenotype of p63-null mice highlighted the major role of p63 in embryonic development, and particularly in the devel- opment of ectodermal lineages (1, 2). Five syndromes in which p63 mutations have been detected include ectrodactyly, ectodermal dysplasia, and cleft lip/palate syndrome (EEC), ankyblepharon, ectodermal dysplasia, and cleft lip/palate syndrome (AEC), limb mammary syndrome, acro-dermato-ungual-lacrimal-tooth syn- drome, and ▇▇▇▇-▇▇▇▇▇▇▇ syndrome. EEC mutations are clus- tered in the DNA-binding domain and AEC mutations are found in the sterile α-motif or transactivation inhibitory domain (3). Although there are some clinical similarities and overlap between the syndromes, the specific location of p63 mutations in the dif- ferent domains of the gene shows a strong genotype-phenotype correlation, and thus different molecular mechanisms behind the various p63-associated syndromes. Clinical and penetrance vari- ability are observed for the same mutation, suggesting that the number of p63 patients could be underestimated (3, 4). More- over, variability of phenotype among syndromes could be a result of specific functional consequences of a single mutation. For ex- ample, two hot-spot mutants, R304W and R204W, both located in the DNA binding domain of the p63 gene, represent EEC syndrome but, based on their analogy with p53 mutations, may act differently because R304W interferes with DNA binding and R204W with global protein structure/stability of p63, influencing the transcription of target genes differently (5, 6). In addition to skin defects, EEC patients suffer from visual morbidity with progressive limbal stem-cell deficiency that leads to severe visual impairments and blindness (7, 8). Therefore, modeling of these diseases is essential to identify abnormalities in molecular processes involving p63, their effects on cell growth and skin development, and for drug screening. In vitro cellular models of rare skin and corneal diseases are obtained by the use of patient-derived primary epidermal cells. Given that p63 is a master regulator of embryonic steps of epithelial development, cellular models that could recapitulate the main steps of skin and corneal epithelial development in vitro are necessary. The re- cently discovered capacity of human somatic cells to be relatively easily reprogrammed into embryonic stem cell-like pluripotent stem cells (iPSC) offers numerous perspectives in therapies by providing patient-specific differentiated cells on demand and novel cellular models for specific pathologies. iPSC technology provide pluripotent stem cells carrying genetic characteristics of patients. These cells have the remarkable ability to recapitulate in vitro the main steps of human embryonic development, and they provide urgently needed tools to generate patient-specific, organotypic disease models. These cellular models may be used for the discovery of novel drugs both in a flexible and highly specific manner, because they facilitate high-throughput com- pound screening and toxicity assays. Finally, unlike patient pri- ▇▇▇▇ cells, iPSC derived from patients’ cells provide researchers with cells with unlimited proliferation capacity. The clinical penetrance of the p63 gene is highly variable, apparently be- cause of other genetic and epigenetic factors (9, 10). Therefore, animal models in which a single EEC mutation is inserted by knock-in may not reproduce the human pathology. Here, we de- rived iPSC lines from healthy control and EEC patients and evaluated their ability to differentiate into epidermal and corneal epithelial cells. Our study demonstrated that they displayed im- paired epithelial commitment that could be partially rescued by a small therapeutic compound. Author contributions: R.S.-F., D.A., and I.P. designed research; R.S.-F., L.S., E.A., ▇.-▇.▇., and I.P. performed research; H.v.B., ▇.▇.▇., and ▇.▇. contributed new reagents/analytic tools; R.S.-F., L.S., F.-▇.▇., D.A., and I.P. analyzed data; and R.S.-F., D.A., and I.P. wrote the paper. Conflict of interest statement: K.G.W. is cofounder, shareholder, and member of the board of ▇▇▇▇▇ ▇▇, a company that develops p53-based cancer therapy including the compound APR-246. This article is a PNAS Direct Submission. 1R.S.-F., L.S., D.A., and I.P. contributed equally to this work. 2To whom correspondence should be addressed. E-mail: ▇▇▇† †Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, U.K., CB2 1EW and ‡Department of Biochemistry, University of Cambridge, Cambridge, U.K., CB2 1GA. §Present address: Biomedical Engineering, University of Glasgow, Glasgow, UK, G12 8LT. ^Present address: Radboud University Nijmegen, Institute for Molecules and ▇▇▇.▇▇▇▇▇▇▇▇▇, @▇▇▇▇▇▇.▇▇. This article contains supporting information online at ▇▇▇.▇▇▇▇.▇▇▇/▇▇▇▇▇▇/▇▇▇▇▇/▇▇▇:▇▇. 1073/pnas.1201753109/-/DCSupplemental. Downloaded from ▇▇▇▇▇://▇▇▇.▇▇▇▇.▇▇▇ by RADBOUD UNIVERSITEIT NIJMEGEN on September 21, 2022 from IP address 195.169.218.33. 2152–2156 | PNAS | February 5, 2013 | vol. 110 | no. 6 ▇▇▇.▇▇▇▇.▇▇▇/▇▇▇/▇▇▇/▇▇.▇▇▇▇/▇▇▇▇.▇▇▇▇▇▇▇▇▇▇ ▇▇▇Results CELL BIOLOGY Derivation of iPSC Lines from WT and EEC Fibroblasts. iPSC lines were obtained by lentiviral infection of primary dermal fibro- blasts isolated from one healthy individual and two EEC patients carrying single point mutations R304W or R204W in the p63 gene. These two mutations located in the DNA binding domain are among the five hotspots accounting for 90% of EEC. Several clones with typical iPSC morphology (Fig. S1A) were expanded and two to three lines for each donor were used for subsequent characterization and experiments (WT or +/+, R204W/+ and R304W/+). Pluripotency of iPSC was confirmed by the expres- sion of various markers such as octamer-binding transcription factor 4 (OCT4), TRA-1-80, and alkaline phosphatase by stain- ing (Fig. S1A) and OCT4, sex-determining region Y box-2 (SOX2), DNA methyltransferase 3b (DNMT3b), and NANOG by quantitative PCR (qRT-PCR) (Fig. S1B). We have previously developed a purely data-driven approach, termed PluriTest, to- ▇▇▇▇ ▇▇ ▇▇▇▇▇▇▇▇empirically defining the human pluripotent state, ▇▇▇ ▇▇▇▇▇▇▇▇▇▇▇because the gold standard germ-line transmission is impossible for human cells (11). ARTICLE ABSTRACT We report a microfluidic dropletBriefly, the PluriTest data model was derived via in- terrogation of large-based approach enabling the measurement scale datasets of chemical reactions of individual enzyme molecules genome-wide somatic and its application to a single-molecule-counting immunoassay. A microfluidic 6 device is used to generate pluripotent expression profiles and manipulate <10 fL droplets at rates of up to 1.3 × 10 per second, about 2 orders of magnitude faster than has previously been reported. The femtodroplets produced with this device can be used to encapsulate single biomolecular complexes tagged rapidly and confidently assess the pluripotency of human cells through bio- informatic analysis of microarray data from new stem-cell prep- arations without the sacrifice of laboratory animals for Teratoma assays (11). All iPSC lines displayed high pluripotency scores, similarly to human embryonic stem cells (hESC) and other fi- broblast-derived iPSC (Fig. S1C). Finally, iPSCWT and iPSCEEC lines were able to form embryoid bodies in suspension and differ- entiate into cell types belonging to the three germ layers upon ad- hesion (Fig. S1D). This finding was confirmed by qRT-PCR analysis on gene expression specific for neuroectodermal (NCAM1), en- dodermal (AFP), and mesodermal (CD31) fates. In addition, EEC- iPSC were able to differentiate into trophoectoderm, as shown by CDX2 expression after 6 d with bone morphogenetic protein-4 (BMP-4) (Fig. S1E). Taken together, these data confirmed that we obtained pluripotent iPSC lines and we next explored their potential to model molecular characteristics of EEC syndrome. Impaired Epidermal Differentiation of EEC-iPSC Lines. EEC patients suffer from impaired skin development. To define whether EEC- iPSC lines could mimic these defects, we optimized protocols to differentiate human iPSC into epidermal cells. Epidermal com- mitment of embryonic stem cells can be induced by BMP-4, which inhibits neural differentiation and promotes epidermal fate of neuroectodermal cells (12–14). Prolonged treatment with BMP-4 and ascorbic acid combined with keratinocyte growth conditions have been shown to promote efficiently hESC differentiation into mature keratinocytes (14). However, applying this protocol to iPSC ture keratinocytes (Fig. S2Ci), as demonstrated by expression of K14 and K10 (Fig. S2C ii and iii), signs of spontaneous stratification (Fig. S2C iv and v), and ability to form typical keratinocyte colonies upon splitting (Fig. S2Cvi). This process demontrates that TGF-β inhibition significantly improved the production of epidermal cells from iPSC lines. iPSCEEC were subjected to epidermal commitment according to the above protocol. In response to SB431542 and BMP-4 during the first 10 d, iPSCEEC initiated ectodermal commitment, as illustrated by typical ectodermal cell morphology similarly to iPSCWT cells (Fig. S2A). However, although iPSCWT cells un- derwent epidermal transition from days 13–15 for the production of keratinocyte-like areas proliferating during the next 10 d, iPSCEEC continued to display the same ectodermal morphology (Fig. S3A). Only a reporter enzyme; their small volume enables number of keratinocyte-like cells could be noticed occasionally. Immunofluorescence staining (Fig. 1A) and quantification by flow cytometry (Fig. S3B) confirmed that both iPSCR304W/+ and iPSCR204W/+ could produce K18+ ecto- dermal progenitors to a similar extent as iPSCWT (42.5% and 46%, respectively). However, iPSCR304W/+ failed to further dif- ferentiate into K14+ epidermal cells (2.2%), compared with iPSCWT (27.5%) (Fig. 1 and Fig. S2B). Similar results have been obtained for iPSCR204W/+ (Fig. 1B). Of note, expression of p63 was up-regulated to the fluorescent product same extent during epidermal commit- ment of both WT and mutated iPSC (Fig. S3C). Impaired Corneal Differentiation of EEC-iPSC Is Rescued by APR-246/ PRIMA-1MET. EEC patients suffer from visual morbidity because of impaired cornea associated with limbal stem-cell deficiency (7). iPSC lines were induced to corneal fate using a slight modifica- tion of a single enzyme molecule protocol designed by ▇▇▇▇ and colleagues for hESC (17). In brief, iPSC lines were seeded on collagen IV in the presence of medium conditioned by human corneal fibroblasts (COF) and treated with BMP-4 between days 0 and 3. As illus- trated by real-time qRT-PCR analysis, human iPSC lines un- derwent sequential differentiation into ectodermal precursors (K18+/Pax6+) at day 4, markers of corneal progenitors (K14+/ p63+/pax6+) appeared at day 8, and markers of terminally dif- ferentiated corneal epithelial (Pax-6+/K3+/K12+) cells were expressed at day 14 (Fig. 2A). Remarkably, at day 14, most of the cells became corneal epithelial cells, as detected by immunoflu- orescence staining (Fig. 2B) and FACS analysis (Fig. 2C). p63 is a putative marker of corneal stem cells, which are located in the limbus, a defined region at the corneal periphery (18). We next challenged iPSCEEC for their ability to be detected within 10 min undergo proper corneal epithelial commitment compared with the iPSCWT. Similar production of onectodermal progenitors (K18+/E-chip incubationcadherin+) was lines appeared inefficient because most of the cells differentiate in a heterogenous manner, with few ectodermal-like cells and with large cystic-like structures (Fig. Our prototype system is validated by detection S2A). We found that extraembry- onic cells expressing CDX2 and CGHα are produced from iPSC in response to a high dose of BMP-4 (Fig. S2B), suggesting that iPSC, contrary to hESC, do not efficiently differentiate into ectodermal lineage because they failed to first spontaneously commit into neu- roectoderm (15). Because inhibition of the TGF-β/nodal pathway promotes lost of pluripotency and neuroectodermal commitment (16), we tested the effect of a biomarker TGF-β inhibitor, SB431542, on the epidermal differentiation of iPSC. Ectodermal differentiation of iPSC was dramatically improved in presence of SB431542. A Day 10 B * * * Morphologically, the colonies lost their pluripotent aspect faster and acquired a homogenous ectodermal phenotype within 7 d (Fig. S2A). Pluripotency markers (Oct4 and Dnmt3b) and tropho- ectoderm markers (CDX2 and CGHα) were reduced but the ker- atinocyte marker K14 was increased (Fig. S2B). Differentiation of iPSC treated with BMP-4, AA, and SB431542 for prostate cancer in buffer, down 30 d led to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte molecules.ma-

