Diffusion of proteins inside crowded structures generated using microemulsions

Di usion o p o eins inside c owded s uc u es gene a ed using mic oemulsions Disse a ion zu E langung des akademischen G ades eines Dok o s de Na u wissenscha en (D . e . na .) im Fach Chemie an de Fakul ä ü Biologie, Chemie und Geowissenscha en de Uni e si ä Bay eu h o geleg on Ralph Neubaue gebo en in Eschenbach Bay eu h, de 27. Feb ua 2013 Die o liegende A bei wu de in de Zei on Augus 2009 bis Janua 2013 an de Uni e si ä Bay eu h am Leh s uhl ü Physikalische Chemie I un e Be euung on He n P o . D . Thomas Hellweg ange e ig . Volls ändige Abd uck de on de Fakul ä ü Biologie, Chemie und Geowissenscha en de Uni e si ä Bay eu h genehmig en Disse a ion zu E langung des akademischen G ades eines Dok o s de Na u wissenscha en (D . e . na ). Disse a ion einge eich am: 27.02.2013 Zulassung du ch die P ü ungskommission: 13.03.2013 Wissenscha liches Kolloquium: 28.10.2013 Am ie ende Dekan: P o . D . Rhe Kempe P ü ungsausschuss: P o . D . S ephan Fö s e (E s gu ach e ) P o . D . Thomas Hellweg (Zwei gu ach e ) P o . D . Jü gen Senke (Vo si z) P o . D . We ne Köhle CONTENTS CONTENTS Con en s 1 Summa y 9 2 Zusammen assung 11 3 In oduc ion 15 4 Theo e ical backg ound 17 4.1 Mic oemulsion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 4.1.1 Su ac an s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 4.1.2 Micelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 4.1.3 S uc u e and dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 4.1.4 The bicon inuous phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 4.1.5 Cu a u e o he in e acial ilm . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 4.1.6 Leng h scales in mic oemulsions . . . . . . . . . . . . . . . . . . . . . . . . . . 22 4.2 Decon amina ion applica ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 4.3 P o eins in mic oemulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 4.4 Fluo escence co ela ion spec oscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 4.4.1 Fluo escence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 4.4.2 FCS se up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 4.4.3 Obse a ion Volume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 4.4.4 Di usion o he luo opho e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 4.4.5 T iple decay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 4.4.6 Anomalous di usion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 4.4.7 Calib a ion o he ins umen . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 4.5 Sca e ing me hods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 4.5.1 Pho on co ela ion spec oscopy . . . . . . . . . . . . . . . . . . . . . . . . . . 34 4.5.2 Se up o he PCS expe imen . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 4.5.3 Small angle neu on and X- ay sca e ing . . . . . . . . . . . . . . . . . . . . . 38 4.5.4 Teubne S ey app oxima ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 4.5.5 D ople s uc u es . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 4.5.6 Neu on-spin echo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 5 CONTENTS CONTENTS 5 Expe imen al sec ion 45 5.1 Used chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 5.2 Sys ems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 5.2.1 Reco ding o phase diag ams . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 5.2.2 P o eins in mic oemulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 5.2.3 P o ein luo escence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 5.3 Expe imen al de ails o he FCS measu emen s . . . . . . . . . . . . . . . . . . . . . . 52 5.4 DLS measu emen s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 5.5 SANS/SAXS measu emen s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 5.5.1 SAXS se up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 5.5.2 SANS se up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 6 Resul s and discussion 57 6.1 Fluo escence measu emen s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 6.1.1 Fluo escen impu i ies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 6.1.2 FCS measu emen s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 6.2 The C9G2based mic oemulsion sys em . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 6.2.1 Phase beha io . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 6.2.2 S uc u e sizes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 6.2.3 GFP+dynamics by FCS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 6.2.4 Mic oemulsion dynamics by DLS . . . . . . . . . . . . . . . . . . . . . . . . . . 74 6.3 The C12G2based mic oemulsion sys em . . . . . . . . . . . . . . . . . . . . . . . . . . 75 6.3.1 Phase beha io . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 6.3.2 Size and shape o he mic oemulsion s uc u es . . . . . . . . . . . . . . . . . 77 6.3.3 GFP+dynamics by FCS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 6.3.4 Mic oemulsion dynamics (FCS) . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 6.3.5 Mic oemulsion dynamics (DLS) . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 6.4 O he mic oemulsion sys ems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 6.5 O he me hods o measu ing p o ein dynamics . . . . . . . . . . . . . . . . . . . . . 91 6.5.1 DFPase NSE measu emen s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 6.5.2 PFG-NMR measu emen s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 6 CONTENTS CONTENTS 7 Conclusion and u u e p ospec s 97 8 Danksagung 99 A Appendix 101 A.1 Al e na i e mic oemulsion sys ems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 A.2 Sol en sensi i e luo escen dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Lis o Figu es 107 Lis o Tables 111 Re e ences 113 Abb e ia ions 125 7 1 SUMMARY 1 Summa y Mic oemulsions a e he modynamically s able mix u es o wa e , oil and su ac an . In he case o mic oemulsions based on suga su ac an s a cosu ac an as an addi ional componen is nec- essa y, mos o en a sho -chain alcohol. In his hesis mainly pu e su ac an s a e used. The ocus is on n-nonyl-β-d-mal osid and n-dodecyl-β-d-mal osid as model sys ems o mic oemul- sions based on echnical g ade su ac an s. Depending on he a io o he componen s mic oemulsions o m di e en s uc u es. In he bicon inuous phase con inuous oil and wa e domains a e p esen sepa a ed by a su ac an /co- su ac an ilm. A highe su ac an amoun s he bicon inuous s uc u e passes o e o a d ople phase. The size o hese s uc u es is in he scale o up o 100nm, he e o e mic oemulsions look anspa en o he human eye. Bicon inuous mic oemulsions a e a p omising ca ie medium o decon amina ion applica ions. Chemical wa a e agen s a e mainly lipophilic, in con as deg ada ion agen s a e hyd ophilic. A mic oemulsion is able o solubilize lipophilic and hyd ophilic molecules. A he in e ace he wa a e agen is close o he deg ada ion agen and can be elimina ed. The p o ein diisop opyl- luo ophospha ase (DFPase) is a p omising candida e o decon amina- ion applica ions and is able o deg ade di e en wa a e agen s. The e o e, he knowledge o he dynamics and p ope ies o enzymes inside a bicon inuous s uc u e is o big impo ance. In his hesis di e en mic oemulsion sys ems based on C9G2o C12G2, wa e , cyclohexane and 1-pen anol a e sys ema ically cha ac e ized by X- ay and neu on sca e ing expe imen s. By using an imp o ed G een Fluo escen P o ein (GFP+) as a coun e pa o DFPase wi h a simila size, he di usion inside a mic oemulsion can be s udied wi h he luo escence co ela ion spec- oscopy (FCS) me hod. He e, luo escen impu i ies in he used componen s a e a p oblem. By he choice o a sui able concen a ion o GFP+and ega ding he dynamics o he mic oemul- sion s uc u e, i could be shown ha he p o ein is able o mo e inside he mic oemulsion s uc u e. Hence, mic oemulsions a e in e es ing model sys ems o p oduce c owding e ec s in a con olled way. This is p obably he mos impo an esul o his hesis. In he C9G2sys em a bicon inuous phase is p esen . A small oil/wa e a ios Φwi h a high wa e amoun big wa e domains exis wi h leng h scales o app oxima ely 10nm, which allows p o ein di usion. Wi h inc easing oil/wa e a io he p o ein di usion is mo e and mo e hinde ed. This esul s in an 9 3 INTRODUCTION In he 1980s, al eady he Ge man a med o ces used mac oemulsions o decon amina ion based on Ma lowe IHF, e