End User Agreement. This publication is distributed under the terms of Article 25fa of the Dutch Copyright Act (Auteurswet) with explicit consent by the author. Dutch law entitles the maker of a short scientific work funded either wholly or partially by Dutch public funds to make that work publicly available for no consideration following a reasonable period of time after the work was first published, provided that clear reference is made to the source of the first publication of the work. This publication is distributed under The Association of Universities in the Netherlands (VSNU) ‘Article 25fa implementation’ pilot project. In this pilot research outputs of researchers employed by Dutch Universities that comply with the legal requirements of Article 25fa of the Dutch Copyright Act are distributed online and free of cost or other barriers in institutional repositories. Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication. You are permitted to download and use the publication for personal purposes. All rights remain with the author(s) and/or copyrights owner(s) of this work. Any use of the publication other than authorised under this licence or copyright law is prohibited. If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please contact the Library through email: ▇▇▇▇▇▇▇▇▇@▇▇▇.▇▇.▇▇, or send a letter to: University Library Radboud University Copyright Information Point PO Box 9100 6500 HA Nijmegen You will be contacted as soon as possible. Ultrarapid Generation Evaluation of Femtoliter Microfluidic Droplets Image Registration in PET/CT of the Liver and Recommendations for Single- Molecule-Counting Immunoassays Jung-uk Shim,†,§,* Optimized Imaging ▇▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇▇▇▇,† ▇, ▇▇▇▇ ▇. ▇▇▇▇▇,† ▇ ▇▇▇▇▇▇, ▇▇▇ ▇. ▇▇▇▇▇▇▇,† ▇, ▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇, ▇▇,‡ ▇▇▇ ▇.▇. Corstens1, ▇▇▇▇ ▇.▇. Ruers2, and ▇▇▇ ▇.▇. Oyen1 1Department of Nuclear Medicine, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands; 2Department of Surgery, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands; and 3Department of Radiology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands Multimodality PET/CT of the liver can be performed with an inte- grated (hybrid) PET/CT scanner or with software fusion of dedi- cated PET and CT. Accurate anatomic correlation and good image quality of both modalities are important prerequisites, re- gardless of the applied method. Registration accuracy is influ- enced by breathing motion differences on PET and CT, which may also have impact on (attenuation correction–related) arti- facts, especially in the upper abdomen. The impact of these is- sues was evaluated for both hybrid PET/CT and software fusion, focused on imaging of the liver. Methods: Thirty patients underwent hybrid PET/CT, 20 with CT during expiration breath- hold (EB) and 10 with CT during free breathing (FB). Ten addi- tional patients underwent software fusion of dedicated PET and dedicated expiration breath-hold CT (SF). The image regis- tration accuracy was evaluated at the location of liver borders on CT and uncorrected PET images and at the location of liver lesions. Attenuation-correction artifacts were evaluated by comparison of liver borders on uncorrected and attenuation- corrected PET images. CT images were evaluated for the pres- ence of breathing artifacts. Results: In EB, 40% of patients had an absolute registration error of the diaphragm in the cranio- caudal direction of .1 cm (range, 216 to 44 mm), and 45% of lesions were mispositioned .1 cm. In 50% of cases, attenuation- correction artifacts caused a deformation of the liver dome on PET of .1 cm. Poor compliance to breath-hold instructions caused CT artifacts in 55% of cases. In FB, 30% had registration errors of .1 cm (range, 24 to 16 mm) and PET artifacts were less extensive, but all CT images had breathing artifacts. As SF allows independent alignment of PET and CT, no registration errors or artifacts of .1 cm of the diaphragm occurred. Conclusion: Hy- brid PET/CT of the liver may have significant registration errors and artifacts related to breathing motion. The extent of these is- sues depends on the selected breathing protocol and the speed of the CT scanner. No protocol or scanner can guarantee perfect image fusion. On the basis of these findings, recommendations were formulated with regard to scanner requirements, breathing protocols, and reporting. Received Sep. 21, 2006; revision accepted Mar. 9, 2007. For correspondence or reprints contact: ▇▇▇▇▇▇ ▇. ▇. ▇▇▇▇,†,^ ▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇,† and ▇▇▇▇▇ ▇▇▇▇▇† †Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, U.K., CB2 1EW and ‡Department of Biochemistry, University of Cambridge, Cambridge, U.K., CB2 1GA. §Present address: Biomedical Engineering, University of Glasgow, Glasgow, UK, G12 8LT. ^Present address: Radboud University Nijmegen, Institute for Molecules and ▇▇▇▇▇▇▇▇▇, MD, Department of Nuclear Medicine (565), Radboud University Nijmegen Medical Center, Postbox ▇▇▇▇▇▇▇▇▇▇▇▇▇▇ ▇▇▇, ▇▇▇▇ ▇▇ ▇▇▇▇▇▇▇▇, ▇▇▇ ▇▇▇▇▇▇▇▇▇▇▇. ARTICLE ABSTRACT We report E-mail: ▇.▇▇▇▇▇@▇▇▇▇▇▇.▇▇▇▇.▇▇ COPYRIGHT ª 2007 by the Society of Nuclear Medicine, Inc. Key Words: PET; PET/CT; accuracy; liver imaging; oncology J Nucl Med 2007; 48:910–919 DOI: 10.2967/jnumed.107.041517 Accurate imaging of liver metastases is important for clinical decision making when considering locoregional therapy, such as partial liver resection or radiofrequency ablation (1,2). These interventions rely on accurate infor- mation about the localization and the extent of tumor sites (3,4). The added value of functional imaging with 18F-FDG PET to conventional anatomic imaging (CT, especially, and MRI) has been well recognized, especially when assessing previous therapeutic interventions (5,6). However, the exact localization of lesions on 18F-FDG PET is limited by a microfluidic dropletrelatively low spatial resolution and a lack of anatomic reference. The obvious benefit of combining the capabil- ities of CT (anatomic reference) and 18F-FDG PET (sensi- tive tumor detection) has led to the practice of correlation of images as obtained by PET and by CT (7–9). Correlation can be performed with mere visual side-by-side evaluation of images acquired by separate scanners or with integrated images provided by either an integrated (hybrid) PET/CT scanner or software image fusion of dedicated PET and CT (10). Regardless of the methodology, the anatomic corre- lation of both image sets must be accurate. This implies that the liver needs to be in the same anatomic position and shape during both CT and PET acquisitions. However, CT and PET are influenced differently by breathing motion. As free breathing is mandatory for PET acquisition, PET has blurring in the lower thoracic and upper abdominal areas. CT acquisition must be adapted to match these images, by scanning during free breathing or timed unforced expiration (10), but neither approach fully eliminates the risk of reg- istration errors between PET and CT (11,12). Furthermore, these registration errors can introduce artifacts on PET images in hybrid PET/CT, where attenuation correction of PET images is based on the CT images. Such artifacts may compromise both clinical interpretation and quantitative eval- uation of PET images (13). Diagnostic imaging requires optimal image quality. In this study, we determined the extent of anatomic registration errors and the occurrence of artifacts in hybrid PET/CT of the liver using a robust technique, during different breathing protocols, and performed a direct comparison with software image fusion of separately acquired PET and CT. Accord- ing to our findings, recommendations were formulated with regard to scanner requirements, breathing protocols, and re- porting. MATERIALS AND METHODS Integrated PET/CT images were acquired with 3 different pro- tocols. Twenty consecutive patients with suspected metastases from colorectal cancer underwent hybrid PET/CT with low-dose CT during expiration breath-hold (EB). Ten other consecutive patients (March 2006), who were referred for various indications and who were unable to comply with breathing instructions for various reasons, underwent hybrid PET/CT with low-dose CT during free breathing (FB). Ten more consecutive patients (between Decem- ber 2002 and November 2003) with suspected metastases from colorectal carcinoma underwent software fusion of dedicated PET and dedicated diagnostic CT acquired during breath-hold (SF). Image Acquisition · Hybrid PET/CT scans were acquired using a Biograph Duo (Siemens Medical Solutions USA, Inc.) containing a 2-slice CT scanner. A low-dose CT scan for localization and attenuation- correction purposes was acquired in the caudocranial direction from the thighs to the base of the skull. Scanning parameters in- cluded 40 mA s, 130 kV, 5-mm slice collimation, 0.8-s rotation time, and pitch of 1.5, reconstructed to 3-mm slices for smooth coronal representation. CT scans were acquired during timed un- forced expiration breath-hold (EB) or during free breathing (FB). Timed expiration breath-hold consisted of free breathing during the first (caudal) part of the scan, a deep inspiration command at the level of the ▇▇▇▇▇ iliaca superior, immediately followed by a command to expire and breath-hold; patients were allowed to re- sume free breathing at the level of the lung tops. The total expi- ration breath-hold time was about 30 s. Free breathing was performed without specific patient instructions. No intravenous contrast was applied. For PET, a 3-dimensional (3D) emission scan of the cen- tral body was acquired during free breathing, 60 min after intra- venous injection of 250 MBq 18F-FDG. The acquisition time per bed position was 4 min for emission only. Uncorrected emission images as well as images with CT-based approach enabling the measurement of chemical reactions of individual enzyme molecules attenuation correction were reconstructed, both using 2 iterations, 8 subsets, and its application to a single5-moleculemm 3D gaussian filter. Dedicated 18F-counting immunoassayFDG PET scans were acquired using an ECAT Exact 47 scanner (Siemens Medical Solutions). A microfluidic 6 device is used 3D emission scan was acquired and reconstructed identical to generate and manipulate <10 fL droplets at rates of up to 1.3 × 10 per secondPET from PET/CT. In addition, about 2 orders of magnitude faster than has previously been reporteda 2-dimensional 68Ge-based transmis- sion scan was acquired for attenuation correction. The femtodroplets produced acquisition time per bed position was 5 min for emission and 3 min for the transmission. Dedicated CT scans were acquired using a Somatom Volume Zoom (Siemens Medical Solutions) 4-slice scanner. Scans of the liver were acquired with 80 mAs, 130 kV, 0.5-s rotation time, and 5-mm slice thickness, during unforced expiration breath-hold. Intravenous contrast was applied; the portal-phase images were selected for image fusion with PET. Image Registration Procedure For hybrid PET/CT, normal image registration quality-assurance procedures were followed as described by the manufacturer. This involved alignment of the PET and CT gantries after maintenance, using a ‘‘crossed-lines’’ phantom. No additional image registration optimization was performed after scanning. Software image reg- istration was performed on a personal computer with image view- ing and registration software, developed in-house, based on the visualization toolkit VTK (14) and the insight segmentation and registration toolkit ITK (15). The procedure has been described in more detail previously (16). In brief, the software allows rigid- body image registration based on 3 translation and 3 rotation parameters. Anatomic registration of PET emission images to CT was pursued using an implementation of the automatic mutual information algorithm, on a 3D volume of interest containing the liver. Image Analysis Image sets from PET and CT were correlated through evalua- tion of borders of the liver and focal lesions within the liver. Mismatches of .10 mm were considered potentially clinically relevant. Mismatches of focal lesions were expressed as 3D vec- tors. For liver borders, this device can be used to encapsulate single biomolecular complexes tagged with approach is not possible, because a reporter enzyme; their small volume enables the fluorescent product unidirectional shift of a single enzyme molecule liver border may be complicated by a (unrecognizable) deformation or rotation that alters the location that represents the top. Selected for landmarks were the tangent points (tops) of 3 liver borders: the diaphragmatic dome, the right liver border, and the caudal tip. 3D ellipsoids were manually positioned to match the curved shapes of the liver borders (Fig. 1); the locations of the tangent points were then derived mathemat- ically. Mismatches were expressed as 1-dimensional distances along the axis of the largest movement (e.g., the craniocaudal direction for the diaphragmatic dome and the caudal tip of the liver; the lat- eral direction for the right lateral liver border). This procedure was performed separately on CT, uncorrected (uPET), and attenuation- corrected (acPET) images, blinded from each other. The localization of liver borders is difficult on uPET and acPET, as the images are blurry. The selected visual cutoff for positioning of a border may be different for uPET and acPET images. The observer-specific systematic bias between localization of liver bor- ders on uPET and acPET was determined by comparing images from dedicated PET, where the position of the liver is theoretically identical on both image sets. The true position of the liver border was assumed to be detected within 10 min between the visual localizations on uPET and acPET. All uPET and acPET measurements were corrected after- ▇▇▇▇ for this bias, using the average measurement difference from the theoretic position. The interobserver variability for manual deter- mination of on-chip incubationpositional differences of tangent points, after correction of the systematic bias, was evaluated in 5 subsequent dedicated PET scans (both uPET and acPET) by 2 experienced observers. Our prototype system is validated by detection • Registration errors: The relative anatomic/positional mis- match of structures (either circumscript lesions or organ bor- ders) as visible on uPET and CT images, expressed as a biomarker for prostate cancer distance in buffer, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte moleculesmillimeters.

End User Agreement. This publication is distributed under the terms of Article 25fa of the Dutch Copyright Act (Auteurswet) with explicit consent by the author. Dutch law entitles the maker of a short scientific work funded either wholly or partially by Dutch public funds to make that work publicly available for no consideration following a reasonable period of time after the work was first published, provided that clear reference is made to the source of the first publication of the work. This publication is distributed under The Association of Universities in the Netherlands (VSNU) ‘Article 25fa implementation’ pilot project. In this pilot research outputs of researchers employed by Dutch Universities that comply with the legal requirements of Article 25fa of the Dutch Copyright Act are distributed online and free of cost or other barriers in institutional repositories. Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication. You are permitted to download and use the publication for personal purposes. All rights remain with the author(s) and/or copyrights owner(s) of this work. Any use of the publication other than authorised under this licence or copyright law is prohibited. If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please contact the Library through email: ▇▇▇▇▇▇▇▇▇@▇▇▇.▇▇.▇▇, or send a letter to: University Library Radboud University Copyright Information Point PO Box 9100 6500 HA Nijmegen You will be contacted as soon as possible. Ultrarapid Generation of Femtoliter Microfluidic Droplets for Single- Molecule-Counting Immunoassays Jung-uk Shim,†,§,* ▇▇▇▇▇▇ ▇. ▇▇▇▇▇, ▇▇▇▇ ▇▇▇▇▇▇, ▇▇▇▇ ▇▇▇▇▇▇▇, and ▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇,† ▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇▇,‡ ▇ 1 Radboud University, The Netherlands 2 University of Strathclyde, Scotland {▇▇▇▇▇▇▇ .▇. ▇. ▇▇▇▇,†,^ ▇▇▇▇▇ .Ghani,▇▇▇▇▇▇▇▇.▇,† ▇▇▇▇▇}@▇▇▇.▇▇▇▇▇▇.▇▇.▇▇ Abstract. This paper provides several induction rules that can be used to prove properties of effectful data types. Our results are semantic in nature and build upon ▇▇▇▇▇▇▇ and ▇▇▇▇▇ ▇▇▇▇▇† †Department ’ fibrational formulation of Chemistry, University of Cambridge, Lensfield Road, Cambridge, U.K., CB2 1EW induction for polynomial data types and ‡Department of Biochemistry, University of Cambridge, Cambridge, U.K., CB2 1GA. §Present address: Biomedical Engineering, University of Glasgow, Glasgow, UK, G12 8LT. ^Present address: Radboud University Nijmegen, Institute for Molecules and ▇▇▇▇its extension to all inductive data types by ▇▇▇▇▇, ▇▇▇▇▇▇, and Fumex. An effectful data type µ(TF ) is built from a functor F that describes data, and a monad T that computes effects. Our main contribution is to derive induction rules that are generic over all functors F and monads T such that µ(TF ) exists. Along the way, we also derive a principle of definition by structural recursion for effectful data types that is similarly generic. Our induction rule is also generic over the kinds of properties to be proved: like the work on which we build, we work in a general fibrational setting and so can accommodate very general notions of properties, rather than just those of particular syntactic forms. We give examples exploiting the generality of our results, and show how our results specialize to those in the literature, particularly those of ▇▇▇▇▇▇▇▇ ▇▇▇, ▇▇▇▇ ▇▇ ▇▇▇▇▇▇▇▇, ▇▇▇ ▇▇▇▇▇▇▇▇▇▇▇. ARTICLE ABSTRACT We report a microfluidic droplet-based approach enabling the measurement of chemical reactions of individual enzyme molecules and its application to a single-molecule-counting immunoassay. A microfluidic 6 device is used to generate and manipulate <10 fL droplets at rates of up to 1.3 × 10 per second, about 2 orders of magnitude faster than has previously been reported. The femtodroplets produced with this device can be used to encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables the fluorescent product of a single enzyme molecule to be detected within 10 min of on-chip incubation. Our prototype system is validated by detection of a biomarker for prostate cancer in buffer, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte moleculesStøvring.