achlo e hylene and calciumhypochlo ide, bu hey a e no s able, ha m ul and a dange o he en i onmen [18]. The e o e mic oemulsions as ca ie media o deg a- da ing agen s a e a dis inguished al e na i e o decon amina ion applica ions. They o e an oil and a wa e phase - a lipophilic and hyd ophilic pa in one sys em. Mo eo e , suga su ac an mic oemulsions can be composed wi h biocompa ible componen s which is ano he big ad an- age o decon amina ion pu poses [19]. Fu he mo e, he empe a u e s abili y is an impo an ac o he use in di e en clima ic egions. S udying he s uc u e o hese sys ems equi es big e o s in heo e ical conside a ions and in expe imen al ac i i ies, many publica ions on his esea ch a ea a e a consequence. Due o he nanome e scale and he e o e he need o expensi e me hods like neu on sca e ing me hods he esea ch is some imes complica ed. The p esen hesis deals wi h he in oduc ion o ano he echnique o obse ing mic oemulsion s uc u e and dynamics, FCS. On he one hand, he di usion o he mic oemulsion s uc u e can be measu ed, namely he collec i e b ea hing mo ion [20], on he o he hand he dynamics o pa icles inside his s uc u e can be in es iga ed. The knowledge o hese p ocesses is impo - an : The p o ein diisop opyl- luo ophospha ase (DFPase) is a amous candida e o decon ami- na ion applica ions, he assignmen in a mic oemulsion sys em is a p omising combina ion [21]. Mo eo e , ano he aim o his pape is he implemen a ion o new mic oemulsion sys ems mainly based on pu e su ac an s ha play a ole as model sys ems o echnical g ade sys ems and show a highe pu i y, which is c ucial o luo escence me hods. Bu FCS is no he only me hod o look inside nano-scale s uc u es. Also small angle sca e - ing (SAS) echniques like small angle X- ay sca e ing (SAXS) and small angle neu on sca - e ing (SANS) a e used o cha ac e ize he s uc u e. neu on spin echo (NSE) and pulsed- ield-g adien nuclea magne ic esonance (PFG-NMR) p o ide in o ma ion on he dynamical beha io o mic oemulsion sys ems. 16 4 THEORETICAL BACKGROUND 4 Theo e ical backg ound The main concep s o mic oemulsions and hei abili ies will be ea ed in his chap e . Fi s ly, he impo an ole o su ac an s and o he in e acial ilm will be discussed, u he se e al di e en me hods a e used o obse e s uc u e and dynamics o mic oemulsion sys ems. To unde s and hese applica ions, he backg ound o hese echniques will be explained in he ol- lowing chap e s. 4.1 Mic oemulsion Mic oemulsions show in e es ing s uc u es, al hough hey look anspa en o he human eye, which is caused by he small s uc u e sizes abou 50 imes lowe han he isible ligh wa e- leng h.. Bu on a close examina ion, hey ea u e o example a sponge-like s uc u e in he nanome e -scale o a sui able mass a io o componen s. Mo eo e , he phase beha iou is e y ich and shows mo e di e en s uc u es [22], which ha e o be discussed in mo e de ail (see chap e 4.1.3). 4.1.1 Su ac an s Amphiphilic molecules wi h a hyd ophobic and a hyd ophilic pa a e called su ac an s o de e - gen s and a e he base o mic oemulsions. No mally, he hyd oca bon esidue is he hyd ophobic pa and he hyd ophilic pa de e mines he kind o he su ac an . Anionic su ac an s, whe e he hyd ophilic pa is anionic ( o example sul a e, sul ona e and phospha e), a e commonly used in washing, and cleaning agen s [23],ca ionic su ac an s ha e a ca ionic hyd ophilic g oup and a e applied in cosme ics, ab ic condi ione s, and sani ize s [24]. In a zwi e ionic su ac an s molecule a posi i e and nega i e cha ge is p esen , because o he skin iendli- ness his de e gen s a e used in shampoos, body ca e p oduc s, ba h addi i es and household de e gen s [25, 26]. Fu he mo e, nonionic su ac an s ha e hyd ophilic pola g oups wi h- ou any cha ge (hyd oxyl-, ca boxyl-, es e -, amide- and polyalkyl-g oups). Also alcylpolyglyco- sides (APG c), known as suga su ac an s a e pa o he non-ionic de e gen s and a e composed o enewable esou ces (suga and a y alcohol can be used as aw ma e ials), hese su ac an s a e he ounda ion o he mic oemulsions used in his wo k. The APG ccomponen s a e Glu- cose and an alkyl- esidue wi h chain leng hs o six up o 14 ca bon a oms. Longe ca bon chains 17 4.1 Mic oemulsion 4 THEORETICAL BACKGROUND implica e a high hyd ophobici y and he o mula ion o a mic oemulsion is di icul , because he su ac an is no soluble in wa e . 4.1.2 Micelles Because su ac an s a e molecules wi h an amphiphilic abili y, hey a e able o o m micelle in solu ion, i a c i ical micell concen a ion (CMC) is eached. Micelles a e a clus e o molecules caused by e e sible agg ega ion. The geome ical o m o such pa icles mus no be sphe e- like, a micelle can also exis as cylind ical, pla e- o od-like objec s. The CMC is he concen a ion o su ac an , when he su ace o he in e ace is comple ely co e ed wi h su ac an molecules, below he CMC he su ace ension o he luid is dec eased, abo e he CMC mo e micelles eme ge and he su ac an has no in luence on he su ace ension anymo e. Micelles can also change mac oscopic pa ame e s like low p ope ies [23]. 4.1.3 S uc u e and dynamics Mic oemulsions based on suga su ac an s a e composed o wa e , oil, suga su ac an (CiGj) and co-su ac an (alcohol). Such sys ems he modynamically s able. Di e en phases occu o ce ain mass a ios o hese componen s. To quan i y he amoun o cons i uen s, he ollowing de ini ions will be used: ac ion o oil: Φ=moil mw+moil (1) ac ion o su ac an : γ=msu mw+moil +msu (2) ac ion o co-su ac an : δ=mcosu mw+moil +msu +mcosu (3) Due o he numbe o ou componen s, he phase beha io can be desc ibed by a phase e a- hed on (compa e ig. 1). When ins ead o a suga su ac an a CiGj-su ac an is used, he e aehed on changes o a phase p ism, whe e he heigh co esponds o he empe a u e axis. Fo CiEj-sys ems, no co-su ac an is necessa y o achie e a 1-phase s uc u e. The empe a u e plays he ole as a uning pa ame e and ac s like he co-su ac an in a CiGjsys em. Fo a cu in he phase e ahed on a a ce ain oil/wa e a io Φ( ig. 1) he phase beha io is illus a ed by a wo-dimensional g aph wi h he axis γand δ( ig. 2). 18 4 THEORETICAL BACKGROUND 4.1 Mic oemulsion wa e oil su ac an co-su ac an Φ δ Figu e 1: Phase e ahed on o a suga -su ac an based mic oemulsion wi h a cu o a wa e /oil a io Φand he esul ing Kahlwei - ish in e e ence o [22]. The esul ing image looks like a ish, known as "Kahlwei - ish". The one phase s uc u es a e loca ed in he ish- ail: bicon inuous, oil- and wa e con inuous, and lamella phase [27]. The phases a e also called Winso -sys ems [1]: Type I wi h a wa e phase whe e he su ac an and oil is solubilised and an oil phase (loca ed below he ish-body). Type II is an oil phase wi h solubilised su ac an and wa e and a wa e excess phase ( egion in he phase diag am abo e he ish body). X γ δ Lα Figu e 2: Kahlwei - ish o a suga su ac an sys em wi h he so called X-poin , ish-head (3-phase egion), ish ail (1-phase) and wa e excess phase below, oil excess phase abo e he ish, in e e ence o [22]. Type III consis s o 3 phases, a wa e phase, a mic oemulsion phase and a oil phase (in he ish- head). In he case o ype IV only one phase is p esen , we e all componen s a e solubilised. The lamella egion is a single phase a ea indica ed by Lαin ig. 2. The Lα-phase and he 19 4.1 Mic oemulsion 4 THEORETICAL BACKGROUND bicon inuous egion a e sepa a ed by a wo phase coexis ence zone [22]. •bicon inuous phase (L3phase): Behind he X-poin , sponge-like s uc u e wi h oil and wa e domains, sepe a ed by he su ac an /co-su ac an in e ace. •lamella phase (Lαphase): Oil and wa e laye s in e up ed by he in e acial ilm. The Lαis a smec ic ype C liquid c ys al. •oil in wa e mic oemulsion: Oil d ople s in wa e wi h a adius o some nm, below he lamella egion. •wa e in oil mic oemulsion: Wa e d ople s in oil, abo e he lamella egion. Wha kind o s uc u e eme ges, depends on he composi ion o he componen s (Φ,γ) and on he shape o he amphiphilic ilm, which is mainly de e mined by he alcohol con en δ. su ac an co-su ac an wa e phase oil phase su ac an co-su ac an (a) (b) Figu e 3: Ske ch o a (a) bicon inuous mic oemulsion and (b) lamella phase A common me hod o in es iga e he s uc u e is measu ing he conduc i i y o a sample [28]. In wa e con inuous s uc u es (oil in wa e ) and in he bicon inuous phase he conduc i i y is ela i ely high: The wa e phase is con inuous o e he whole sample. On he o he hand o wa e in oil phases, he conduc i i y is low. Fo lamella s uc u es he conduc i i y is lowe han in he bicon inuous phase , i he conduc i i y is measu ed pe pendicula o he in e acial ilm. Howe e , i he conduc i i y can be measu ed in plane o he lamellae i will be highe compa ed o he bicon inuous phase [29]. The su ac an ilm and he oil phase hinde s he conduc ion pe pendicula o he lamellae. 20 4 THEORETICAL BACKGROUND 4.1 Mic oemulsion 4.1.4 The bicon inuous phase This phase occu s close o he X-poin in he ish ail o a Kahlwei - ish phase diag am ( igu e 2). The s uc u e can be imagined as a sponge-like pa e n and can be isualized by eeze ac u e elec on mic oscopy (FFEM) ( [30]). The bicon inuous phase o e s a huge in e nal in e ace, which is bene icial o decon amina ion applica ions. The oil and wa e componen exis as in e laced con inuous phases, sepa a ed by a su ac an /co-su ac an ilm. The bicon inuous s uc u e appea s p e e ably a equal oil/wa e ac ion, meaning Φ=0.5 [31]. Bicon inuous phases can be de ec ed by elec ical conduc i i y measu emen s [32]and leng h scales can be ob ained by SANS and SAXS expe imen s (chap e 4.1.6). The dynamic o such s uc u es is o en called “collec i e b ea hing mo ion” [33,34]and can be measu ed by NSE, dynamic ligh sca e ing (Dynamic Ligh Sca e ing (DLS)) and PFG-NMR me hods. 4.1.5 Cu a u e o he in e acial ilm The in e acial ilm in he mic oemulsion sys ems s udied he e consis s o a CiGjsu ac an (hy- d ophilic) and a co-su ac an (alcohol, hyd ophobic su ac an ). The cu a u e Ho he ilm depends on he amoun o co-su ac an a he in e ace. Because o he smalle co-su ac an head g oup compa ed o he suga su ac an , Hchanges om posi i e o nega i e alues when co-su ac an is added. inc easing δ H < 0 H = 0 H > 0 non-ionic su ac an co-su ac an Figu e 4: Non-ionic su ac an ilm in a suga su ac an mic oemulsion: The cu a u e H depends on he amoun o co-su ac an δ(hyd ophobic), in e e ence o [22]. Hcan be calcula ed wi h he local cu a u es c1=1 R1and c2=1 R2, loca ed a a poin o he 21 4.1 Mic oemulsion 4 THEORETICAL BACKGROUND su ac an ilm (wi h R1and R2: adius o he cu a u es): H=1 2(c1+c2)(4) Fo H>0, oil in wa e d ople s a e o med. When H eaches nega i e alues, he s uc u e changes o wa e -in-oil d ople s. I H=0, he e is no cu a u e o he ilm, he Gaussian cu a- u e Khas o be conside ed: K=c1·c2(5) When Kis nega i e, he bicon inuous s uc u e appea s, o posi i e alues he su ace is plana and he s uc u e is lamella . This cu a u e can be a ec ed wi h he ype and amoun o he su ac an and co-su ac an , empe a u e and addi ion o polyme s. In such a bicon inuous s uc u e, he leng h scales can be desc ibed acco ding o S ey [35]by he cha ac e is ical leng h ξ: ξ= u 2 c2 1+c2 2 (6) In expe imen s i was obse ed ha ξis app oxima ely hal he pe iodici y d[36], which is a alue o he domain size o he oil and wa e domain. 4.1.6 Leng h scales in mic oemulsions Because o he s uc u al dimensions in he nano-me e scale, a mic oemulsion looks anspa en o he human eye. Hence, he s uc u e canno be obse ed by op ical echniques. Ligh sca - e ing me hods would e eal he bulk-dynamics o he s uc u e, he e o e echnologies wi h smalle wa eleng hs ha e o be used: X- ay o neu on sca e ing. SAXS and SANS expe imen s yield he dimensions o mic oemulsion sys ems (compa e sca e ing heo ies in chap e 4.5). Ano he possibili y is FFEM, o such measu emen s he sample has o be ozen e y as . Bu in a eezing p ocess, he s uc u e o he sys em migh be changing and he appea ing s uc u es migh no e lec he eal s a e a no mal empe a u es. Dimensions in a bicon inuous s uc u e a e cha ac e ized in e ms o co ela ion leng hs ξand domain sizes d(equa ions 44, 45). These dimensions depend on he chain-leng h o he used su ac an and on he mass a ios Φand γ. Fo suga su ac an sys ems, he co ela ion leng h is be ween se e al nanome e s up o nea ly 100nm. Fo Φ olume =0.5 he oil and wa e domains show a simila size - a smalle Φ, he wa e domain size inc eases and dec eases o Φ>0.5. 22 4 THEORETICAL BACKGROUND 4.2 Decon amina ion applica ions 4.2 Decon amina ion applica ions Chemical wa a e agen s a e a big dange in imes when e o is a acks a e a daily issue in se e al egions on ea h. The e o e i ge s mo e and mo e impo an ha e ec i e decon ami- na ion media a e a ailable. A couple o ea u es a e equi ed o a adequa e decon amina ion sys em: Abili y o pene a e di e en kinds o su aces, non oxic, s o able, nonco osi e and cheap o ab ica e. Mic oemulsions can be composed o a ious di e en componen s and o e in he sui able composi ion all o hese p econdi ions. Mos oxic chemicals and wa a e agen s ha e hyd ophobic abili ies. Bu in con as he majo - i y o deg ada ion agen s a e hyd ophilic. In a mic oemulsion, bo h ea u es a e p esen wi h a s uc u e on he nanome e scale. Fu he mo e, a decon amina ion medium should be able o we su aces. The human skin o coa ings o mili a y equipmen a e hyd ophobic, he e o e a luid in o m o a mic oemulsion can pene a e such su aces o b ing he decon amina ion agen close o he wa a e subs ance. Ne e heless, also o hyd ophilic su aces, he media should be capable o we ing hese su - aces. En i onmen al compa ibili y is ano he impo an ea u e o decon amina ion media. On he one hand, when media a e sp ead on mili a y ehicles, hey come also in con ac wi h he en- i onmen . On he o he hand, when media a e applied on soldie s, i mus no be oxic o ha m ul. A i s app oach is he composi ion o biodiesel based mic oemulsions [37]and sys- ems a e cu en ly de eloped wi h componen s app o ed o cosme ic applica ions. Fo a low-cos p oduc ion i is impo an , ha he necessa y amoun o su ac an is as small as possible. The e o e he X-poin has o be shi ed o small γ. E iciency boos ing by block co-polyme s plays a majo ole o exis ing mic oemulsion sys ems, wi h only small amoun s o block copolyme s a much bigge amoun o su ac an can be eplaced [38–40]. Enzymes wi h decon amina ing abili ies solubilised in he wa e phase o a mic oemulsion a e p omising candida es o decon amina ion pu poses. Especially he enzyme DFPase (see igu e 5) o he squid Loligo ulga is is able o de oxi y ne e agen s [18,21,22]. A ecen ly p esen ed decon amina ion p ocess wo ks as ollows: In a i s s ep he wa a e agen s a e solubilised by he oil phase o he mic oemulsion, whe e hey can ge o he in e ace o come nea he deg ada ion agen s which a e sol ed in he wa e phase and he decon amina ion can 23 4.2 Decon amina ion applica ions 4 THEORETICAL BACKGROUND ake place [41]. Figu e 5: X- ay s uc u e o DFPase o he squid Loligo ulga is [42], wi h a molecula weigh o 35kDa.[43] Ano he impo an abili y o mic oemulsions is he s uc u al s abili y o e a huge empe a u e ange (-20◦C o 50◦C) [44,45], which is a c ucial ea u e o applica ions in he mili a y ield. The leng h scales o he wa e and oil domains s ay nea ly cons an de e mined by SANS and he bending elas ic cons an is empe a u e independen measu ed in NSE expe imen s. 24 4 THEORETICAL BACKGROUND 4.3 P o eins in mic oemulsions 4.3 P o eins in mic oemulsions In he las subsec ion he use o p o eins o decon amina ion applica ions was discussed al eady. Knowing he dynamics o such pa icles inside a bicon inuous phase o a mic oemulsion is im- po an o designing e icien decon amina ion media. One ques ion is, i he enzyme is mo ing inside e.g. he wa e domain o i i s icks o he su ac an in e ace. The dynamics o DFPase wi h a hyd odynamic adius o 2.34nm [34]a e ha d o obse e wi h inhouse-me hods like pho on co ela ion spec oscopy. Mo eo e , he mic oemulsion s uc u e sca e s ligh as well and o e lays he signal o he p o ein. The e o e, we use p o eins compa able in size, which a e luo escen and can be di ec ly obse ed in a mo e simple way wi h luo escence co ela ion spec oscopy. G een Fluo escen P o ein (GFP) is a amous p o ein used o many applica ions in science by aking ad an age o he luo escen beha io wi hou he need o labeling (chap e 5.2.3). Besides he ques ions ela e o he use o mic oemulsions as eac ion medium o en- zymes, i is also possible o use he mic oemulsion as model sys em o he s udy o sub-di usi e beha io . The domain sizes in a bicon inuous mic oemulsion o he olume ac ion in a d ople sys em can be easily con olled. Hence, such a sys em can be used o gene a e c owding in a well de ined way. 4.4 Fluo escence co ela ion spec oscopy This me hod exploi s he luo escence abili y o molecules and is used o measu e pa icle dy- namics down o he nanome e scale [46]. The ad an ages a e ha one can decide, wha dy- namic is measu