End User Agreement. This publication is distributed under the terms of Article 25fa of the Dutch Copyright Act (Auteurswet) with explicit consent by the author. Dutch law entitles the maker of a short scientific work funded either wholly or partially by Dutch public funds to make that work publicly available for no consideration following a reasonable period of time after the work was first published, provided that clear reference is made to the source of the first publication of the work. This publication is distributed under The Association of Universities in the Netherlands (VSNU) ‘Article 25fa implementation’ pilot project. In this pilot research outputs of researchers employed by Dutch Universities that comply with the legal requirements of Article 25fa of the Dutch Copyright Act are distributed online and free of cost or other barriers in institutional repositories. Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication. You are permitted to download and use the publication for personal purposes. All rights remain with the author(s) and/or copyrights owner(s) of this work. Any use of the publication other than authorised under this licence or copyright law is prohibited. If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please contact the Library through email: ▇▇▇▇▇▇▇▇▇@▇▇▇.▇▇.▇▇, or send a letter to: University Library Radboud University Copyright Information Point PO Box 9100 6500 HA Nijmegen You will be contacted as soon as possible. Ultrarapid Generation of Femtoliter Microfluidic Droplets for Single- Molecule-Counting Immunoassays Jung-uk Shim,†,§,* ▇▇▇ ▇▇▇ ▇▇▇ ▇▇▇▇▇, ▇▇▇▇ ▇. ▇▇▇▇▇▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇,† ▇▇, ▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇▇,‡ (✉), ▇▇▇▇▇ ▇▇ ▇▇▇▇▇▇▇ ▇. ▇. ▇▇▇▇,†,^ , ▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇,† , and ▇▇▇▇▇ ▇▇▇▇▇† †Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, U.K., CB2 1EW and ‡Department of Biochemistry, University of Cambridge, Cambridge, U.K., CB2 1GA. §Present address: Biomedical Engineering, University of Glasgow, Glasgow, UK, G12 8LT. ^Present address: Radboud University Nijmegen, Institute for Molecules and ▇▇▇▇▇▇▇▇▇▇▇ 1 Netherlands National Communication Security Agency (NLNCSA), The Hague, The Netherlands {ebo.laan,▇▇▇.▇▇▇▇▇▇▇▇▇▇}@▇▇▇▇▇▇.▇▇ Digital Security Group, Radboud University, Nijmegen, The Netherlands {erikpoll,▇▇▇▇▇}@▇▇.▇▇.▇▇, ▇▇▇▇▇@▇▇▇▇▇▇▇▇▇▇▇▇▇▇ ▇.▇▇, ▇▇▇▇ ▇▇ ▇▇▇▇▇▇▇▇, ▇▇▇ @▇▇▇▇▇▇▇▇▇▇.▇▇▇ Abstract. ARTICLE ABSTRACT We report a microfluidic dropletThe current Java Card platform does not seem to allow for fast implementations of hash-based approach enabling signature schemes. While the measurement under- lying implementation of chemical reactions of individual enzyme molecules and its application to a single-molecule-counting immunoassay. A microfluidic 6 device is used to generate and manipulate <10 fL droplets at rates of up to 1.3 × 10 per second, about 2 orders of magnitude faster than has previously been reported. The femtodroplets produced with this device the cryptographic primitives provided by the API can be used fast, thanks to encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables implementations in native code or in hard- ware, the fluorescent product cumulative overhead of a single enzyme molecule the many separate API calls results in prohibitive performance for many common applications. In this work, we present an implementation of XMSSMT on the current Java Card platform, and make suggestions how to be detected within 10 min of on-chip incubation. Our prototype system is validated by detection of a biomarker for prostate cancer improve this platform in buffer, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte moleculesfuture versions.

End User Agreement. This publication is distributed under the terms of Article 25fa of the Dutch Copyright Act (Auteurswet) with explicit consent by the author. Dutch law entitles the maker of a short scientific work funded either wholly or partially by Dutch public funds to make that work publicly available for no consideration following a reasonable period of time after the work was first published, provided that clear reference is made to the source of the first publication of the work. This publication is distributed under The Association of Universities in the Netherlands (VSNU) ‘Article 25fa implementation’ pilot project. In this pilot research outputs of researchers employed by Dutch Universities that comply with the legal requirements of Article 25fa of the Dutch Copyright Act are distributed online and free of cost or other barriers in institutional repositories. Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication. You are permitted to download and use the publication for personal purposes. All rights remain with the author(s) and/or copyrights owner(s) of this work. Any use of the publication other than authorised under this licence or copyright law is prohibited. If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please contact the Library through email: ▇▇▇▇▇▇▇▇▇@▇▇▇.▇▇.▇▇, or send a letter to: University Library Radboud University Copyright Information Point PO Box 9100 6500 HA Nijmegen You will be contacted as soon as possible. Ultrarapid Generation Enhanced transcription rates in membrane-free protocells formed by coacervation of Femtoliter Microfluidic Droplets for Single- Molecule-Counting Immunoassays Jung-uk Shim,†,§,* cell lysate ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇,† ▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇, ▇▇,‡ ▇▇ ▇▇▇▇▇▇▇, ▇▇▇▇▇ ▇. ▇. ▇▇▇▇,†,^ ▇▇, ▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇,† and , ▇▇▇▇▇ ▇▇▇▇▇† †Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, U.K., CB2 1EW and ‡Department of Biochemistry, University of Cambridge, Cambridge, U.K., CB2 1GA. §Present address: Biomedical Engineering, University of Glasgow, Glasgow, UK, G12 8LT. ^Present address: Radboud University Nijmegen, Institute for Molecules and ▇▇▇▇▇▇▇▇▇, ▇▇▇▇▇▇▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇▇▇▇, ▇▇▇▇▇▇ ▇▇▇▇▇▇▇, ▇▇▇▇ ▇. ▇▇▇▇, and ▇▇▇▇▇▇▇ ▇. ▇. Huck1 Institute for Molecules and Materials, Radboud University Nijmegen, ▇▇▇▇ ▇▇, ▇▇▇▇▇▇▇▇, ▇▇▇ ▇▇▇▇▇▇▇▇▇▇▇ Edited by ▇▇▇▇▇ ▇. ARTICLE ABSTRACT ▇▇▇▇▇▇▇, California Institute of Technology, Pasadena, CA, and approved June 10, 2013 (received for review December 28, 2012) Liquid–liquid phase transitions in complex mixtures of proteins and other molecules produce crowded compartments supporting in vitro transcription and translation. We report developed a microfluidic dropletmethod based on picoliter water-based approach enabling in-oil droplets to induce coacervation in Escherichia coli cell lysate and follow gene expression under crowded and noncrowded conditions. Coacervation creates an ar- tificial cell-like environment in which the measurement rate of chemical reactions of individual enzyme molecules and its application mRNA production is increased significantly. Fits to the measured transcription rates show a single-molecule-counting immunoassay. A microfluidic 6 device is used to generate and manipulate <10 fL droplets at rates of up to 1.3 × 10 per second, about 2 two orders of magnitude faster than has previously been reportedlarger binding constant between DNA and T7 RNA polymerase, and five to six times larger rate constant for transcription in crowded environments, strikingly sim- ilar to in vivo rates. The femtodroplets produced with this device effect of crowding on interactions and kinetics of the fundamental machinery of gene expression has a di- rect impact on our understanding of biochemical networks in vivo. Moreover, our results show the intrinsic potential of cellular com- ponents to facilitate macromolecular organization into membrane- free compartments by phase separation. microdroplets | macromolecular crowding P rotocells are minimal compartmentalized systems exhibiting key characteristics of cellular function, including metabolism and replication (1, 2). Lipid vesicles are considered the pro- totypical protocell as they can be used to encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables the fluorescent product of a single enzyme molecule to be detected within 10 min of on-chip incubationform functional microscopic spherical assemblies suited for in vitro gene expression (3, 4). Our prototype system Compartmentalization via lipid bilayers is validated by detection of a biomarker for prostate cancer in buffer, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets considered essential for the counting emergence of individual analyte molecules.cells (4), but there are alternative models based on liquid–liquid phase transitions that lead to the emer- gence of compartments (5, 6). Compartmentalization is but one characteristic, as protocells ideally also mimic the highly crowded interior of living cells, which have total macromolecule concen- trations in excess of 300 g/L (7). Examples in which compart- mentalization and high local concentrations are obtained concurrently, include DNA brushes (8), aqueous two-phase sys- tems (9), and liquid coacervates (10). Phase separation or co- acervation occurs in a wide range of polymer and protein solutions, often triggered by changes in temperature or salt concentration, or by the addition of coacervating agents (11). The (complex) coacervate droplets that are formed in such sys- tems present macromolecularly crowded, aqueous, physical compartments, 1–100 μm in diameter (12). Recent work has identified similar liquid phase transitions in vivo in the formation of intracellular non–membrane-bound compartments exhibiting liquid-like properties, slowed down diffusion, and strongly inter- acting macromolecular components (13, 14). Well-studied exam- ples are the intracellular localization of DNA or RNA and proteins

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Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication. You are permitted to download and use the publication for personal purposes. All rights remain with the author(s) and/or copyrights owner(s) of this work. Any use of the publication other than authorised under this licence or copyright law is prohibited. If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please contact the Library through email: ▇▇▇▇▇▇▇▇▇@▇▇▇.▇▇.▇▇, or send a letter to: University Library Radboud University Copyright Information Point PO Box 9100 6500 HA Nijmegen You will be contacted as soon as possible. Ultrarapid Generation of Femtoliter Microfluidic Droplets for Single- MoleculePublished on 20 June 2014. Downloaded by Radboud University Nijmegen on 9/5/2022 7:47:52 AM. ChemComm COMMUNICATION Cite this: Chem. Commun., 2014, 50, 8930 Received 28th April 2014, Accepted 20th June 2014 DOI: 10.1039/c4cc03167a ▇▇▇.▇▇▇.▇▇▇/▇▇▇▇▇▇▇▇ Mechanically strong, fluorescent hydrogels from zwitterionic, fully p-Counting Immunoassays Jung-uk Shim,†,§,* conjugated polymers† ▇▇▇▇▇ ▇. ▇▇▇▇▇▇,a ▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇▇,† *▇▇ ▇▇▇▇ ▇. ▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇,† a ▇▇▇▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇▇▇,‡ cf ▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇▇▇▇,c ▇▇▇▇▇▇ ▇▇▇▇▇▇ and ▇▇▇▇▇▇▇ ▇. ▇. ▇▇▇▇,†,^ ▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇,† and ▇▇▇▇▇ ▇▇▇▇▇† †Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, U.K., CB2 1EW and ‡Department of Biochemistry, University of Cambridge, Cambridge, U.K., CB2 1GA. §Present address: Biomedical Engineering, University of Glasgow, Glasgow, UK, G12 8LT. ^Present address: Radboud University Nijmegen, Institute for Molecules and ▇▇▇▇▇▇▇▇▇, ▇▇▇▇▇▇▇▇▇▇▇▇▇▇ ▇▇▇, ▇▇▇▇ ▇▇ ▇▇▇▇▇▇▇▇, ▇▇▇ ▇▇▇▇▇▇▇▇▇▇▇. ARTICLE ABSTRACT We report a microfluidic droplet-based approach enabling the measurement of chemical reactions of individual enzyme molecules and its application to a single-molecule-counting immunoassay. A microfluidic 6 device is used to generate and manipulate <10 fL droplets at rates of *ae Mechanically strong supramolecular hydrogels (up to 1.3 × 10 per second98.9% water content) were obtained by the combination of a rigid, about 2 orders of magnitude faster than has previously been reportedfully p-conjugated polymer backbone and zwitterionic side chains. The femtodroplets produced gels were characterized by SAXS, SEM and rheology measurements and are fluorescent, stimuli responsive (temperature, salts) and bind DNA. Self-assembled, non-covalently cross-linked polymer hydrogels show dynamically reversible responses to external stimuli such as mechanical forces, temperature or ionic strength, and can have self-healing abilities.1 Various reversible bonding interactions such as hydrogen-bonds, p–p-stacking and charge transfer interactions have been exploited for non-covalent polymer cross-linking,1a–d,2 however, the mechanical performances of theses ‘‘honey-like’’ materials are often not ideal. Hydrogels with this device can be used to encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables the fluorescent product improved mechanical properties have been obtained through use of a single enzyme molecule host–guest binding interactions,1c,3 bundling into filaments of stiff polymers that mimick natural hydrogels from collagen or fibrin,4 or Coulombic interactions.5 For instance, mixing of poly-anions and poly-cations yields mechanically strong self-assembled materials,5 although poly-cationic species are known to be detected within 10 min of ontoxic. Furthermore, multi- component gel preparation requires an accurately controlled mixing-chip incubation. Our prototype system is validated by detection of ratio and can lead to undesired phase-separation.1c,6 Single-component hydrogels from zwitterionic polymers are much more biocompatible, e.g. implanted zwitterionic gels did not cause a biomarker for prostate cancer in buffer, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely highforeign-speed generation of monodisperse femtoliter droplets for the counting of individual analyte molecules.body reaction.7