ed by speci ic labeling and ha ime anges om mic oseconds up o 100ms can be co e ed. Bu no only di usion imes, also concen a ion can be measu ed by FCS. In his sec ion i s he e ec o luo escence is discussed and a e wa ds he heo e ical backg ounds will be co e ed. 4.4.1 Fluo escence When a molecule is exci a ed in o a highe ene gy s a e, he ene gy can be eleased again in di e en ways. The as es p ocess is he ib a ion elaxa ion wi h a hal -li e pe iod o 10−12 s, hen he molecule can all back o he g ound s a e by emi ing ligh , which is called luo es- cence [47], o i can pe o m in e -sys em c ossing in o a iple s a e ( igu e 6), called phospho- 25 4.4 Fluo escence co ela ion spec oscopy 4 THEORETICAL BACKGROUND The model can be adap ed o mo e componen s wi h di e en size and di usion imes, which a e de ec ed simul aneously: g2( ) = 1+1 N1+T 1−Te− τT· N X i=1  1 (1+ τi )·Ç1+ ( 0 z0)2· τi  (19) kiis he a io o pa icle i. The di usion imes τiha e o di e su icien ly om each o he , o be dis inguished using a i acco ding o equa ion 19, no mally by a ac o o 2. The highe he numbe o di using componen s in he equa ion, he mo e pa ame e s in luence he i . The e o e a maximum o h ee componen s is easonable, when o he bounda y condi ions like pa icle a io a e known. 4.4.6 Anomalous di usion In case o con inemen o c owding e ec s, he di usion can be anomalous. In his case, he mean squa e displacemen σ is no linea in ime anymo e: <s2>=σ =D· α(20) The anomalous di usion exponen αhas o be ega ded in equa ion 16, ha leads o [56] g2( ) = 1+1 N1+T 1−Te− τT·1 1+ ( τd )α·Ç1+ (S2· τd )α(21) I α < 1, sub-di usi e beha io is obse ed. Smalle αmeans mo e con inemen o he di using pa icle. In he bicon inuous phase o a mic oemulsion, he di usion o a pa icle inside he wa e domains is hinde ed by he su ac an /co-su ac an in e ace. 4.4.7 Calib a ion o he ins umen τDis he ime a pa icle needs o pass he obse a ion olume τD= 2 0 4D(22) Wi h a dye, whe e Dis known om he li e a u e (o en luo escein o he 488nm beampa h, hodamine 6G o 514nm beampa h and hodamine B o 534nm beampa h), 0can be cal- cula ed and τDob ained by a i o he au oco ela ion cu e. Wi h 0and he i pa ame e s, also z0can be calcula ed. Then Scan be hold cons an o measu emen s wi h an unknown sample [51]. 32 4 THEORETICAL BACKGROUND 4.5 Sca e ing me hods 4.5 Sca e ing me hods S uc u es and dynamics o mic oemulsions can be in es iga ed using di e en sca e ing me h- ods. In p inciple, sca e ing is he in e ac ion o a pa icle wi h an objec . Pho ons wi h di e en ene gies (o wa eleng hs) a e sca e ed a objec s o a ious size: In ligh sca e ing expe i- men s, di e ences o he e ac i e index ( o example oil ↔wa e ) and he size o pa icles in luence he sca e ed ligh . To esol e s uc u es, he used wa eleng h has o be in he dimen- sion o he s uc u e size. I he wa eleng h is smalle (e.g. X- ays) he pho on ene gy is bigge and pho on is sca e ed by he molecule elec ons: The e o e, in X- ay sca e ing applica ions he dis ibu ion o he elec ons is c ucial. Ano he commonly used echnique is neu on sca e ing, neu ons ha e no elec ic cha ge and a e able o pe mea e ma e and in e ac wi h he nucleus o a oms. Ligh and X- ay sca e ing echniques can be accomplished a comme cially a ailable o sel de- signed equipmen . Neu on sca e ing expe imen s ha e o be pe o med a a esea ch acili y, no mally he neu on sou ce is a nuclea eac o and he neu on de ec ion is a sophis ica ed and expensi e echnology. Fo X- ay sca e ing applica ions, a esea ch acili y wi h a synch o on sou ce is he supe io op ion, because he lux and he quali y o he de ec o is be e compa ed o inhouse-ins umen s. 33 4.5 Sca e ing me hods 4 THEORETICAL BACKGROUND 4.5.1 Pho on co ela ion spec oscopy This echnique, also called Dynamic Ligh Sca e ing (DLS), exploi s he abili y o small pa i- cles o s uc u es o sca e isible ligh . In con as o s a ic ligh sca e ing me hods, pho on co ela ion spec oscopy (PCS) yields in o ma ion abou di usion p ocesses. DLS is pa icula ly sui able o measu e he dynamics o mac o-molecules, p o ides in o ma ion abou he di usion coe icien and also he polydispe si y o pa icles. ki ks ϴq Figu e 11: wa e ec o ~q in a ligh sca e ing expe imen wi h incoming wa e ~ ks, sca e ed wa e ~ ks and sca e ing angle Θ Impo an o ligh sca e ing expe imen s is he wa e ec o ~q(compa e igu e 11): q=|~q|=4πn0 λsin Θ 2(23) wi h: n0: e ac i e index o he sol en , λ: wa eleng h o he sca e ed ligh , Θ: sca e ing angle The no malized elec ic ield au oco ela ion unc ion is gi en by: g1( ) = <E∗(~q, )E(~q, + 0)> <E(~q,0)>(24) In expe imen s mos ly only he sca e ed in ensi y is accessible by a ins umen . Thus g1( )can no be measu ed di ec ly. The in ensi y Iis de ec ed ime- and q- dependen o calcula e he au oco ela ion unc ion G2( ), which measu es he co ela ion a he ime 0wi h i sel a a la e ime + 0: G2(~q, ) =<I(~q, + 0)I(~q, )>=lim T→∞ 1 TZT 0 I( + 0)I( 0)d 0(25) The au oco ela ion unc ion ca be no malized: g2(~q, ) = G2(~q, ) <I(~q, 0)>2(26) 34 4 THEORETICAL BACKGROUND 4.5 Sca e ing me hods g1 ( ) 0 0.2 0.4 0.6 0.8 1 ime / s 1e-05 0.0001 0.001 0.01 0.1 1 Figu e 12: Au oco ela ed measu emen da a om a pho on co ela ion spec oscopy measu emen . Time-axis is loga i hmic. The measu ed in ensi y p o ides no in o ma ion abou he elec ical ield ~ E, bu he Siege - ela ion connec s he in ensi y au oco ela ion and g1( ): g2( ) = 1+β|g1( )|2(27) βdepends on he used measu emen ins umen . In a monodispe se sys em (e.g. sphe ical pa icles), g1( )can be calcula ed by using a monoexponen ial unc ion: g1( ) = e−Dq2 =e−Γ (28) The di usion coe icien Dgi es in o ma ion abou he pa icle adius. Fo polydispe se sys ems, a dis ibu ion unc ion G(Γ)is implemen ed, which gi es in o ma ion abou polydispe si y: g1( ) = Z∞ 0 G(Γ)e−Γ d (29) G(Γ)can be ega ded as a sum o single con ibu ions [57]: G(Γ) = N X i=0 aiδ(Γ−Γi)(30) applied o eq. 28: g1( ) = N X i=1 aie−Γi (31) The e a e di e en possibili ies o compu e he dynamics o a polydispe se sys em. One is he me hod o cumulan s [58]. Ano he sui able app oach o his p oblem wi h mo e species is he 35 4.5 Sca e ing me hods 4 THEORETICAL BACKGROUND me hod o he in e se Laplace ans o ma ion. The CONTIN p og am [59],[60]exploi s his p ocedu e and can be used o analyze measu ed ligh sca e ing da a. The ou pu o CONTIN is a alue o Γi, which depends on q. Wi h Γ=Dq2(32) a linea eg ession when Γis plo ed as a unc ion o q2(qcalcula ed by eq. 23) leads o D. When sphe ical pa icles a e obse ed (which is no he case in a bicon inuous s uc u e), he hyd odynamic adius RHcan be es ima ed by he S okes-Eins ein equa ion: D=kBT 6πηRH (33) He e, ηis he iscosi y. Wi h equa ion 33 he hyd odynamic adius o e.g. p o eins can be es ima ed (compa e chap e 5.2.2). 36 4 THEORETICAL BACKGROUND 4.5 Sca e ing me hods 4.5.2 Se up o he PCS expe imen The PCS measu emen s we e pe o med on an ALV-5000 [61]ins umen . In ig. 13 he se up o pho on co ela ions spec oscopy is shown. The ligh om a Nd:YVO4lase (g een, 532nm) o a Helium-Neon lase ( ed, 633nm) passes a neu al densi y il e (ND il e , o adjus he lase in ensi y), a lens and a Glan Thompson P ism and is sca e ed by he sample. The sample loca ed in a empe a u e con olled ba h illed wi h oluene sca e s he lase ligh . Nd:YVO4 M1 M2 sample lens GP co ela o ALV ND il e θ GP PMT pho on coun e HeNe 20°C he mos a Figu e 13: ALV-5000 Pho on Co ela ion Spec oscopy se up, Mi: mi o , GP: Glan Thompson P ism, PMT: Pho o Mul iplie Tube Toluene is used o ma ching he e ac i e index o he glass cu e e. The sca e ed ligh is de ec ed by a pho omul iplie moun ed on a goniome e , whe e he sca e ing angle Θand he e o e qcan be adjus ed. Wi h a pho on coun e and a co ela o , he signal o he PMT is egis e ed by he compu e wi h he ALV so wa e. 37 4.5 Sca e ing me hods 4 THEORETICAL BACKGROUND 4.5.3 Small angle neu on and X- ay sca e ing Fo SAS expe imen s, he sca e ing in ensi y and he sca e ing ec o qa e impo an pa am- e e s. The sca e ing in ensi y om a single pa icle depends on he sca e ed ampli ude F(q): I(q) = |F(q)|2(34) F(q)is he Fou ie ans o m o he mass dis ibu ion (SANS) o elec on dis ibu ion (SAXS) ρ( ): F(q) = ZV ρ( )e−iq d (35) ⇒I(q) =  1 VZV ρ( )e−iq d  2 (36) In he case o Niden ical pa icles, he in ensi y is gi en by: I(q) = N V(ρp−ρ0)2V2 p 1 VpZV ρ( )e−iq d  2 (37) wi h ρ0: mass/elec on densi y o he sol en , ρp: mass/elec on densi y o he pa icle, VP: pa icle olume, V: illumina ed olume, ρ0−ρ0: sca e ing con as and he o m ac o P(q) =  1 VpRVρ( )e−iq d  2 Bu also in e ac ions be ween he pa icles ha e o be conside ed, he o al sca e ing in ensi y is now calcula ed by [62](Fk(q): elec ical ield con ibu ion o pa icle k): I(q) = 1 V N X k=1¬Fk(q) 2¶+1 V*N X k=1 N X j=1 j6=k Fk(q)F∗ j(q)e−iq( k− j)+(38) When he pa icles a e monodispe se and sphe ical, he in ensi y can be simpli ied o: I(q) = n¬Fk(q) 2¶       1+*N X k=1 N X j=1 j6=k e−iq( k− j)+       (39) I(q) = nP(q)S(q)(40) The i s e m P(q)( o m ac o ) e lec s he con ibu ion o one pa icle, he second e m, S(q) (s uc u e ac o ) is based on he con ibu ion o he pa icle in e ac ion: spa ial a angemen 38 4 THEORETICAL BACKGROUND 4.5 Sca e ing me hods o he pa icles ela i e o an a bi a y o igin. S(q) = 1 in a dilu e solu ion, whe e he pa icles do no “ eel” hei neighbo s. 4.5.4 Teubne S ey app oxima ion SANS and SAXS sca e ing measu emen s o a bicon inuous phase show a ypical b oad peak when I(q)is plo ed. M. Teubne and R. S ey de eloped a heo y o desc ibe his peak [36], based on an o de expansion o he Landau ee ene gy F, which is gi en by: F=Z (ψ,∇ψ,∆ψ)d3 (41) ee ene gy densi y : =a0+a1ψ+a2ψ2+a3ψ3+a4ψ4+··· +c1(∇ψ)2+c2(∆ψ)2+... (42) In he case o mic oemulsions, a2>0, c1<0 and c2>0, all o he pa ame e s =0. This yields he sca e ing in ensi y dis ibu ion: I(q) = 8π/ξ (q−¯ q)2c2/V a2+c1q2+c2q4+bkg (43) wi h: (q−¯ q)2: mean squa e luc ua ion o he sca e ing densi y ρ. domain size (quasi pe iodic epea dis ance o he oil and wa e domains): d 2π=  1 2a2 c21/2 −c1 4c2  −1/2 (44) and he co ela ion leng h ξ(dispe sion o d): ξ=  1 2a2 c21/2 +c1 4c2  −1/2 (45) Fo i ing SANS and SAXS da a, eq. 43 will be simpli ied, he cons an s in he nume a o a e summa ized in k: k=8π/ξ < n2> /V(46) di iding eq. 43 by kc2: 39 4.5 Sca e ing me hods 4 THEORETICAL BACKGROUND I(q) = 1 a2 kc2+c1q2 kc2+q4 k =1 2+ 1q2+ 2q4(47) When ξis calcula ed simila o eq. 45, k is elimina ed and he esul is equal o eq. 45: ξ=  1 2 2 21/2 + 1 4 2  −1/2 =  1 2a2k kc21/2 +c1k 4kc2  −1/2 =  1 2a2 c21/2 +c1 4c2  −1/2 (48) Hence, he e a e only h ee pa ame e s le (wi h an addi ional backg ound pa ame e ) o i ing SANS and SAXS cu es wi h eq. 47. 4.5.5 D ople s uc u es D ople s uc u es can be app oxima ed by a i ing model o polydispe se ha d sphe es. Ac- co ding o equa ion 40 he sca e ing in ensi y o a sample can be calcula ed by ega ding he s uc u e ac o and o m ac o . In he case o sphe es, he o m ac o can be calcula ed by (η: sca e ing leng h densi y di e - ence be ween pa icle and ma ix, R: Radius o he sphe es): P(q,R) = 4 3πR3η·3sin(qR)−qRcos(qR) (qR)3(49) In mic oemulsion sys ems he d ople s a e polydispe se, he e o e a Schul z-Zimm SZ(R)dis- ibu ion can be assumed (Ra: scaling pa ame e - maximum o he dis ibu ion o la ge k, k=1/σ2,σ. a iance, Γ(k): Gamma- unc ion): SZ(R) = N Ra R Ra k−1kkexp(−k∗R/Ra) Γ(k)(50) Rega ding a monodispe se app oxima ion o he ha d sphe e s uc u e ac o S(q,R)(gi en by a ha d sphe e po en ial which depends on he olume ac ion o he sphe es and he sphe e adius), he in ensi y can be calcula ed by: I(q) =<P(q,R)2>S(q)(51) These calcula ions wi h he abo e men ioned assump ions can be done using he SAS i [63]o he GIFT [64,65]so wa e. 40 4 THEORETICAL BACKGROUND 4.5 Sca e ing me hods 4.5.6 Neu on-spin echo Wi h his echnique he dynamics o pa icles o s uc u es can be measu ed based on neu on sca e ing [66,67]. The accessible ime ange is 0.001 o 250ns a a wa eleng h o 6 o 25 A . selec o beam magne ic ield 1 magne ic ield 2 sample analyse pola ize de ec o π/2 lippe π lippe π/2 lippe Figu e 14: Simpli ied se up o a NSE expe imen in e e ence o [68]. A pola ized neu on beam is wa eleng h selec ed and en e s a magne ic ield B1(leng h L1) which is o ien a ed pe pendicula o he neu on pola iza ion. In he magne ic ield 1 he neu- ons unde go La mo p ecession. A e he magne ic ield, he neu on spins ha e a speci ic phase depending on hei eloci y . The La mo equency ωLis de ined by: ωL=γB(52) and he neu on spin ϕ( ): ϕ( ) = ωL =ωL1B1 (53) The pola iza ion Pxin he x-di ec ion is calcula ed by: Px=cosϕ(54) In he case o elas ic sca e ing in he sample, he neu ons en e he second magne ic ield B2 (leng h L2), which is o ien ed an ipa allel o B1. A e B2, he spin phase is: ϕ=γL1B1 −L2B2 (55) I L1B1=L2B2, he spin phase is 0 and Px=1 (maximum alue), which is called he spin echo poin . When he sca e ing o he sample is inelas ic, he kine ic ene gy o he neu ons changes a e 41 5.2 Sys ems 5 EXPERIMENTAL SECTION aining he p o ein dynamics is neu on spin echo. Bu he di icul y he e is he need o con as a ia ion. To measu e only he dynamics o he p o ein, he wa e - and oil-phase, su ac an and co-su ac an ha e o be deu e a ed. Deu e a ed su ac an s a e e y expensi e and no a ailable o e e y ype o suga su ac an . Hence, he me hod o choice is FCS. The componen s o he mic oemulsion don’ need o be deu e a ed and compa ed o o he me hods, quick measu emen s a e possible. Bu he main p oblem wi hFCS is he need o a luo escen pa icle. No mally, a luo escen label is bound o he ace pa icle, which should be obse ed. Howe e , he ideal way o ealizing he FCSexpe imen is he usage o a pa icle, which shows luo escence i sel . This minimizes he p oblem o an incomple e labeling, whe e unbound dye-molecules a e le and ep esen an addi ional dynamical componen . This complica es he i ing p ocess o he au oco ela ion unc ion. con ined di usion a achemen a he in e ace wa e phase oil phase su ac . co-su . p o ein luo escen amphiphil Figu e 16: Model o pa icles in he wa e domain o a bicon inuous mic oemulsion. Fluo escen su ac an s a ach a he in e ace, hyd ophilic p o eins a e dissol ed in he wa e phase. An app op ia e choice o his wo k is he GFP (compa e igu e 17), pa icula ly GFP+. This p o- ein was ound in he jelly ish Aequo ea ic o ia, which was i s ex ac ed a 1962 [77]. GFP+ shows a 320 imes highe luo escence in ensi y han he wild ype p o ein [78]. Compa ed o DFPase wi h a molecula weigh o 35kDa, GFP+has app oxima ely 27kDa, he e- o e he size is simila . The exci a ion wa eleng h wi h maximum abso p ion is λGFPexc =490nm, he emission maximum is λGFPem =510nm. GFP shows quan um yields up o 0.77 [79]and is s able up o 65 ◦C[80]. 48 5 EXPERIMENTAL SECTION 5.2 Sys ems (a) (b) Figu e 17: X- ay s uc u es o (a) G een Fluo escen P o ein [75](b) mChe y P o ein [76]. These molecules a e used as ace pa icles. An al e na i e a ailable p o ein wi h simila size is mChe y om he co al “Discosoma sp” [81] wi h a molecula weigh o ≈27 kDa [76], i shows luo escence a highe wa eleng hs han GFP+( ig. 18). The e o e i can be used wi h he 543nm beampa h. 49 5.2 Sys ems 5 EXPERIMENTAL SECTION 5.2.3 P o ein luo escence To cha ac e ize he used luo escing pa icles, luo escence spec oscopy measu emen s we e pe o med on a Jasco FP-6500 spec ome e . In addi ion o no mal I(λ) luo escence in ensi y measu emen s a a ixed exci a ion wa eleng h, so called 3D-measu emen s a e possible. A 3D-measu emen s di e en exci a ion wa eleng hs a e used. The esul o hese measu emen s a e depic ed using a con ou plo . The eby, he maximum exci a ion/emission can be ob ained easily. ∆λexc was 5nm in all 3D-measu emen s. The 3D plo s ( ig. 19 and 20) o he luo escence in ensi y measu emen s o GFP+and mChe y indica e, ha he op imum a ailable FCS lase -exci a ion-wa eleng h is he A gon-Ion 488nm line o GFP+and he 543nm HeNe line o mChe y. The luo escence spec a o he exci a ion wi h he lase co esponding wa eleng h a e plo ed in igu e 18. Bo h p o eins a e sligh ly smalle han he DFPase used o decon amina ion applica ions. Bu hei main ad an age, he luo escence, gi es he oppo uni y o obse e he dynamics inside he wa e phase o a bicon inuous phase. no malized in ensi y 0 0.2 0.4 0.6 0.8 1 emission wa eleng h / nm 500 550 600 650 700 GFP+ mChe y Figu e 18: Fluo escence in ensi y o GFP+wi h an exci a ion wa eleng h o 490nm and he maxi- mum in ensi y a 512nm and mChe y wi h an exci a ion wa eleng h o 545nm and he maximum in ensi y a 604nm. 50 5 EXPERIMENTAL SECTION 5.2 Sys ems exci a ion wa eleng h / nm emission wa eleng h / nm 620 600 580 560 540 520 500 480 460 no malized in ensi y 0 0.2 0.4 0.6 0.8 1 350 400 450 500 550 Figu e 19: Fluo escence con ou plo o he p o ein GFP+, wi h an exci a ion maximum λexc-max = 490nm and emission maximum λem−max =510nm emission wa eleng h / nm 700 680 660 640 620 600 580 560 540 520 no malized in ensi y 0 0.2 0.4 0.6 0.8 1 exci a ion wa eleng h / nm 500 550 600 