End User Agreement. This publication is distributed under the terms of Article 25fa of the Dutch Copyright Act (Auteurswet) with explicit consent by the author. Dutch law entitles the maker of a short scientific work funded either wholly or partially by Dutch public funds to make that work publicly available for no consideration following a reasonable period of time after the work was first published, provided that clear reference is made to the source of the first publication of the work. This publication is distributed under The Association of Universities in the Netherlands (VSNU) ‘Article 25fa implementation’ pilot project. In this pilot research outputs of researchers employed by Dutch Universities that comply with the legal requirements of Article 25fa of the Dutch Copyright Act are distributed online and free of cost or other barriers in institutional repositories. Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication. You are permitted to download and use the publication for personal purposes. All rights remain with the author(s) and/or copyrights owner(s) of this work. Any use of the publication other than authorised under this licence or copyright law is prohibited. If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please contact the Library through email: ▇▇▇▇▇▇▇▇▇@▇▇▇.▇▇.▇▇, or send a letter to: University Library Radboud University Copyright Information Point PO Box 9100 6500 HA Nijmegen You will be contacted as soon as possible. Ultrarapid Generation View Article Online / Journal Homepage / Table of Femtoliter Microfluidic Droplets Contents for Single- Molecule-Counting Immunoassays Jung-uk Shim,†,§,* this issue PAPER ▇▇▇.▇▇ ▇. ▇.▇▇▇/▇▇▇▇▇▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇,† | Journal of Materials Chemistry Columnar mesophases from half-discoid platinum cyclometalated metallomesogens{{ ▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇▇,‡ ▇▇▇, ▇▇▇▇ ▇. ▇. ▇▇▇▇,†,^ ▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇,† and ▇▇▇▇▇ ▇▇▇▇▇† †Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, U.K., CB2 1EW and ‡Department of Biochemistry, University of Cambridge, Cambridge, U.K., CB2 1GA. §Present address: Biomedical Engineering, University of Glasgow, Glasgow, UK, G12 8LT. ^Present address: Radboud University Nijmegen, Institute for Molecules and ▇▇▇▇▇▇▇▇▇, ▇▇▇▇▇▇▇▇▇ ▇▇▇▇, ▇▇▇▇▇ ▇▇¨ller and ▇▇▇▇▇▇▇ ▇▇▇, ▇▇▇▇ ▇▇ . ▇▇▇▇▇▇▇▇, ▇▇▇ ▇▇▇▇▇▇▇▇▇▇▇* Published on 26 November 2007. ARTICLE ABSTRACT We report a microfluidic dropletDownloaded by Radboud University Nijmegen on 5/13/2022 12:44:19 PM. DOI: 10.1039/b714291a A series of liquid crystals have been synthesized and studied based upon mononuclear ortho- platinated rod-based approach enabling the measurement of chemical reactions of individual enzyme molecules like heteroaromatic and its application to a single1,3-molecule-counting immunoassay. A microfluidic 6 device is used to generate and manipulate <10 fL droplets at rates of up to 1.3 × 10 per second, about 2 orders of magnitude faster than has previously been reporteddiketonate ligands. The femtodroplets produced with this device can be used liquid crystalline properties of these molecules were investigated using polarized light optical microscopy, differential scanning calorimetry, single crystal X-ray diffraction, and powder X-ray diffraction. Increasing the number of alkyl chains attached to encapsulate single biomolecular the 1,3-diketonate units resulted in a transition from lamellar (SmA) to hexagonal columnar phases (Colh). The 2-thienylpyridine units were previously unexplored in metallomesogenic complexes tagged with a reporter enzyme; their small volume enables and these studies extend the fluorescent product utility of a single enzyme molecule to be detected within 10 min of onortho-chip incubation. Our prototype system is validated by detection of a biomarker for prostate cancer in buffer, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte molecules.platinated

End User Agreement. This publication is distributed under the terms of Article 25fa of the Dutch Copyright Act (Auteurswet) with explicit consent by the author. Dutch law entitles the maker of a short scientific work funded either wholly or partially by Dutch public funds to make that work publicly available for no consideration following a reasonable period of time after the work was first published, provided that clear reference is made to the source of the first publication of the work. This publication is distributed under The Association of Universities in the Netherlands (VSNU) ‘Article 25fa implementation’ pilot project. In this pilot research outputs of researchers employed by Dutch Universities that comply with the legal requirements of Article 25fa of the Dutch Copyright Act are distributed online and free of cost or other barriers in institutional repositories. Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication. You are permitted to download and use the publication for personal purposes. All rights remain with the author(s) and/or copyrights owner(s) of this work. Any use of the publication other than authorised under this licence or copyright law is prohibited. If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please contact the Library through email: ▇▇▇▇▇▇▇▇▇@▇▇▇.▇▇.▇▇, or send a letter to: University Library Radboud University Copyright Information Point PO Box 9100 6500 HA Nijmegen You will be contacted as soon as possible. Ultrarapid Generation of Femtoliter Microfluidic Droplets for Single- MoleculeDOI 10.1007/s00330-Counting Immunoassays Jung014-uk Shim,†,§,* 3301-z UROGENITAL Correlation between dynamic contrast-enhanced MRI and quantitative histopathologic microvascular parameters in organ-confined prostate cancer ▇▇▇▇▇▇▇▇ ▇. ▇▇▇ ▇▇▇▇▇▇▇ • ▇▇▇▇▇▇ ▇. ▇. ▇. ▇▇▇ ▇▇▇ ▇▇▇▇ • ▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇ • ▇▇▇▇-▇▇▇ ▇▇▇▇▇▇▇ • ▇. ▇▇▇▇▇▇ ▇▇▇▇▇▇ • ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇▇ • ▇▇▇▇▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇▇▇▇,† ▇▇▇▇▇ -van de Kaa Received: 19 February 2014 / Revised: 21 May 2014 / Accepted: 27 June 2014 / Published online: 18 July 2014 Ⓒ European Society of Radiology 2014 Abstract Objectives To correlate pharmacokinetic parameters of 3-T dynamic contrast-enhanced (DCE-)MRI with histopathologic microvascular and lymphatic parameters in organ-confined prostate cancer. Methods In 18 patients with unilateral peripheral zone (pT2a) tumours who underwent DCE-MRI prior to radical prostatec- tomy (RP), the following pharmacokinetic parameters were assessed: permeability surface area volume transfer constant (Ktrans), extravascular extracellular volume (Ve) and rate con- stant (Kep). In the RP sections blood and lymph vessels were visualised immunohistochemically and automatically exam- ined and analysed. Parameters assessed included microvessel density (MVD), area (MVA) and perimeter (MVP) as well as lymph vessel density (LVD), area (LVA) and perimeter (LVP). Results A negative correlation was found between age and Ktrans and Kep for tumour (r=−0.60, p=0.009; r=−0.67, p= 0.002) and normal (r=−0.54, p=0.021; r=−0.46, p=0.055) tissue. No correlation existed between absolute values of mi- crovascular parameters from histopathology and DCE-MRI. In contrast, the ratio between tumour and normal tissue (correcting for individual microvascularity variations) significantly corre- lated between Kep and MVD (r=0.61, p=0.007) and MVP (r= ▇. ▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇,† ▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇ . ▇. ▇▇▇,‡ ▇▇▇▇▇▇▇ . ▇. ▇. ▇▇▇▇,†,^ ▇▇▇ ▇▇▇ ▇▇▇▇▇▇▇▇▇,† and ▇▇▇▇▇ ▇▇▇▇▇† †Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, U.K., CB2 1EW and ‡Department of Biochemistry, University of Cambridge, Cambridge, U.K., CB2 1GA. §Present address: Biomedical Engineering, University of Glasgow, Glasgow, UK, G12 8LT. ^Present address: Radboud University Nijmegen, Institute for Molecules and ▇▇▇▇▇▇▇▇▇, ▇▇▇▇▇▇▇▇▇▇▇▇▇▇ ▇▇▇, ▇▇▇▇ ▇▇ ▇▇▇▇▇▇▇▇, ▇▇▇ ▇▇▇▇▇▇▇▇▇▇▇. ARTICLE ABSTRACT We report a microfluidic droplet-based approach enabling the measurement of chemical reactions of individual enzyme molecules and its application to a single-molecule-counting immunoassay. A microfluidic 6 device is used to generate and manipulate <10 fL droplets at rates of up to 1.3 × 10 per second, about 2 orders of magnitude faster than has previously been reported. The femtodroplets produced with this device can be used to encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables the fluorescent product of a single enzyme molecule to be detected within 10 min of on-chip incubation. Our prototype system is validated by detection of a biomarker for prostate cancer in buffer, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte molecules.

End User Agreement. This publication is distributed under the terms of Article 25fa of the Dutch Copyright Act (Auteurswet) with explicit consent by the author. Dutch law entitles the maker of a short scientific work funded either wholly or partially by Dutch public funds to make that work publicly available for no consideration following a reasonable period of time after the work was first published, provided that clear reference is made to the source of the first publication of the work. This publication is distributed under The Association of Universities in the Netherlands (VSNU) ‘Article 25fa implementation’ pilot project. In this pilot research outputs of researchers employed by Dutch Universities that comply with the legal requirements of Article 25fa of the Dutch Copyright Act are distributed online and free of cost or other barriers in institutional repositories. Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication. You are permitted to download and use the publication for personal purposes. All rights remain with the author(s) and/or copyrights owner(s) of this work. Any use of the publication other than authorised under this licence or copyright law is prohibited. If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please contact the Library through email: ▇▇▇▇▇▇▇▇▇@▇▇▇.▇▇.▇▇, or send a letter to: University Library Radboud University Copyright Information Point PO Box 9100 6500 HA Nijmegen You will be contacted as soon as possible. Ultrarapid Generation Organic & Published on 23 May 2014. Downloaded by Radboud University Nijmegen on 9/6/2022 9:28:56 AM. Biomolecular Chemistry PAPER Cite this: Org. Biomol. Chem., 2014, 12, 5031 Received 1st April 2014, Accepted 23rd May 2014 DOI: 10.1039/c4ob00694a ▇▇▇.▇▇▇.▇▇▇/▇▇▇ Synthesis of Femtoliter Microfluidic Droplets for Single- Molecule-Counting Immunoassays Jung-uk Shim,†,§,* DIBAC analogues with excellent SPAAC rate constants† ▇▇▇▇▇▇▇ ▇. ▇▇▇▇▇▇, ▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇,† ▇▇, ▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇▇,‡ ▇, ▇▇▇▇▇▇ ▇. ▇. ▇▇▇▇,†,^ ▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇,† and ▇▇▇▇▇ ▇▇▇▇▇† †Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, U.K., CB2 1EW and ‡Department of Biochemistry, University of Cambridge, Cambridge, U.K., CB2 1GA. §Present address: Biomedical Engineering, University of Glasgow, Glasgow, UK, G12 8LT. ^Present address: Radboud University Nijmegen, Institute for Molecules and ▇▇▇▇▇▇▇▇▇, ▇▇▇▇▇▇ ▇▇▇▇▇▇. ▇▇▇ ▇▇▇, ▇▇▇▇ ▇▇ ▇▇▇▇▇▇▇▇, ▇▇▇ ▇. ▇. ▇▇▇ Hest and ▇▇▇▇▇▇ P. J. T. Rutjes* In search for increased reactivity in strain-promoted azide alkyne cycloadditions (SPAAC), the synthesis of new and more reactive cyclooctynes is of pivotal importance. To identify cyclooctynes with enhanced reactivity, without loss of stability, the synthesis and kinetic analysis of new dibenzoazacyclooctyne (DIBAC) analogues were conducted. Starting from iodobenzyl alcohol analogues and ortho-ethynylaniline various substituted dihydrodibenzo[b,f ]azocines were produced. Subsequent bromination and elimination proved to be difficult depending on the aromatic substitution pattern, yielding chloro-, bromo-, and methoxy-substituted DIBACs in moderate yield. In the elimination reaction towards nitro- and Br,Cl-DIBAC, the corresponding cyclooctene was obtained instead of the cyclooctyne. Additionally, a dimethoxy-substituted DIBAC analogue was prepared following an alternative route involving light- induced deprotection of a cyclopropenone derivative. In total, four DIBAC analogues were successfully prepared showing excellent rate constants in the SPAAC reaction ranging from 0.45 to 0.9 M−1 s−1, which makes them comparable to the fastest cyclooctynes currently known. Introduction Selective bioorthogonal ligation strategies for the investigation of biological processes and biomolecule modification have become increasingly important in the last decade. Currently, the azide function is the most commonly applied reactive group in bioorthogonal chemistry being utilised in the ▇▇▇▇- ▇▇▇▇▇▇ ligation,1 the Cu(I)-catalysed azide–alkyne cycloaddition (CuAAC),2,3 and the strain-promoted azide–alkyne cyclo- addition (SPAAC).4,5 The advantages of SPAAC over the ▇▇▇▇- ▇▇▇▇▇▇ ligation include, depending on the cyclooctyne used, an increased reactivity and, as opposed to phosphines, the stability under ambient conditions. Unlike CuAAC, SPAAC avoids the use of a toxic Cu(I)-catalyst. Since the first application of SPAAC in a biological system,6 a wide range of cyclooctynes and dibenzocyclooctynes has been developed.5,7,8 Each cyclooctyne displays specific advan- tageous characteristics, e.g. excellent rate constant,9–11 good water solubility,12,13 synthetic ease,14 and/or fluorogenic pro- perties, but nevertheless requires improvement.15,16 In particu- lar hydrophilicity, reactivity, stability, and selectivity are key † Electronic supplementary information (ESI) available: Experimental proce- dures, spectroscopic characterization, 1H and 13C NMR spectra of all compounds. See DOI: 10.1039/c4ob00694a aspects for a wider applicability of the strained alkynes in a biological context. The fastest dibenzocyclooctyne currently known is BARAC (2) with a rate constant of 0.9 M−1 s−1. It was recently shown that the reactivity of BARAC could be tuned by the introduction of substituents on the aromatic rings.17 BARAC, however, has the disadvantage that it is susceptible to ▇▇▇▇▇▇▇ addition by thiols.10 Another interesting cyclooctyne is DIBAC (1)9 (also referred to as ADIBO18 or Aza-DBCO),19 designed in analogy to DIBO,20 displaying a rate constant of 0.3 ± 0.1 M−1 s−1. Unlike BARAC, DIBAC shows no ▇▇▇▇▇▇▇ addition product when reacted with glutathione, even at elevated temperatures. ARTICLE ABSTRACT In addition, DIBAC showed complete shelf-stability when stored in neat form at −20 °C, and was stored in aqueous solution for over a year at 4 °C without noticeable loss of reactivity. Presum- ably, it is this optimal combination of reactivity and stability which has made DIBAC the most commonly used cyclooctyne for SPAAC applications. We report envisioned that the addition of substituents on the aro- matic rings of DIBAC would lead to an increase in reactivity while retaining stability. Based on previous studies,17,21 we anticipated that electron-withdrawing groups could have a microfluidic dropletsub- stantial positive effect on the reactivity. In addition, we expected a change in reactivity based on the positioning of substituents on the aromatic rings. To investigate these hypotheses we aimed to prepare a series of DIBAC analogues (3a–f, Fig. 1) fol- lowing a previously reported synthetic route9 and investigate the rate constants in SPAAC reactions with benzyl azide. Published on 23 May 2014. Downloaded by Radboud University Nijmegen on 9/6/2022 9:28:56 AM. Fig. 1 DIBAC (1), BARAC (2) and target DIBAC-based approach enabling analogues 3a–f. Scheme 1 Retrosynthetic analysis of the measurement synthesis of chemical reactions DIBAC analogues 3a–e. Retrosynthesis of individual enzyme molecules the proposed DIBAC analogues 3a–e involved acylation of dihydrodibenzoazocine 4, bromination and its application subsequent elimination (Scheme 1). The key intermediate 4 was envisaged to a singlebe prepared from Z-moleculeolefin 5 in two steps, which in turn was prepared from 2-counting immunoassayethynylaniline (7) and sub- stituted iodobenzyl alcohol derivatives 6. A microfluidic 6 device is used to generate and manipulate <10 fL droplets at rates different strategy was envisaged for DIBAC analogue 3f. Synthesis of up to 1.3 × 10 per second, about 2 orders of magnitude faster than has previously been reported. The femtodroplets produced with this device can be used to encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables the fluorescent product of a single enzyme molecule to be detected within 10 min of on-chip incubation. Our prototype system is validated by detection of a biomarker for prostate cancer in buffer, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte molecules.cyclooctene intermediates