650 Figu e 20: Fluo escence con ou plo o he p o ein mChe y, wi h an exci a ion maximum λexcmax =590nm and emission maximum λemmax =600nm 51 5.3 Expe imen al de ails o he FCS measu emen s 5 EXPERIMENTAL SECTION 5.3 Expe imen al de ails o he FCS measu emen s The FCS measu emen s we e pe o med wi h he Zeiss Con oco 2 FCS-sys em. Usually a 0.5ml sample was p epa ed and 60µl we e ans e ed in o a sample holde , composed o wo s eele pla es (one pla e wi h a co e glass window, hickness 0.15mm), compa e igu e 21, al eady in oduced om H. Ze l [82]a he same FCS-sys em. The pla es a e ixed o each o he by sc ews and an O- ing p e en s he sample om e apo a ing. Fo measu emen s wi h GFP+, he exci a ion wa eleng h was 488nm (a gon-ion) and o oc adecyl- hodamin B he wa eleng h was 543nm (helium-neon). s eel pla e co e glass sample Figu e 21: FCS sample chambe , composed o wo s eel pla es wi h a glued on co e glass. g2( ) 1 2 3 4 5 / μs 1 100 1e+04 Figu e 22: Calib a ion o he 543nm beampa h wi h he dye hodamin B The beam pa h o he FCS ins umen has o be adjus ed ou inely. The e o e, he pinhole is shi ed in he x-, y- and z-di ec ion. This is done in h ee s eps, i s x, hen y, hen z, while he espec i e o he alues a e hold cons an . The in ensi y is measu ed du ing he pinhole is shi ed and he posi ions yielding he maximum alues a e used o he measu emen s (compa e ig. 23). 52 5 EXPERIMENTAL SECTION 5.4 DLS measu emen s (a) (b) (c) Figu e 23: Con oco 2 pinhole adjus men wi h hodamin B, (a) x-di ec ion, (b) y-di ec ion and (c) z-di ec ion. The alue wi h he maximum in ensi y is held cons an o he measu emen s. To de e mine he adius o he obse a ion olume, a measu emen is pe o med wi h a dye o known di usion coe icien 22, o he 488nm beampa h his yields o 0=254nm and o he 543nm beampa h 0=261nm. 5.4 DLS measu emen s Fo DLS measu emen s 1mm samples we e p epa ed and illed in a glass cu e e which is cleaned by e hanol o a oid sca e ing om dus pa icles. Measu emen s a e pe o med a ele en di - e en angles Θin 10◦s eps be ween 40◦and 140◦wi h a measu emen ime o h ee minu es pe angle a a lase wa eleng h o 543nm. The lase in ensi y was adjus ed o each a su icien coun a e (≈200kHz) o e he whole angula ange. The ob ained au oco ela ion cu es a e ea ed wi h he CONTIN algo i hm which esul s in di e en Γ o e e y Θ.qcan be calcula ed om Θand a Γ(q2)plo i ed linea ly, leads o he di usion coe icien D. 53 5.5 SANS/SAXS measu emen s 5 EXPERIMENTAL SECTION 5.5 SANS/SAXS measu emen s The s uc u al dimensions in a mic oemulsion a e on he nanome e scale. The e o e, only a ew possibili ies exis o in es iga e hese s uc u es. Wi h pho on co ela ion spec oscopy he hyd odynamic adius o geome ically simple objec s like sphe es would be easy o measu e. Bu in he bicon inuous phase he s uc u e is e y complex. One possibili y o obse e such small s uc u es is cons i u ed by elec on mic oscopy. Fo ha he sample has o be ozen. The eezing p ocess happens no ins an ly, he s uc u e o he sample may change. Hence, a echnique is necessa y which enables he obse a ion o he s uc u e in si u. SAXS is a sca e ing me hod wi h a wa eleng h much lowe han isible ligh , he mic oemulsion s uc u es can be measu ed. X- ay adia ion is sca e ed by he elec on shell o he a oms. In a SANS measu emen neu ons a e sca e ed by he a om co es. Due o ha , one can play wi h he deu e a ion o he sample: Ei he he oil o wa e componen is deu e a ed, hen he s uc u es o he wa e /oil domains shine ou (bulk dynamics), o he wa e and oil componen s a e deu e a ed, hen he su ac an in e ace is domina ing he signal ( ilm dynamics). Based on expe ience, when he wa e componen o a mic oemulsion is eplaced by D2O, he 1-phase boa de shi s o lowe δ- alues, which was al eady obse ed o CiEjmic oemulsions [83]. This ci cums ance has o be ega ded when a sample is composed o neu on sca e ing. Fo d ople mic oemulsions i was al eady shown by Huang and Wu [84], ha he eplacemen o H2O by D2O does no change he s uc u e sizes o he d ople s. The heo e ical basis o SANS and SAXS on he bicon inuous phase was in oduced by M. Teub- ne and R. S ey [36]. The model which desc ibes he sca e ing beha io o a bicon inuous phase is depic ed in 4.5.4. The sca e ing in ensi y depending on he sca e ing ec o qpic- u es a b oad peak, whe e he domain size dand co ela ion leng h ξcan be ob ained by i ing he da a wi h he Teubne -S ey equa ion. The i s we e pe o med wi h he Q iplo [85]so - wa e using he Nelde -Mead Simplex, which gene a es a sequence o simplexes wi h dec easing diame e and accumula e a ound he desi ed minimum [86]. 5.5.1 SAXS se up The SAXS measu emen s we e pe o med on a SAXSLAB Ganesha ins umen [87]( ig. 24) a a sample - de ec o dis ance o 1.2m. I is an inhouse SAXS sys em which o e s a mode n 54 5 EXPERIMENTAL SECTION 5.5 SANS/SAXS measu emen s high esolu ion 2D-Pila us 300k de ec o , a o a ing anode, a pinhole collima ion sys em and a mul iple sample holde wi h au oma ic alignmen . The X- adia ion sca e ed by he sample in an angle o Θen e s an e acua ed olume, whe e a de ec o can be mo ed o une he q- ange and esolu ion. The de ec ed 2D in ensi y “image” is a e aged adially o ob ain he in ensi y da a depending on q. mo able 2D de ec o x- ay sou ce Θ beams op sample holde e acua ed ube beam Figu e 24: Se up o he Ganesha SAXS sys em. By mo ing he de ec o he obse ed sca e ing angle Θcan be adjus ed. In he sample holde mo e samples can be measu ed in a ow. 5.5.2 SANS se up A neu on sca e ing ins umen is compa able o a X- ay sca e ing se up. In mos cases he neu on sou ce is a scien i ic nuclea eac o , whe e se e al beamlines a e p o ided wi h neu- ons o di e en eloci ies depending on he expe imen . In a small angle measu emen , he dis ance be ween sample and de ec o is up o 40m (D11 /ILL), he desi ed q- ange can be uned by he dis ance. The se up is shown in igu e 25. x/y de ec o acuum sample collima ion sli s (adjus able) eloci y selec o beam shu e choppe neu on beam Figu e 25: P incipal se up o a SANS expe imen using he example o he PAXY ins umen (LLB Saclay /F ance, [88]) 55 5.5 SANS/SAXS measu emen s 5 EXPERIMENTAL SECTION No mally he in ensi y is measu ed o h ee di e en dis ances o ob ain a good esolu ion o e he whole q- ange. The da a e alua ion is undamen ally compa able o SAXS sca e ing, he esul is an in ensi y I(q)plo which can be app oxima ed wi h an app op ia e model. 56 6 RESULTS AND DISCUSSION 6 Resul s and discussion 6.1 Fluo escence measu emen s Fi s o all, he p oblem o luo escen impu i ies usingsuga su ac an sys ems will be discussed. A e wa ds, he s uc u es o mic oemulsion sys ems will be cha ac e ized by SAS measu emen s and inally, di usion measu emen s o GFP+inside he wa e phase o di e en bicon inuous s uc u es a e shown. 6.1.1 Fluo escen impu i ies A cen al complexi y in he wo k wi h mic oemulsions based on a suga su ac an is he exis- ence o luo escen impu i ies. Suga su ac an s and co-su ac an s exhibi a weak luo escen ac i i y in he isible ange. FCS and luo escence spec oscopy measu emen s show, ha he concen a ion o he impu i y-molecules is in he ange o 10−7−10−8mol/l. This is a p oblem, because i ’s an app op ia e concen a ion o FCS-measu emen s. emission wa eleng h / nm 750 700 650 600 550 500 no malized in ensi y 0 0.2 0.4 0.6 0.8 1 exci a ion wa eleng h / nm 450 500 550 600 650 700 Figu e 26: Con ou plo o he luo escence in ensi y o 1-pen anol in he exci a ion ange om 400nm o 800nm wi hou luo escen label. In ig. 26 he luo escence spec um o 1-pen anol is shown. No luo escen label was added o he sample, bu s ill a b oad luo escen ac i i y nea ly o e he whole isible ange is de ec able. The lines in he 3D plo e eal Raman sca e ing, no luo escence. 