End User Agreement. This publication is distributed under the terms of Article 25fa of the Dutch Copyright Act (Auteurswet) with explicit consent by the author. Dutch law entitles the maker of a short scientific work funded either wholly or partially by Dutch public funds to make that work publicly available for no consideration following a reasonable period of time after the work was first published, provided that clear reference is made to the source of the first publication of the work. This publication is distributed under The Association of Universities in the Netherlands (VSNU) ‘Article 25fa implementation’ pilot project. In this pilot research outputs of researchers employed by Dutch Universities that comply with the legal requirements of Article 25fa of the Dutch Copyright Act are distributed online and free of cost or other barriers in institutional repositories. Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication. You are permitted to download and use the publication for personal purposes. All rights remain with the author(s) and/or copyrights owner(s) of this work. Any use of the publication other than authorised under this licence or copyright law is prohibited. If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please contact the Library through email: ▇▇▇▇▇▇▇▇▇@▇▇▇.▇▇.▇▇, or send a letter to: University Library Radboud University Copyright Information Point PO Box 9100 6500 HA Nijmegen You will be contacted as soon as possible. Ultrarapid Generation of Femtoliter Microfluidic Droplets for Single- Molecule-Counting Immunoassays Jung-uk Shim,†,§,* Actin and serum response factor transduce physical cues from the microenvironment to regulate epidermal stem cell fate decisions ▇▇▇▇ ▇. ▇▇▇▇▇▇▇▇▇, ▇▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇,† ▇, ▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇▇,‡ ▇▇, ▇▇▇▇▇ ▇. ▇. ▇-▇▇▇ ▇▇▇▇,†,^ , ▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇, ▇▇,† ▇▇▇▇▇ ▇.▇. Huck2,4 and ▇▇▇▇▇ ▇. ▇▇▇▇† †Department ▇,3,5 Epidermal homeostasis depends on a balance between stem cell renewal and differentiation and is regulated by extrinsic signals from the extracellular matrix (ECM)1,2. A powerful approach to analysing the pathways involved is to engineer single-cell microenvironments in which individual variables are precisely and quantitatively controlled3–5. Here, we employ micropatterned surfaces to identify the signalling pathways by which restricted ECM contact triggers human epidermal stem cells to initiate terminal differentiation. On small (20 μm diameter) circular islands, keratinocytes remained rounded, and differentiated at higher frequency than cells that could spread on large (50 μm diameter) islands. Differentiation differentiation, whereas overexpression of MAL stimulated SRF activity and involucrin expression. SRF target genes FOS and JUNB were also required for differentiation: c-Fos mediated serum responsiveness, whereas JunB was regulated by actin and MAL. Our findings demonstrate how biophysical cues Human interfollicular epidermis comprises multiple layers of kerati- nocytes. Proliferation takes place in the basal cell layer, attached to the underlying extracellular matrix known as the basement membrane. Cells that leave the basal layer undergo a programme of terminal differentia- tion as they move towards the tissue surface. Differentiated cells are con- tinually shed from the epidermis and replaced through proliferation of stem cells in the basal layer. Several human epidermal stem cell markers have been described, including elevated levels of β1 (ref. 2) and α6 (ref. 6) integrins, and expression of Delta-like1 (Dll1; ref. 7), Lrig1 (ref. 8) and p63 (ref. 9). Terminal differentiation is characterized by withdrawal from the cell cycle and expression of a number of proteins, including involu- crin, periplakin and transglutaminase 1, which subsequently become incorporated into the epidermal cornified envelope10,11. To investigate the role of cell–ECM interactions in regulating the dif- ferentiation of human epidermal stem cells, micropatterned, polymer brush substrates were developed by micro-contact printing and surface- initiated polymerization12,13 (Fig. 1a). We constructed circular islands (20–50 μm diameter) composed of type I collagen, surrounded by a background that was resistant to protein adsorption (Fig. 1a, b). When keratinocytes were seeded onto these substrates, cells adhered specifi- cally to the collagen islands within 1 h and displayed increasingly spread morphologies with increasing island size (Fig. 1b). The adherent cells were negative for involucrin and positive for Ki67 (Fig. 1c–e). After 24 h, the number of involucrin-positive cells increased significantly on the smallest islands, and there was an inverse correlation between the number of differentiated cells and adhesive area (Fig. 1c, d and Supplementary Information, Fig. S1a, b). There was a decrease in Ki67 expression on all island sizes after 24 h, with the greatest decrease found on the smallest islands (Fig. 1c, e). S phase cells were not present on 20 μm islands, but were found on larger islands (Supplementary Information, Fig. S1c). Blocking DNA synthesis did not alter the effect of island size on involucrin expression (Supplementary Information, Fig. S1d), indicating that differentiation is regulated independently of cell-cycle exit3,14. These results demonstrate that engineered substrates can modulate the adhesive interactions of single human keratinocytes and induce terminal differentiation by restricting adhesive area. To examine whether micropatterned substrates selectively cap- tured stem cells, basal cells (low forward scatter and side scatter) were flow-sorted according to surface β1 integrin expression2 (Fig. 2a). Keratinocytes with low β1 integrin expression did not adhere to the 1Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR, U.K. 2Melville Laboratory for Polymer Synthesis, Department of Chemistry, University of Cambridge, Lensfield RoadCambridge, CB2 1EW, U.K. 3CRUK Cambridge Research Institute, ▇▇ ▇▇ ▇▇▇▇▇ Centre, ▇▇▇▇▇▇▇▇ Way, Cambridge, U.K.CB2 0RE, CB2 1EW and ‡Department of Biochemistry, University of Cambridge, Cambridge, U.K., CB2 1GA. §Present address: Biomedical Engineering, University of Glasgow, Glasgow, UK, G12 8LT. ^Present address: Radboud U.K. 4Radboud University Nijmegen, Institute for Molecules and ▇▇▇▇▇▇▇▇▇Materials, ▇▇▇▇▇▇▇▇▇▇▇▇▇▇ ▇▇▇, ▇▇▇▇ ▇▇ ▇▇▇▇▇▇▇▇, ▇▇▇ ▇▇▇▇▇▇▇▇▇▇▇. ARTICLE ABSTRACT We report 5Correspondence should be addressed to F.M.W. (e-mail: ▇▇▇▇▇.▇▇▇▇@▇▇▇▇▇▇.▇▇▇.▇▇). Received 23 February 2010; accepted 13 May 2010; published online 27 June 2010; DOI: 10.1038/ncb Br O O (CH2)11 S a microfluidic dropletBromoisobutyrate b 20 µm 30 µm 50 µm Gold-based approach enabling coated coverslip Micro-contact printing with patterned silicone stamp Patterned monolayer of initiator Oligo(ethylene glycol) methacrylate (Average Mr = 300) O n O x O n O O O O Br O O (CH2)11 S CuCl/CuBr2 Bipyridine (CH2)11 S Adsorption of ECM on islands Involucrin positive (percentage) 60 * Involucrin Ki67 DNA 1 h 50 24 h 30 20 10 0 0 500 1000 1500 2000 2500 Adhesive area (µm2) 24 h 100 Ki67 positive (percentage) 80 60 40 20 0 * Adhesive area (µm2) Figure 1 Regulation of keratinocyte shape and differentiation on micropatterned substrates. (a) Overview of the measurement micropatterning strategy. (b) Immunofluorescence microscopy images of chemical reactions type I collagen (top) and phase-contrast microscopy images of individual enzyme molecules primary human keratinocytes (bottom) on 20, 30 and its application to a single-molecule-counting immunoassay. A microfluidic 6 device is used to generate and manipulate <10 fL droplets at rates of up to 1.3 × 10 per second, about 2 orders of magnitude faster than has previously been reported. The femtodroplets produced with this device can be used to encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables the fluorescent product of a single enzyme molecule to be detected within 10 min of on-chip incubation. Our prototype system is validated by detection of a biomarker for prostate cancer in buffer, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte molecules.50 μm

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Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication. You are permitted to download and use the publication for personal purposes. All rights remain with the author(s) and/or copyrights owner(s) of this work. Any use of the publication other than authorised under this licence or copyright law is prohibited. If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please contact the Library through email: ▇▇▇▇▇▇▇▇▇@▇▇▇.▇▇.▇▇, or send a letter to: University Library Radboud University Copyright Information Point PO Box 9100 6500 HA Nijmegen You will be contacted as soon as possible. Ultrarapid Generation of Femtoliter Microfluidic Droplets Lab on a Chip TECHNICAL INNOVATION Cite this: Lab Chip, 2016, 16, 65 Received 14th July 2015, Accepted 24th November 2015 DOI: 10.1039/c5lc00823a ▇▇▇.▇▇▇.▇▇▇/▇▇▇ Biocompatible fluorinated polyglycerols for Single- Molecule-Counting Immunoassays Jung-uk Shim,†,§droplet microfluidics as an alternative to PEG- based copolymer surfactants† ▇▇▇▇ ▇▇▇▇▇▇,* a ▇▇▇▇▇▇ ▇. ▇▇▇▇▇,‡b ▇▇▇▇▇ ▇▇▇▇▇▇▇▇,† a ▇▇▇▇▇ ▇▇▇▇▇▇▇,c ▇▇▇▇▇ ▇. ▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇,† ▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇▇,‡ c ▇▇▇▇▇▇▇ ▇. ▇. ▇▇▇▇,†,^ ▇ and ▇▇▇▇▇▇ ▇▇▇▇*a In droplet-based microfluidics, non-ionic, high-molecular weight surfactants are required to stabilize drop- let interfaces. One of the most common structures that imparts stability as well as biocompatibility to water-in-oil droplets is a triblock copolymer surfactant composed of perfluoropolyether (PFPE) and poly- ethylene glycol (PEG) blocks. However, the fast growing applications of microdroplets in biology would benefit from a larger choice of specialized surfactants. PEG as a hydrophilic moiety, however, is a very lim- ited tool in surfactant modification as one can only vary the molecular weight and chain-end functionalization. In contrast, linear polyglycerol offers further side-chain functionalization to create cus- ▇▇▇▇▇,† -tailored, biocompatible droplet interfaces. Herein, we describe the synthesis and ▇▇▇▇▇ ▇▇▇▇▇† †Department characterization of Chemistrypolyglycerol-based triblock surfactants with tailored side-chain composition, University and exemplify their applica- tion in cell encapsulation and in vitro gene expression studies in droplet-based microfluidics. Introduction Droplet-based microfluidics has attracted much attention since the first monodisperse droplets were produced inside microfluidic polyurethane chips in 2001.1 This technology is based on production of Cambridgepico- to nano-liter volume droplets at high throughput rates (typically 1–10 kHz) and their subse- quent manipulation in an automated or semi-automated manner. The small droplet size greatly reduces reagent vol- umes and provides a powerful tool for single gene, Lensfield Roadcell, Cambridgeor organism isolation and analysis.2–7 To make this technology applicable, U.K.cross-contamination between droplets should be minimized or eliminated completely. The use of fluorocarbon oils as a continuous phase is advantageous as it provides hydrophobic and lipophobic properties8 thus significantly reducing the solubility of biochemical compounds and their diffusion between flowing droplets. Furthermore, CB2 1EW perfluorinated fluids exhibit high gas solubility, which is important for cell sur- vival in droplets,9 and ‡Department they cause less swelling of Biochemistrymicro- channels in PDMS-based microfluidic devices than hydrocar- bon oils.10 Since preventing droplet coalescence is crucial for any droplet-based application, University surfactants are used to provide droplet stability.11 The degree to which the surfactant orga- nizes at the interface between the immiscible water and fluorous oil phases, such as in a water-in-oil (W/O) emulsion, can be quantified by its reduction of Cambridgethe interfacial tension at the oil water interface, Cambridgeγcmc, U.K.where CMC is the critical micel- lar concentration.12 Efficient surfactants in droplet micro- fluidics reduce γcmc of a fluorous oil/water mixture below 20 mN m−1.13 In addition to the surfactant's interfacial activity, CB2 1GAsteric Published on 24 November 2015. §Present address: Biomedical Engineering, University of Glasgow, Glasgow, UK, G12 8LTDownloaded on 23/10/2017 15:00:01. ^Present address: repulsion prevents droplet coalescence.11 Oligomeric b Radboud University NijmegenUniversity, Institute for Molecules and ▇▇▇▇▇▇▇▇▇Materials (IMM), Physical- Organic Chemistry, ▇▇▇▇▇▇▇▇▇▇▇▇▇▇ ▇▇▇, ▇▇▇▇ ▇▇ ▇▇▇▇▇▇▇▇, ▇▇▇ ▇▇▇▇▇▇▇▇▇▇▇ c School of Engineering and Applied Sciences, Department of Physics, Harvard University, ▇▇ ▇▇▇▇▇▇ ▇▇▇▇▇▇, ▇▇▇▇▇▇▇▇▇, ▇▇ ▇▇▇▇▇, ▇▇▇ † Electronic supplementary information (ESI) available: Experimental details, used materials, surfactants synthesis and fabrication of microfluidic devices. See DOI: 10.1039/c5lc00823a ‡ Current address: ▇▇. ARTICLE ABSTRACT We report ▇▇▇▇▇▇ ▇▇▇▇▇▇, Department of Nanostructured Materials and Leibniz Research Cluster (LRC), Leibniz-Institut für Polymerforschung Dres- den e. V., 01069 Dresden, Germany. E-mail: ▇▇▇▇▇▇@▇▇▇▇▇.▇▇ perfluoropolyethers (PFPEs), when used as large hydrophobic building blocks of copolymer surfactants, were found to be soluble in fluorocarbon oils and sufficiently large to provide good steric stabilization by forming a microfluidic dropletdense PFPE layer on the outer droplet surface.14 PolyIJperfluoropropylene glycol)-carboxylates, commercially available as “Krytox” by DuPont®, have emerged as a ▇▇▇▇- dard PFPE moiety. However, the charged carboxylate group of Krytox interacts with oppositely charged biomolecules, which causes the encapsulated biomacromolecules to lose their activity and agglomerate at the droplet interface.15 Consequently, the carboxylic head group has to be substituted with different nonpolar hydrophilic head groups such as the ammonium salt of carboxy-based approach enabling PFPE, poly-L-lysine, dimorpholinophosphate, and polyIJethylene glycol) (PEG).16 The ammonium salt and poly-L-lysine causes cell lysis, while the measurement latter two performed well. The block copolymer of chemical reactions PEG and perfluoropolyether was further optimized by Holtze et al. who produced a number of individual enzyme molecules PEG–PFPE2 surfactants from PEG and its application to a singlePFPE chains of different lengths. The combination of 600 g mol−1 PEG with 6000 g mol−1 PFPE performed best in droplet formation and long-moleculeterm droplet stability.17,18 The reduced protein adsorption of this non-counting immunoassay. A microfluidic 6 device is ionic surfac- tant with molecular weights usually ranging from 2000–13 000 g mol−1 has been used to generate screen enzymes,19,20 mamma- lian cells,6,21–23 bacteria,24 and manipulate <10 fL droplets at rates viruses in microdroplets.25 Its biocompatibility is attributed to the PEG-block forming a bio- logically inert interior surface in the water droplets. PEG–PFPE2 triblock copolymers appear to be the most applied surfactants in fluorous droplet microfluidics nowa- days. They are commercially available26 or synthesized from Krytox and amino-functionalized polyethers. However, the PEG block in between the PFPEs as a hydro- philic moiety offers very limited opportunity for further chemical modification. While custom-made surfactant mole- cules have been used, for instance, to create droplet inter- faces for controlled immobilization27 and to promote chemi- cal reactions28 or protein crystallization by functional hydrophilic moieties, one can only vary the molecular weight of up PEG in PEG–PFPE2.29 Polyglycerols represent a class of biocompatible and multihydroxy-functional polymers that may be considered as a multifunctional analog of PEG.30,31 Recently, we introduced a novel class of biocompatible surface coating32,33 as an alternative to 1.3 × 10 per secondPEG. Linear poly- glycerol (LPG) derivatives form resistant layers to inhibit the uncontrolled adsorption of fibrinogen, about 2 orders pepsin, albumin, and lysozyme and showed even less adsorption of magnitude faster human plasma protein than has previously been reporteda PEG-modified surface. The femtodroplets produced with this device can be used Additionally, cell adhe- sion experiments on linear polyglycerol LPG(OH) and polyIJmethyl glycerol) LPG(OMe) modified surfaces showed a similar cell resistance to encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables the fluorescent product that of a single enzyme molecule to be detected within 10 min of onPEG-chip incubationmodified surface. Our prototype system is validated by detection of Therefore we employed LPG(OMe) and LPG(OH) as a biomarker for prostate cancer building block in buffer, down to a concentration of 46 fM. This work demonstrates a highly flexible triblock copolymer and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte molecules.introduced LPG-

End User Agreement. This publication is distributed under the terms of Article 25fa of the Dutch Copyright Act (Auteurswet) with explicit consent by the author. Dutch law entitles the maker of a short scientific work funded either wholly or partially by Dutch public funds to make that work publicly available for no consideration following a reasonable period of time after the work was first published, provided that clear reference is made to the source of the first publication of the work. This publication is distributed under The Association of Universities in the Netherlands (VSNU) ‘Article 25fa implementation’ pilot project. In this pilot research outputs of researchers employed by Dutch Universities that comply with the legal requirements of Article 25fa of the Dutch Copyright Act are distributed online and free of cost or other barriers in institutional repositories. Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication. You are permitted to download and use the publication for personal purposes. All rights remain with the author(s) and/or copyrights owner(s) of this work. Any use of the publication other than authorised under this licence or copyright law is prohibited. If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please contact the Library through email: ▇▇▇▇▇▇▇▇▇@▇▇▇.▇▇.▇▇, or send a letter to: University Library Radboud University Copyright Information Point PO Box 9100 6500 HA Nijmegen You will be contacted as soon as possible. Ultrarapid Generation of Femtoliter Microfluidic Droplets for Single- Molecule-Counting Immunoassays Jung-uk Shim,†,§,* ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇,† ▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇▇,‡ ▇▇▇(✉), ▇▇▇▇ ▇. ▇. ▇▇▇▇,†,^ ▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇,† and ▇▇▇▇▇ ▇▇▇▇▇† †Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, U.K., CB2 1EW and ‡Department of Biochemistry, University of Cambridge, Cambridge, U.K., CB2 1GA. §Present address: Biomedical Engineering, University of Glasgow, Glasgow, UK, G12 8LT. ^Present address: Radboud University Nijmegen, Institute for Molecules and ▇▇▇▇▇▇▇▇▇, ▇▇▇▇▇▇ ▇▇▇▇´e1, and ▇▇▇▇▇▇▇▇▇ ▇▇▇, ▇▇▇▇ ▇▇ ▇▇▇▇▇▇▇▇1 Department of Computer Science, ▇▇▇ University College London, London, UK ▇▇▇▇▇▇▇▇▇▇▇▇▇▇@▇▇▇▇▇.▇▇▇ 2 Institute for Computing and Information Sciences, Radboud University Nijmegen, Nijmegen, The Netherlands In this paper, we show how to generalize a known active automata learning algorithm—▇▇▇▇▇▇▇’s L*—to Reo automata. ARTICLE ABSTRACT We report use recent cat- egorical insights on ▇▇▇▇▇▇▇’s original algorithm to devise this general- ization, which turns out to require a microfluidic droplet-based approach enabling the measurement change of chemical reactions of individual enzyme molecules and its application to a single-molecule-counting immunoassay. A microfluidic 6 device is used to generate and manipulate <10 fL droplets at rates of up to 1.3 × 10 per second, about 2 orders of magnitude faster than has previously been reported. The femtodroplets produced with this device can be used to encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables the fluorescent product of a single enzyme molecule to be detected within 10 min of on-chip incubation. Our prototype system is validated by detection of a biomarker for prostate cancer in buffer, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte moleculesbase category.

End User Agreement. This publication is distributed under the terms of Article 25fa of the Dutch Copyright Act (Auteurswet) with explicit consent by the author. Dutch law entitles the maker of a short scientific work funded either wholly or partially by Dutch public funds to make that work publicly available for no consideration following a reasonable period of time after the work was first published, provided that clear reference is made to the source of the first publication of the work. This publication is distributed under The Association of Universities in the Netherlands (VSNU) ‘Article 25fa implementation’ pilot project. In this pilot research outputs of researchers employed by Dutch Universities that comply with the legal requirements of Article 25fa of the Dutch Copyright Act are distributed online and free of cost or other barriers in institutional repositories. Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication. You are permitted to download and use the publication for personal purposes. All rights remain with the author(s) and/or copyrights owner(s) of this work. Any use of the publication other than authorised under this licence or copyright law is prohibited. If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please contact the Library through email: ▇▇▇▇▇▇▇▇▇@▇▇▇.▇▇.▇▇, or send a letter to: University Library Radboud University Copyright Information Point PO Box 9100 6500 HA Nijmegen You will be contacted as soon as possible. Ultrarapid Generation Original article Correction of Femtoliter Microfluidic Droplets for Single- Molecule-Counting Immunoassays Jung-uk Shim,†,§,* an image size difference between positron emission tomography (PET) and computed tomography (CT) improves image fusion of dedicated PET and CT ▇▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇▇▇▇,† ▇, ▇▇▇▇ ▇. ▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇,† ▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇▇,‡ , ▇▇▇▇▇▇▇ ▇. ▇. ▇▇▇▇,†,^ ▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇,† and ▇▇▇▇▇ ▇▇▇▇▇† †Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, U.K., CB2 1EW and ‡Department of Biochemistry, University of Cambridge, Cambridge, U.K., CB2 1GA. §Present address: Biomedical Engineering, University of Glasgow, Glasgow, UK, G12 8LT. ^Present address: Radboud University Nijmegen, Institute for Molecules and .X. ▇▇▇▇▇▇▇▇▇, ▇▇▇▇▇▇▇▇ ▇.A.M. ▇▇▇▇▇▇▇ ▇▇▇, ▇▇▇▇ ▇▇▇▇ ▇▇▇▇▇▇▇▇, ▇▇▇▇▇ ▇.▇. ▇▇▇▇▇▇▇▇▇ and ▇▇▇ ▇.▇. Oyena Subsequently, 13 patients with cancer in the head/neck area underwent both CT and [18F]fluorodeoxyglucose PET significantly smaller. Image fusion using original images demonstrated an average registration error of 2.7 mm. This error was decreased to 1.4 mm after size correction of the PET images, a significant improvement of 48% (P < 0.001). CT before performing high-accuracy rigid-body image fusion. Nucl Med Commun 27:515–519 ◯c 2006 Lippincott in a custom-made mask for external beam radiotherapy, with multimodality markers for positional reference. The image size of PET relative to CT was determined by evaluating the distances between the markers in multiple directions in both scans. Rigid-body image fusion was performed using the markers as landmarks, with and without correction of the calculated image size difference. image size of 2.0% in the transverse plane and 0.8% along the longitudinal axis, the PET images being ▇▇▇▇▇▇▇▇ & ▇▇▇▇▇▇▇. ARTICLE ABSTRACT We report Nuclear Medicine Communications 2006, 27:515–519 Keywords: image fusion, positron emission tomography (PET), positron emission tomography /computed tomography (PET/CT) Departments of aNuclear Medicine, bRadiotherapy and cRadiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands. Correspondence to ▇▇▇▇▇▇ ▇. ▇▇▇▇▇ MD, Radboud University Nijmegen Medical Centre, Department of Nuclear Medicine (565), Postbox 9101, 6500 HB Nijmegen, The Netherlands. Tel: + ▇▇-▇▇-▇▇▇▇▇▇▇; fax: + ▇▇-▇▇-▇▇▇▇▇▇▇; e-mail: ▇.▇▇▇▇▇@▇▇▇▇▇▇.▇▇▇▇.▇▇ Received 4 August 2005 Accepted 7 March 2006 Introduction Image fusion of positron emission tomography (PET) and computed tomography (CT) can improve the diagnostic value and diagnostic accuracy in oncological imaging of the head and neck area [1–3]. Image fusion may also be applied to incorporate functional information in external beam radiation treatment [4,5]. When performing image fusion, a microfluidic droplethigh accuracy in anatomical registration of the images is required, because incorrect registration may induce diagnostic errors, such as erroneous localization or characterization of the lesions [6]. In particular, when using image fusion for the definition of target volumes in intensity-based approach enabling modulated radiation therapy (IMRT), the measurement required accuracy is high as the error in dose delivery is in the range of chemical reactions only 2–3 mm [7]. Errors in image registration may influence the outcome of individual enzyme molecules therapy and its application 0143-3636 ◯c 2006 Lippincott ▇▇▇▇▇▇▇▇ & ▇▇▇▇▇▇▇ the level of complications of external beam radiation therapy. For software image fusion of dedicated PET and CT, an accuracy of better than 2 mm has been demonstrated using phantoms [8]. The accuracy that can be achieved in patients will probably be lower as a result of complicating factors, such as small positioning errors, motion artefacts, the time interval between scans and limited compar- ability between scans due to a single-molecule-counting immunoassayvisualization of different structures and processes on PET and CT. Furthermore, differences may exist in image size. In this article, real image size is defined as the discrepancy between the measured size of an object on an image and the true size of that object. Furthermore, relative differences in image size may occur between scanning modalities. The image 516 Nuclear Medicine Communications 2006, Vol 27 No 6 Fig. 1 Software image fusion of positron emission tomography (PET) and computed tomography (CT). A microfluidic 6 device slice through the head is used to generate and manipulate <10 fL droplets shown at rates the level of up to 1.3 × 10 per second, about 2 orders two multimodality fiducial markers positioned in front of magnitude faster than has previously been reported. The femtodroplets produced with this device can be used to encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables the fluorescent product of a single enzyme molecule to be detected within 10 min of on-chip incubation. Our prototype system is validated by detection of a biomarker for prostate cancer in buffer, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte moleculesears.