57 6.2 The C9G2based mic oemulsion sys em 6 RESULTS AND DISCUSSION 0 0.05 0.10 0.15 0 0.05 0.10 0.15 amoun o su ac an (γ) amoun o co-su ac an (δ) bicon . Φ = 0.1 Φ = 0.2 Φ = 0.3 Φ = 0.4 Φ = 0.5 Φ = 0.6 Φ = 0.7 Figu e 33: Phase diag ams o he C9G2sys em o di e en oil/wa e a ios Φ, he connec ion o he X-poin s desc ibe a pa abolic ajec o y. Table 1: Composi ion o he C9G2mic oemulsion samples o SAS and FCS measu emen s oil/wa e a io αsu ac an amoun γalcohol con en δ 0.1 0.08 0.035 0.2 0.11 0.055 0.3 0.12 0.075 0.4 0.14 0.10 0.5 0.12 0.105 0.6 0.12 0.145 0.7 0.11 0.17 6.2.2 S uc u e sizes Leng h scales by SANS The s uc u e o he p esen ed C9G2sys em was in es iga ed by sca e ing me hods applied o he samples shown in able 1. The SANS measu emen s we e pe o med in Saclay/F ance a 64 6 RESULTS AND DISCUSSION 6.2 The C9G2based mic oemulsion sys em he LLB (Labo a oi e Léon B illouin) and in Be lin a he HZB (Helmhol z-Zen um-Be lin). The in ensi y cu es shown in igu e 34 exhibi a s uc u e peak o oil/wa e a ios o 0.3 o 0.5. The Teubne -S ey app oxima ion is only sui able o olume ac ions close o 0.5, which applies o he Φ=0.3 o 0.5 samples. Then he co ela ion leng h and domain size can be calcula ed om he i pa ame e s, discussed in chap e 4.5.4. The esul s a e shown in able 2. I(q) 1 100 10,000 q / ÅÅ-1 0.01 0.1 0.3 0.4 0.5 Figu e 34: SANS in ensi y measu emen o C9G2mic oemulsions wi h a ying Φ. The da a was ea ed wi h he Teubne -S ey app oxima ion. The i ing esul s a e ep esen ed by he solid lines. As a consequence o he deu e a ed wa e phase, ξ e lec s he co ela ion leng h o he oil domains. ξinc eases o ising Φ, in e u n, he wa e domains sh ink ( ig. 35). The domain size ds ays nea ly cons an a ound 25nm, which e lec s he epea ing leng h o he oil and he wa e domains oge he . The wa e domain size could be es ima ed by sub ac ing he co ela ion leng h om he domain size, bu hen he su ac an in e ace is no ega ded. Table 2: S uc u e sizes o C9G2-mic oemulsions de e mined by SANS oil/wa e a io Φ olume a io co ela ion leng h ξ/nm domain size d/nm 0.3 0.35 7.7 25.9 0.4 0.46 8.2 24.7 0.5 0.56 9.3 26.7 Also o smalle alues o ΦSANS measu emen s we e pe o med, he in ensi y cu es a e shown 65 6.2 The C9G2based mic oemulsion sys em 6 RESULTS AND DISCUSSION co ela ion leng h /nm 7 7,5 8 8,5 9 9,5 10 oil/wa e a io Φ 0,25 0,3 0,35 0,4 0,45 0,5 0,55 Figu e 35: Co ela ion leng h which esul s om he Teubne -S ey app oxima ion o he C9G2 sys em depending on he oil/wa e a io Φ. The inc ease wi h ising Φmeans g owing oil domains, he e o e sh inking wa e domains. in ig. 36. No peak a ises in he I(q)plo , he e o e Teubne -S ey model is no app op ia e. Ano he way o ex ac in o ma ion om he measu emen s a smalle Φwi hou s uc u e peak is o ake ad an age o he Guinie law: The SANS da a in a double loga i hmic plo ollows a linea dec ease a low q- alues (Guinie - egime) [94]. Φ = 0.1 I(q) / a.u. 0 100 200 300 400 q / ÅÅ-1 0.01 0.1 Φ = 0.2 I(q) / a.u. 0 200 400 600 800 q / ÅÅ-1 0.01 0.1 Figu e 36: SANS in ensi y measu emen o C9G2mic oemulsions wi h a iing Φ. The da a canno be i ed wi h he Teubne -S ey model. 66 6 RESULTS AND DISCUSSION 6.2 The C9G2based mic oemulsion sys em Table 3: Resul s om he scaling analysis in ig. 37 o he sys em C9G2wi h he esul ing slope depending on Φ Φslope ms uc u e 0.1 -0.64 ellipsoidal 0.2 -0.25 ellipsoidal/sphe ical 0.7 -0.71 ellipsoidal/cylind ical log(I) -1 0 1 2 3 4 5 log(q) -2.5 -2 -1.5 -1 -0.5 oil/wa e a io Φ 0.7 0.2 0.1 Figu e 37: Double loga i hmic plo o he SANS measu emen o C9G2mic oemulsions wi h a ying Φ. The slope in he low q- ange indica es he shape o obse ed s uc u e. The slope in he Guinie - egime shows, wha s uc u e is p esen in his mic oemulsion samples. Because o he oil/wa e a io oil d ople s inside a con inuous wa e phase o small alues o Φand wa e d ople s inside an oil con inuous phase o high alues o Φoccu . The loga i hmic plo s o he in ensi y cu es a e shown in ig. 37. While a slope m=−1 indica es cylind i- cal objec s, m=0 means sphe ical pa icles and a slope mo 0 o -1 implies ellipsoidal objec s. In he C9G2sys em mis app ox. −1 o Φ=0.1 and 0.7 and nea 0 o Φ=0.2, compa e able 3. Leng h scales by SAXS In addi ion o he SANS obse a ions, also SAXS in es iga ions we e pe o med o e i y he SANS esul s. The elec on densi y is c ucial o he sca e ing p ocess o X- ays. The elec on 67 6.2 The C9G2based mic oemulsion sys em 6 RESULTS AND DISCUSSION dis ibu ion is di e en o wa e domains, oil domains and he su ac an ilm. In con as o neu on sca e ing expe imen s, whe e he deu e a ion is impo an o he sca e ing con as , he elec on densi y canno be a ied wi hou changing he sol en s and he e o e changing he s uc u e o he mic oemulsion sys em. I(q) 1 100 1e+04 1e+06 1e+08 q / nm-1 0.1 1 0.1 1 oil/wa e a io Φ 0.7 0.6 0.5 0.4 0.3 0.2 0.1 Figu e 38: SAXS measu emen s o se ies o he di e en mic oemulsion C9G2samples wi h di e en Φ. The lines ep esen i s acco ding o he Teubne -S ey app oxima ion, which is no possible o Φ0.6 and 0.7. The in ensi y o he Φ0.2 ... 0.7 was shi ed o cla i y easons. Fo he measu emen s samples iden ical o SANS we e used ( able 1). The in ensi y cu es shown in igu e 38 a e again i ed wi h he Teubne -S ey app oxima ion, he esul s a e sum- ma ized in able 4. Compa ed o he SANS esul s o he analogous samples, he domain sizes ob ained om he SAXS measu emen s a e simila . The co ela ion leng hs di e because he deu e a ion o he wa e phase in he SANS samples implies, ha he co ela ion leng h alues apply o he oil domains. Howe e , in a SAXS measu emen , he oil o wa e domains canno be 68 6 RESULTS AND DISCUSSION 6.2 The C9G2based mic oemulsion sys em Table 4: S uc u e sizes o C9G2-mic oemulsions de e mined by i ing he SAXS da a wi h he Teubne -S ey app oxima ion. oil/wa e a io Φ olume a io co ela ion leng h ξ/nm domain size d/nm 0.1 0.12 4.55 15.11 0.2 0.24 6.11 18.45 0.3 0.35 9.77 24.21 0.4 0.46 10.27 22.85 0.5 0.56 9.25 23.79 clea ly sepa a ed. The SAXS- esul end in igu e 39 is in good acco dance o he SANS esul s, he e is only a small de ia ion due o he di e en sca e ing con as s. ξ, d / nm 0 5 10 15 20 25 30 oil/wa e a io Φ 0 0.1 0.2 0.3 0.4 0.5 ξSAXS ξSANS dSAXS dSANS Figu e 39: C9G2mic oemulsion domain sizes d and co ela ion leng hs ξde i ed om he Teubne - S ey app oxima ion compa ed o he SANS esul s o chap e 6.2.2. 69 6.2 The C9G2based mic oemulsion sys em 6 RESULTS AND DISCUSSION 6.2.3 GFP+dynamics by FCS The co ela ion leng hs o nea ly 10nm o he oil phase (comp. able 4) imply a simila alue o he wa e domains a Φ=0.5. These leng hs a e s ill big enough o allow di usion o GFP+wi h a diame e o app oxima ely 5nm. Howe e , he domain size o he wa e domains is al eady su icien ly low o p oduce a no able con inemen o c owding e ec o he p o ein. Hence, o he analysis i he da a he model in oduced by Weiss has o be used [56]. g2 ( ) 1 1.2 1.4 1.6 1.8 ime / μs 10 100 1,000 1e+04 1e+05 1e+061e+06 GFP+ in bu e GFP+ in Φ=0.2 mic oemulsion GFP+ in Φ=0.35 mic oemulsion Φ=0.2 mic oemulsion dynamics Figu e 40: Compa ison o FCS au oco ela ion cu es o GFP+in a C9G2mic oemulsion. GFP+ shows a sligh ly slowe di usion inside he wa e phase o he mic oemulsion, bu as e han he mic oemulsion dynamics. The p o ein is non-pola and mainly hyd ophilic. The e o e i is con ined o he wa e domains o he mic oemulsion. Figu e 40 shows au oco ela ion cu es o he C9G2sys em. The p o ein is able o mo e inside he wa e domains, and he di usion is slowe in a bicon inuous s uc u e (Φ0.3), han in a wa e con inuous s uc u e (Φ0.1). As expec ed, he di usion is hinde ed by he con inemen o he wa e domains. C owding e ec s ake place o Φ0.1, whe e oil-in-wa e s uc u es (see able 3) appea , which al eady esul s in a slowed di usion a his low alue o Φcompa ed o GFP+in bu e . 70 6 RESULTS AND DISCUSSION 6.2 The C9G2based mic oemulsion sys em g2 ( ) 1 1.2 1.4 1.6 1.8 ime / μs 10 100 1,000 1e+04 1e+05 oil/wa e a io Φ 0.1 0.2 0.35 0.5 0.6 0.7 Figu e 41: GFP+FCS au oco ela ion cu es inside he wa e domain o he C9G2mic oemulsion o di e en Φ. Wi h ising Φ he cu es mo e o bigge imescales. The lines indica e i s acco ding o equa ion 21. g2( ) 1 1.5 2 ime / μs 1 10 100 1,000 1e+04 1e+05 1e+061e+06 0.1 0.2 0.3 0.4 0.5 Figu e 42: No malized FCS au oco ela ion cu es o he C9G2mic oemulsion o di e en Φla- beled wi h oc adecyl- hodamin B. Wi h ising Φ he cu es mo e o smalle imescales. Oc adecyl- hodamin B is amphiphilic and inco po a es in he in e ace. The lines indica e i s acco ding o equa ion 16. au oco ela ion cu es shi ing o bigge imescales ( igu e 42). In ig. 43 he di usion imes τsub o GFP+a e plo ed. The da a was i ed wi h an anomalous di usion model (equa ion 21). 