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Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication. You are permitted to download and use the publication for personal purposes. All rights remain with the author(s) and/or copyrights owner(s) of this work. Any use of the publication other than authorised under this licence or copyright law is prohibited. If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please contact the Library through email: ▇▇▇▇▇▇▇▇▇@▇▇▇.▇▇.▇▇, or send a letter to: University Library Radboud University Copyright Information Point PO Box 9100 6500 HA Nijmegen You will be contacted as soon as possible. Ultrarapid Generation of Femtoliter Microfluidic Droplets for Single- Molecule-Counting Immunoassays Jung-uk Shim,†,§,* This article was downloaded by: [Radboud Universiteit Nijmegen] On: 04 November 2013, At: 06:10 Publisher: Routledge Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, ▇▇▇-▇▇ ▇. ▇▇▇▇▇▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇,† ▇▇▇▇▇▇▇ ▇▇▇▇▇▇, ▇▇▇▇,‡ ▇▇ ▇▇▇ ▇▇▇, ▇▇ Publication details, including instructions for authors and subscription information: ▇▇▇▇://▇▇▇.▇▇▇▇▇▇▇▇▇▇▇.▇▇▇/loi/hijt20 ▇▇▇▇▇ ▇. ▇▇▇▇▇▇ a , ▇▇▇▇▇ ▇. ▇. ▇▇▇▇,†,^ ▇ b & ▇▇▇▇▇ ▇▇▇▇▇▇▇ b a Institute for Management Research , Radboud University Nijmegen , The Netherlands b Department of Methodology and Statistics , Tilburg University , The Netherlands Published online: 31 May 2013. To cite this article: ▇▇▇,† and ▇▇ ▇. ▇▇▇▇▇▇ , ▇▇▇▇▇ ▇. ▇. ▇▇▇▇▇ & ▇▇▇▇▇ ▇▇▇▇▇† †Department ▇▇ (2013) On the Shortcomings of ChemistryShortened Tests: A Literature Review, University International Journal of CambridgeTesting, Lensfield Road13:3, Cambridge223-248, U.K., CB2 1EW and ‡Department of Biochemistry, University of Cambridge, Cambridge, U.K., CB2 1GA. §Present addressDOI: Biomedical Engineering, University of Glasgow, Glasgow, UK, G12 8LT. ^Present address10.1080/15305058.2012.703734 To link to this article: Radboud University Nijmegen, Institute for Molecules and ▇▇▇▇://▇▇.▇▇▇.▇▇▇/10.1080/15305058.2012.703734 PLEASE SCROLL DOWN FOR ARTICLE ▇▇▇▇▇▇ & ▇▇▇▇▇▇▇ makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, ▇▇▇▇▇▇ & ▇▇▇▇▇▇▇, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. 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Terms & Conditions of access and use can be found at ▇▇▇▇://▇▇▇.▇▇▇▇▇▇▇▇▇▇▇▇▇▇ .▇▇▇/page/terms-and-conditions Downloaded by [Radboud Universiteit Nijmegen] at 06:10 04 November 2013 ⃝ International Journal of Testing, 13: 223–248, 2013 Copyright C ▇▇▇▇▇▇ ▇▇ & ▇▇▇▇▇▇▇▇▇ Group, ▇▇▇ ▇▇▇▇▇▇▇▇▇▇▇. ARTICLE ABSTRACT We report a microfluidic dropletLLC ISSN: 1530-based approach enabling the measurement of chemical reactions of individual enzyme molecules and its application to a single5058 print / 1532-molecule-counting immunoassay. A microfluidic 6 device is used to generate and manipulate <10 fL droplets 7574 online DOI: 10.1080/15305058.2012.703734 Downloaded by [Radboud Universiteit Nijmegen] at rates of up to 1.3 × 10 per second, about 2 orders of magnitude faster than has previously been reported. The femtodroplets produced with this device can be used to encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables the fluorescent product of a single enzyme molecule to be detected within 10 min of on-chip incubation. Our prototype system is validated by detection of a biomarker for prostate cancer in buffer, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte molecules.06:10 04 November 2013

End User Agreement. This publication is distributed under the terms of Article 25fa of the Dutch Copyright Act (Auteurswet) with explicit consent by the author. Dutch law entitles the maker of a short scientific work funded either wholly or partially by Dutch public funds to make that work publicly available for no consideration following a reasonable period of time after the work was first published, provided that clear reference is made to the source of the first publication of the work. This publication is distributed under The Association of Universities in the Netherlands (VSNU) ‘Article 25fa implementation’ pilot project. In this pilot research outputs of researchers employed by Dutch Universities that comply with the legal requirements of Article 25fa of the Dutch Copyright Act are distributed online and free of cost or other barriers in institutional repositories. Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication. You are permitted to download and use the publication for personal purposes. All rights remain with the author(s) and/or copyrights owner(s) of this work. Any use of the publication other than authorised under this licence or copyright law is prohibited. If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please contact the Library through email: ▇▇▇▇▇▇▇▇▇@▇▇▇.▇▇.▇▇, or send a letter to: University Library Radboud University Copyright Information Point PO Box 9100 6500 HA Nijmegen You will be contacted as soon as possible. Ultrarapid Generation Original article The number of Femtoliter Microfluidic Droplets for Single- Molecule-Counting Immunoassays Jung-uk Shim,†,§,* ▇▇▇▇multinucleated trophoblastic giant cells in the basal decidua is decreased in retained placenta ▇ ▇. ▇▇▇▇▇▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇,† ▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇▇,‡ 1 I ▇▇▇▇▇▇▇,2 ▇ ▇ ▇ ▇ ▇. ▇ ▇. ▇▇▇▇,†,^ 1 ▇ ▇ ▇▇▇▇▇▇▇▇▇,1 ▇ ▇▇▇ ▇▇▇ ▇▇▇▇,3 J Bulten3 1 Department of Obstetrics and Gynaecology, Erasmus Medical Centre, Rotterdam, The Netherlands; 2 Department of Blood Transfusion and Transplantation Immunology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; 3 Department of Pathology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands Correspondence to: ▇▇▇▇▇▇ ▇ ▇▇▇ ▇▇▇▇▇▇▇▇▇,† and ▇▇▇▇▇ ▇▇▇▇▇† †Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, U.K., CB2 1EW and ‡Department of Biochemistry, University of Cambridge, Cambridge, U.K., CB2 1GA. §Present address: Biomedical Engineering, University of Glasgow, Glasgow, UK, G12 8LT. ^Present address: Radboud University Nijmegen, Institute for Molecules and ▇▇▇▇▇▇▇▇▇, Erasmus Medical Centre, PO Box 2040, 3000 CA Rotterdam, The Netherlands; ▇.▇▇▇▇▇▇▇▇▇▇▇▇▇▇ ▇▇▇, ▇▇▇▇ ▇▇ @ ▇▇▇▇▇▇▇▇, .▇▇▇ ▇▇▇▇▇▇▇▇▇▇▇. ARTICLE Accepted 5 May 2009 ABSTRACT We report Aims: Retained placenta (RP) is a microfluidic droplet-based approach enabling the measurement major cause of chemical reactions of individual enzyme molecules and its application to a single-molecule-counting immunoassay. A microfluidic 6 device is used to generate and manipulate <10 fL droplets at rates of up to 1.3 × 10 per second, about 2 orders of magnitude faster than has previously been reportedobstetric haemorrhage. The femtodroplets produced with this device can be used aim of the study was to encapsulate single biomolecular complexes tagged with obtain a reporter enzyme; their small volume enables better understanding of the fluorescent product of a single enzyme molecule mechanisms that cause some placentas to be detected within 10 min of on-chip incubation. Our prototype system is validated by detection of a biomarker for prostate cancer in bufferbecome retained, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte moleculeswhile most are not.

End User Agreement. This publication is distributed under the terms of Article 25fa of the Dutch Copyright Act (Auteurswet) with explicit consent by the author. Dutch law entitles the maker of a short scientific work funded either wholly or partially by Dutch public funds to make that work publicly available for no consideration following a reasonable period of time after the work was first published, provided that clear reference is made to the source of the first publication of the work. This publication is distributed under The Association of Universities in the Netherlands (VSNU) ‘Article 25fa implementation’ pilot project. In this pilot research outputs of researchers employed by Dutch Universities that comply with the legal requirements of Article 25fa of the Dutch Copyright Act are distributed online and free of cost or other barriers in institutional repositories. Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication. You are permitted to download and use the publication for personal purposes. All rights remain with the author(s) and/or copyrights owner(s) of this work. Any use of the publication other than authorised under this licence or copyright law is prohibited. If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please contact the Library through email: ▇▇▇▇▇▇▇▇▇@▇▇▇.▇▇.▇▇, or send a letter to: University Library Radboud University Copyright Information Point PO Box 9100 6500 HA Nijmegen You will be contacted as soon as possible. Ultrarapid Generation of Femtoliter Microfluidic Droplets for Single- Molecule-Counting Immunoassays Jung-uk Shim,†,§,* ▇▇▇▇▇ ▇. .▇▇▇.▇▇▇▇▇▇▇,† ▇/▇▇▇▇ ▇. ▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇,† ▇Letter Panchromatic “Dye-Doped” Polymer Solar Cells: From Femtosecond Energy Relays to Enhanced Photo-Response ▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇,†,‡ ▇. ▇▇▇ ▇▇▇▇▇▇▇ ▇▇▇▇▇,‡ ▇▇▇▇▇▇▇▇▇▇ ▇▇▇▇▇▇,§ ▇▇▇▇▇▇▇ ▇▇▇▇,∥ ▇▇▇▇▇▇▇ ▇. ▇. ▇▇▇▇,†,^ ∥,⊥ ▇▇▇▇▇▇▇ ▇. ▇. ▇▇▇▇▇▇▇,‡,§ ▇▇▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇,‡ ▇▇▇▇▇▇ ▇▇▇▇▇▇▇,§ ▇▇▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇,*,‡ and ▇▇▇▇▇ ▇. ▇▇▇▇▇▇*,† †Clarendon Laboratory, Department of Physics, Oxford University, Parks Road, Oxford, OX13PU, U.K. ‡Center for Nano Science and Technology @Polimi, Istituto Italiano di Tecnologia, ▇▇▇ ▇▇▇▇▇▇▇▇ ▇▇▇▇▇† †Department ▇▇ 70/3, 20133 Milano, Italy §IFN-CNR, Dipartimento di Fisica, Politecnico di Milano, ▇▇▇▇▇▇ ▇. ▇▇ ▇▇▇▇▇, 32, 20133 Milano, Italy ∥Melvile Laboratory of Polymer Synthesis, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, U.K.CB2 1TN, CB2 1EW and ‡Department of Biochemistry, University of Cambridge, Cambridge, U.K., CB2 1GA. §Present address: Biomedical Engineering, University of Glasgow, Glasgow, UK, G12 8LT. ^Present address: Radboud University Nijmegen, Institute U.K. ⊥Institute for Molecules and ▇Materials, Radboud University ▇▇▇▇▇▇▇▇, ▇▇▇▇▇▇▇▇▇▇▇▇▇▇ ▇▇▇, ▇▇▇▇ ▇▇ ▇▇▇▇▇▇▇▇, ▇▇▇ ▇▇▇▇▇▇▇▇▇▇▇▇ *S Supporting Information ABSTRACT: There has been phenomenal effort synthesizing new low-band gap polymer hole-conductors which absorb into the near-infrared (NIR), leading to >10% efficient all-organic solar cells. ARTICLE ABSTRACT We report However, organic light absorbers have relatively narrow bandwidths, making it challenging to obtain panchromatic absorption in a microfluidic dropletsingle organic semiconductor. Here, we demonstrate that (poly[2,6-(4,4-bis-(2-ethylhexyl)-4H- cyclopenta[2,1-b;3,4-b0]dithiophene)-alt-4,7-(2,1,3-benzothiadia-zole)] (PCPDTBT) can be “photo-sensitized” across the whole visible spectrum by “doping” with a visible absorbing dye, the (2,2,7,7-tetrakis(3-hexyl-5-(7-(4-hexylthiophen-2-yl)benzo[c][1,2,5]- thiadiazol-4-yl)thiophen-2-yl)-9,9-spirobifluorene) (spiro-TBT). Through a comprehen- sive sub-12 femtosecond−nanosecond spectroscopic study, we demonstrate that extremely efficient and fast energy transfer occurs from the photoexcited spiro-TBT to the PCPDTBT, and ultrafast charge injection happens when the system is interfaced with ZnO as a prototypal electron-acceptor compound. The visible photosensitization can be effectively exploited and gives panchromatic photoresponse in prototype polymer/oxide bilayer photovoltaic diodes. This concept can be successfully adopted for tuning and optimizing the light absorption and photoresponse in a broad range of polymeric and hybrid solar cells. SECTION: Energy Conversion and Storage; Energy and Charge Transport S emiconducting polymers are attracting a growing interest as active materials for clean power generation: they offer excellent light harvesting capabilities and good charge carrier mobility.1−4 However, in contrast to inorganic absorbers, the energy bands are relatively narrow, and the low band gap polymers tend to incompletely absorb light in the visible region of the spectrum. Although this opens aesthetic possibilities for applications such as building integrated photovoltaics, it limits the overall solar light absorbed and hence the efficiency of a polymer-based approach enabling solar cell.3 In addition, in contrast to inorganic absorbers, a heterojunction is required between the measurement light absorbing polymer and an electron acceptor in order to ionize the photoinduced excitons.4−6 For all organic solar cells, panchromatic absorption is achieved by employing an electron acceptor that also absorbs visible light.7−9 However, despite significant effort on developing n-type light absorbing polymers and molecules, solar cells incorporating the (6,6)-phenyl-C70- butyric acid methyl ester (C70-PCBM),10,11 or derivatives thereof remain twice as efficient as those incorporating the next best electron acceptor. Fullerene derivatives, especially the larger molecules such as C70-PCBM, are reportedly challenging to isolate and purify and produced at a relatively low yield, and in addition, C70PCBM is limited in its own spectral width. Nanostructured hybrid architectures, where the polymer is infiltrated into a metal oxide scaffold, employ a transparent n- type oxide as the electron acceptor.12−14 For this system, the light harvesting capacity of chemical reactions of individual enzyme molecules and its application to a single-molecule-counting immunoassay. A microfluidic 6 device is used to generate and manipulate <10 fL droplets at rates of up to 1.3 × 10 per second, about 2 orders of magnitude faster than has previously been reported. The femtodroplets produced with this device the polymer can be used enhanced by a surface adsorbed dye, as a fusion between dye-sensitized solar cells (DSSCs) and organic photovoltaics (OPV).12,23 However, extremely careful engineering of the interface is required to encapsulate single biomolecular complexes tagged with a reporter enzyme; ensure good charge generation from both the dye and the polymer phases.15−20 Alternatively, in order to achieve intense panchromatic absorption from an organic system, additional dyes can be employed as “light harvesting antennas”, and transfer their small volume enables captured photon energy to the fluorescent product of a single enzyme molecule to be detected within 10 min of on-chip incubationorganic component responsible for charge generation in the solar cell. Our prototype system is validated by detection of a biomarker for prostate cancer in bufferReceived: December 23, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte molecules.2012 Accepted: January 15, 2013 Published: January 15, 2013

End User Agreement. This publication is distributed under the terms of Article 25fa of the Dutch Copyright Act (Auteurswet) with explicit consent by the author. Dutch law entitles the maker of a short scientific work funded either wholly or partially by Dutch public funds to make that work publicly available for no consideration following a reasonable period of time after the work was first published, provided that clear reference is made to the source of the first publication of the work. This publication is distributed under The Association of Universities in the Netherlands (VSNU) ‘Article 25fa implementation’ pilot project. In this pilot research outputs of researchers employed by Dutch Universities that comply with the legal requirements of Article 25fa of the Dutch Copyright Act are distributed online and free of cost or other barriers in institutional repositories. Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication. You are permitted to download and use the publication for personal purposes. All rights remain with the author(s) and/or copyrights owner(s) of this work. Any use of the publication other than authorised under this licence or copyright law is prohibited. If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please contact the Library through email: ▇▇▇▇▇▇▇▇▇@▇▇▇.▇▇.▇▇, or send a letter to: University Library Radboud University Copyright Information Point PO Box 9100 6500 HA Nijmegen You will be contacted as soon as possible. Ultrarapid Generation Abdominal Imaging Abdom Imaging (2015) 40:2306–2312 DOI: 10.1007/s00261-015-0442-8 Liver parenchyma at the site of Femtoliter Microfluidic Droplets for Single- Molecule-Counting Immunoassays Jung-uk Shim,†,§,* ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇,† ▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇▇,‡ ▇hypodense parafissural pseudolesion contains increased collagen ▇▇▇▇▇▇ ▇. ▇. ▇▇▇▇▇▇▇▇▇,†,^ 1 ▇▇▇▇ ▇▇¨ ster,2 ▇▇▇▇▇▇▇ ▇▇▇▇▇▇,1 ▇▇▇,† and ▇▇▇ ▇. ▇. ▇. ▇▇▇ ▇▇▇ ▇▇▇▇▇† †Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, U.K., CB2 1EW and ‡Department of Biochemistry, University of Cambridge, Cambridge, U.K., CB2 1GA. §Present address: Biomedical Engineering, University of Glasgow, Glasgow, UK, G12 8LT. ^Present address: Radboud University Nijmegen, Institute for Molecules and ▇▇▇▇▇▇▇▇▇, ▇▇▇▇▇,2 ▇▇▇▇▇▇▇▇▇ ▇▇▇, ▇▇▇▇ ▇▇ . ▇▇▇▇▇▇▇▇▇ 1Department of Radiology and Nuclear Medicine, ▇▇▇ ▇▇▇▇▇▇▇▇▇▇▇Radboud University Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands 2Department of Pathology, Radboud University Medical Centre, Nijmegen, The Netherlands Abstract‌ Purpose: To identify a histological substrate explaining the hypodense pseudolesion in the liver at the right side of the falciform ligament and the correlation with CT radiodensity. ARTICLE ABSTRACT We report Materials and methods: Tissue specimens were obtained from the right (pseudolesion) and left (control) side of the falciform ligament at the level of the left portal vein, in deceased adults during autopsy. Radiodensity was measured at the same locations at CT. Digital image analysis determined the amount of collagen and fat in histological sections, and the number of portal triads and central veins were counted. Glycogen content was visually assessed by the area percentage of the histo- logical section. Results: Specimens from 17 patients showed a microfluidic droplet-based approach enabling the measurement of chemical reactions of individual enzyme molecules and its application to a single-molecule-counting immunoassay. A microfluidic 6 device is used to generate and manipulate <10 fL droplets at rates of up to 1.3 × 10 per second, about 2 orders of magnitude faster than has previously been reported. The femtodroplets produced with this device can be used to encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables the fluorescent product of a single enzyme molecule to be detected within 10 min of on-chip incubation. Our prototype system is validated by detection of a biomarker for prostate cancer 39% increase in buffer, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets collagen for the counting site of individual analyte moleculesthe pseudolesion compared to the contralateral side (p = 0.08). No significant differences were found for the amount of fat, glycogen, portal triads, or central veins. In one patient a pseudolesion was visible on CT, and this contained 52% more collagen than the control side.

End User Agreement. This publication is distributed under the terms of Article 25fa of the Dutch Copyright Act (Auteurswet) with explicit consent by the author. Dutch law entitles the maker of a short scientific work funded either wholly or partially by Dutch public funds to make that work publicly available for no consideration following a reasonable period of time after the work was first published, provided that clear reference is made to the source of the first publication of the work. This publication is distributed under The Association of Universities in the Netherlands (VSNU) ‘Article 25fa implementation’ pilot project. In this pilot research outputs of researchers employed by Dutch Universities that comply with the legal requirements of Article 25fa of the Dutch Copyright Act are distributed online and free of cost or other barriers in institutional repositories. Research outputs are distributed six months after their first online publication in the original published version and with proper attribution to the source of the original publication. You are permitted to download and use the publication for personal purposes. All rights remain with the author(s) and/or copyrights owner(s) of this work. Any use of the publication other than authorised under this licence or copyright law is prohibited. If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please contact the Library through email: ▇▇▇▇▇▇▇▇▇@▇▇▇.▇▇.▇▇, or send a letter to: University Library Radboud University Copyright Information Point PO Box 9100 6500 HA Nijmegen You will be contacted as soon as possible. Ultrarapid Generation of Femtoliter Microfluidic Droplets for Single- Molecule-Counting Immunoassays Jung-uk Shim,†,§▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇▇,* 1 ▇▇▇▇▇▇▇ ▇▇▇▇,2 ▇▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇▇▇▇▇,† 2 ▇▇▇▇-▇▇▇▇▇ ▇▇▇▇▇,3,4 ▇▇▇▇ ▇▇▇▇▇▇▇,3 ▇▇▇▇-▇▇▇ ▇▇▇▇▇▇,4 ▇▇▇▇▇▇ ▇▇▇▇▇▇,5 ▇▇▇▇▇▇▇ ▇. ▇▇▇▇▇,† ▇1,5 ▇▇▇▇ ▇. ▇▇▇▇▇▇▇,† ▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇▇,‡ ▇▇▇▇▇▇▇ ▇. ▇. ▇▇▇▇,†,^ ▇2 and ▇▇▇▇ ▇▇▇▇▇▇▇▇▇,† ▇ 1Faculty of Medical and ▇▇▇▇▇ ▇▇▇▇▇† †Department of ChemistryHuman Sciences, Manchester Academic Health Sciences Centre, University of CambridgeManchester, Lensfield RoadManchester, CambridgeUnited Kingdom. 2Department of Human Genetics, U.K.Nijmegen Centre for Molecular Life Sciences, CB2 1EW and ‡Department Radboud University Nijmegen Medical Centrum, Nijmegen, The Netherlands. 3Department of Biochemistry, University of CambridgeLausanne, CambridgeEpalinges, U.K.Switzerland. 4Cutaneous Biology Research Center, CB2 1GAMassachusetts General Hospital, Charlestown, Massachusetts, USA. §Present address: Biomedical Engineering5Faculty of Life Sciences, University of GlasgowManchester, GlasgowManchester, UK, G12 8LTUnited Kingdom. ^Present address: Radboud University Nijmegen, Institute for Molecules and ▇▇▇▇▇▇▇▇▇, ▇▇▇▇▇▇▇▇▇▇▇▇▇▇ ▇▇▇, ▇▇▇▇ ▇▇ ▇▇▇▇▇▇▇▇, ▇▇▇ ▇▇▇▇▇▇▇▇▇▇▇. ARTICLE ABSTRACT We report Cleft palate is a microfluidic droplet-based approach enabling the measurement of chemical reactions of individual enzyme molecules and its application to a single-molecule-counting immunoassay. A microfluidic 6 device is used to generate and manipulate <10 fL droplets at rates of common congenital disorder that affects up to 1.3 × 10 per second, about 2 orders of magnitude faster than has previously been reported1 in 2,500 live human births and results in considerable morbidity to affected individuals and their families. The femtodroplets produced etiology of cleft palate is complex, with both genetic and environmental factors implicated. Mutations in the transcription factor–encoding genes p63 and interferon regulatory factor 5 (IRF6) have individually been identified as causes of cleft palate; however, a relationship between the key transcription factors p53 and IRF5 has not been determined. Here, we used both mouse models and human primary keratinocytes from patients with cleft palate to demonstrate that IRF6 and p63 interact epistatically during development of the secondary palate. Mice simultaneously carry- ing a heterozygous deletion of p63 and the Irf6 knockin mutation R84C, which causes cleft palate in humans, displayed ectodermal abnormalities that led to cleft palate. Furthermore, we showed that p53 transactivated IRF6 by binding to an upstream enhancer element; genetic variation within this device can be used enhancer element is associ- ated with increased susceptibility to encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables the fluorescent product of a single enzyme molecule to be detected within 10 min of on-chip incubationcleft lip. Our prototype system is validated by detection of findings therefore identify p53 as a biomarker for prostate cancer in buffer, down to key regulatory molecule during palate development and provide a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets mechanism for the counting cooperative role of individual analyte moleculesp53 and IRF5 in orofacial development in mice and humans.

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Ultrarapid Generation of Femtoliter Microfluidic Droplets for Single- Molecule-Counting Immunoassays Jung-uk Shim,†,§,* ARTICLE Monitoring a Reaction at Submillisecond Resolution in Picoliter Volumes ) ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇,† ▇▇▇▇▇ ▇. ▇▇▇▇▇▇▇,† ▇▇†,‡ ▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇▇,‡ ▇▇▇▇▇▇▇ ▇. ▇. ▇▇▇▇,†,^ ‡, ▇▇▇▇▇▇▇ ▇. ▇▇▇▇▇▇,*,§ and ▇▇▇▇▇▇▇ ▇▇▇▇▇▇▇▇▇▇*,† and ▇▇▇▇▇ ▇▇▇▇▇† †Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom ‡Department of Chemistry, University of Cambridge, Lensfield RoadCambridge CB2 1EW, CambridgeUnited Kingdom §Laboratoire d'Hydrodynamique and Department of Mechanics (LadHyX), U.K.Ecole Polytechnique, CB2 1EW and ‡Department of BiochemistryCNRS, University of CambridgeF-91128 Palaiseau, CambridgeCedex, U.K., CB2 1GA. §Present addressFrance bS Supporting Information ABSTRACT: Biomedical Engineering, University of Glasgow, Glasgow, UK, G12 8LT. ^Present address: Radboud University Nijmegen, Institute for Molecules and ▇▇▇▇▇▇▇▇▇, ▇▇▇▇▇▇▇▇▇▇▇▇▇▇ ▇▇▇, ▇▇▇▇ ▇▇ ▇▇▇▇▇▇▇▇, ▇▇▇ ▇▇▇▇▇▇▇▇▇▇▇. ARTICLE ABSTRACT We report a microfluidic dropletWell-based approach enabling the measurement of chemical reactions of individual enzyme molecules and its application to a single-molecule-counting immunoassay. A microfluidic 6 device is established rapid mixing techniques such as stopped-flow have been used to generate and manipulate <10 fL droplets at rates of up to 1.3 × 10 per second, about 2 orders of magnitude faster than has previously been reported. The femtodroplets produced with this device can be used to encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables push the fluorescent product of a single enzyme molecule to be detected within 10 min of on-chip incubation. Our prototype system is validated by detection of a biomarker dead time for prostate cancer in buffer, kinetic experiments down to a few milliseconds. However, very fast reactions are difficult to resolve below this limit. We now outline an approach that provides access to ultrafast kinetics but does not rely on active mixing at all. Here, the reagents are compartmentalized into water-in-oil emulsion microdroplets (diameter ∼50 μm) that are statically arrayed in pairs, resting side-by-side in a well feature of a poly- (dimethylsiloxane) (PDMS) device. A reaction between the contents of two droplets arrayed in such a holding trap is initiated by droplet fusion that is brought about by electro- coalescence and known to occur on a time scale of about 100 μs. A reaction between the reactants (Fe3+ and SCN-) is monitored by image analysis measuring the product formation in the newly merged drop in both space and time, by use of a fast camera. A comparison of the concentration field of 46 fM. This work demonstrates the reaction product with the output of a highly flexible reaction-diffusion system of equations yields a rate constant k ∼ 3 × 10 M s . Since reaction and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for diffusion are formally included in the counting of individual analyte molecules.mathematical model,