71 6.2 The C9G2based mic oemulsion sys em 6 RESULTS AND DISCUSSION Fo small Φ he di usion is nea ly as as as in dilu e solu ion. τsub / μs 500 1,000 1,500 oil/wa e a io Φ 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 GFP+ in ME ME dynamics GFP+ in ME (2 comp) Figu e 43: Subdi usion imes τsub o GFP+in he wa e phase o a C9G2mic oemulsion, calcula ed om he i o he FCS au oco ela ion cu es. In he ange Φ=0.3 o 0.5, he s uc u e changes o bicon inuous and he di usion is hinde ed by he sponge like phase. A Φ=0.6 and 0.7 τsub is cons an and e lec s he dynamics o he mic oemulsion s uc u e - his is an indica o o he con inemen o he p o ein, i seems o be s uck in he mic oemulsion and pa icipa es in he collec i e mo ion o i . When compa ing he au oco ela ion cu es o he mic oemulsion dynamics and he GFP+au oco ela ion cu es in a mic oemulsion, he GFP+cu es look mo e shallow. This is an indica o o anomalous di usion [95]. The anomalous di usion exponen αcan also be ex ac ed om he i . Fig. 44 shows he anomalous di usion exponen . A small Φαis nea ly 1, he p o ein is ha dly con ined by he mic oemulsion s uc u e, which is mo e d ople -like (sligh ly elonga ed d ops, see SANS esul s) han bicon inuous o Φ=0.1 and 0.2. When he s uc u e is bicon inuous s a ing a Φ=0.3, αdec eases apidly and indica es, ha he p o ein is con ined by he wa e domains. F om Φ=0.6 on, αinc eases again, which shows, ha he p o ein ge s now s uck in he s uc u e and pa icipa es in he di usi e b ea hing mo ion o he mic oemulsion ma ix. 72 6 RESULTS AND DISCUSSION 6.2 The C9G2based mic oemulsion sys em α 0.5 0.6 0.7 0.8 0.9 1 oil/wa e a io Φ 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Figu e 44: Anomalous di usion pa ame e α o GFP+in he C9G2sys em. Wi h an α < 1 subdi - usion is obse ed, especially o Φ>0.2. 73 6.3 The C12G2based mic oemulsion sys em 6 RESULTS AND DISCUSSION o 0.56. A Φ0.6, he adius and he olume ac ion suddenly d op o much smalle alues. This is due o he inapp op ia e i o a s uc u al change o wa e d ople s in an oil con inuous phase. R / nm 4 6 8 10 12 14 oil/wa e a io Φ 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 SAXS SANS olume ac ion 0.2 0.3 0.4 0.5 0.6 oil/wa e a io 0 0.2 0.4 0.6 0.8 (a) (b) Figu e 52: Resul s o he GIFT analysis o he C12G2mic oemulsion samples, (a) sphe e adius R (SANS and SAXS alues), inc eases due o he ising amoun o oil and dec eases o Φ0.6 and 0.7 (b) olume ac ion o he sphe es (SAXS), inc eases as well up o Φ=0.5. The SAXS d ople adius esul s a e in good ag eemen wi h he SANS ou comes, excep o he Φ0.1 alue. This could be connec ed o he di e se sca e ing con as s and he di e en analysis me hods. The polydispe si y is nea ly cons an a app oxima ely 20%, which is in good ag eemen wi h he esul s o compa able CiEjsys ems [98]. 80 6 RESULTS AND DISCUSSION 6.3 The C12G2based mic oemulsion sys em 6.3.3 GFP+dynamics by FCS The SAS esul s indica e, ha he s uc u e is no bicon inuous, he oil componen is loca ed as d ople s in a wa e con inuous phase, especially o Φ<0.6. This should allow GFP+di usion inside he wa e -(bu e -) phase. Bu wi h a olume a io ac ion up o 50%, he su ounding o he p o ein is c owded. Figu e 53 shows he si ua ion o a p o ein di using in he wa e con inuous phase o a mic oemulsion. wa e phase oil phase su ac . co-su . p o ein luo escen amphiphil hinde ed di usion Figu e 53: Di usion model o he C12G2sys em, whe e he s uc u e is no bicon inuous bu d ople - like. The oil d ople s in a con inuous wa e phase a e polydispe se and bigge han he p o ein size. A Φ0.1 he adius is compa able o he GFP+hyd odynamic adius, he di usion should be nea ly unhinde ed. Fo ising Φ he d ople s g ow and me ge wi h o he d ople s. The sys em ge s c owded and hampe s he di usion o he p o ein. To obse e he GFP+dynamics, FCS measu emen s we e pe o med. 81 6.3 The C12G2based mic oemulsion sys em 6 RESULTS AND DISCUSSION no malized g2 ( ) 1 1.2 1.4 1.6 1.8 ime / μs 10 100 1,000 1e+04 1e+05 GFP in bu e GFP in Φ 0.1 ME GFP in Φ 0.3 ME Φ 0.1 ME Φ 0.3 ME Figu e 54: FCS au oco ela ion da a o GFP+in a C12G2mic oemulsion, compa ed o GFP+in bu e and he pu e mic oemulsion dynamics. The cu es we e no malized o compa ison easons. The mic oemulsion shows much slowe dynamics han GFP+inside he wa e domain. Hence, he p o ein is mobile. no malized g2 ( ) 1 1.2 1.4 1.6 1.8 ime / μs 10 100 1,000 1e+04 1e+05 oil/wa e a io Φ 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Figu e 55: GFP+FCS au oco ela ion cu es inside he wa e domain o he C12G2mic oemulsion o di e en Φ. As seen in igu e 54, he GFP+measu emen s show a hinde ed di usion o GFP+inside he mic oemulsion compa ed o GFP+in bu e : The di usion is hinde ed by c owding and con- inemen e ec s. Howe e , unde hese condi ions he p o ein mo es s ill signi ican ly as e 82 6 RESULTS AND DISCUSSION 6.3 The C12G2based mic oemulsion sys em compa ed o he slow collec i e mode (b ea hing mode) o he mic oemulsion. di usion ime / ms 0,5 1 1,5 oil/wa e a io Φ 0 0,2 0,4 0,6 0,8 GFP+ dilu e GFP+ in ME ME dynamics Figu e 56: Compa ison o he FCS di usion imes in he mic oemulsion sys em C12G2 o GFP+ inside he wa e domain o he mic oemulsion, GFP+in bu e and he mic oemulsion dynamics. In igu e 55 he FCS measu emen s wi h samples a di e en Φ( able 5). In gene al, he au o- co ela ion cu es mo e o highe imes wi h inc easing Φ. This clea ly shows ha he dynamic is slowed down. The i esul s a e summa ized in igu e 56. Fo small Φ(0.1 and 0.2), GFP+ shows a good mobili y because o small oil d ople s in a con inuous wa e phase. These d ople s hinde he di usion o he p o ein no signi ican ly. Fo high Φ(0.6 and 0.7), he p o ein dy- namics ge simila o he mic oemulsion dynamics. This clea ly shows, ha GFP+is s uck and can only mo e wi h he collec i e b ea hing mo ion o he mic oemulsion s uc u e. As seen in ig. 57, αis smalle han 1, which indica es subdi usi e beha io . α o he 1- componen i dec eases un il a minimum a Φ0.5, he p o ein ge s mo e and mo e con ined by he sh inking wa e domains. A Φ0.6, αinc eases again, which indica es, ha he p o ein is s uck in he bicon inuous s uc u e leading o he eco e y o Fickian di usion. The b ea hing mo ion o he mic oemulsion s uc u e is a di usi e mo ion., Hence, αcon e ges o 1. 83 6.3 The C12G2based mic oemulsion sys em 6 RESULTS AND DISCUSSION anomalous di . exp. α 0,6 0,7 0,8 0,9 oil/wa e a io Φ 0,2 0,4 0,6 0,8 1-componen i 2-componen i Figu e 57: Anomalous di usion exponen α o GFP+in he C12G2sys em. Wi h an α < 1 subdi - usion is obse ed. The FCS da a can also be app oxima ed wi h a 2 componen model, o ega d he mic oemulsion dynamics, which is measu ed also, due o he luo escen impu i ies, αbeha es simila . Fo small oil/wa e a ios αis nea ly one. This means only mino c owding e ec s, un il Φ=0.4 i dec eases o a minimum. Then α ises again because o he di usi e mic oemulsion dynamics. Bu he 2-componen i is no eally alid om α=0.5 up o highe alues. The e he p o ein is s uck and he di usion shows only one cha ac e is ic ime decay. These anomalous di usion exponen esul s a e in he same ange compa ed o in es iga ions o p o eins in c owded solu ions [99], he e he au ho s ound α alues om 1 o 0.75. 84 6 RESULTS AND DISCUSSION 6.3 The C12G2based mic oemulsion sys em 6.3.4 Mic oemulsion dynamics (FCS) Knowing ha he s uc u e o he C12G2samples a Φ=0.1 −0.6 is d ople -like, mo e can be ex ac ed om he FCS measu emen s. The di usion ime ob ained om he FCS measu e- men s allows o compu e he di usion coe icien . By using he S okes-Eins ein equa ion also he hyd odynamic adius o he d ople s can be calcula ed. Figu e 58 shows he no malized FCS au oco ela ion cu es. no malized g2 ( ) 1 1.2 1.4 1.6 1.8 2 ime / μs 1 10 100 1,000 1e+04 1e+05 1e+061e+06 oil/wa e a io Φ 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Figu e 58: FCS au oco ela ion cu es o he mic oemulsion dynamics o di e en oil/wa e a ios Φ. The lines ep esen a 1-componen i . Because o he high polydispe si y o he sys em he ob ained alues o he adius o he d ople s a e only app oxima ed esul s. The da a could also be i ed wi h a 2- o 3-componen model. Bu o a con incable i mo e pa ame e s like polydispe si y o he dis ibu ion o di usion imes should be known. The esul s o he hyd odynamic adius compa ed o alues ob ained by o he me hods a e plo ed in igu e 59. 85 6.3 The C12G2based mic oemulsion sys em 6 RESULTS AND DISCUSSION 6.3.5 Mic oemulsion dynamics (DLS) In he SAS expe imen s a d ople mic oemulsion s uc u e was iden i ied. The e o e samples can also be s udied by dynamic ligh sca e ing. Wi h he esul ing appa en di usion coe i- cien DDLS, a hyd odynamic adius RHcan be calcula ed wi h equa ion 33. The S okes-Eins ein equa ion is no ully alid in his case because o polydispe se and in e ac ing sphe es. Bu he hyd odynamic adius can be es ima ed and he e o e i is called appa en hyd odynamic adius. The sca e ing con as o pho ons in he isible ange is cons i u ed by he e ac ion index di - e ence in he sys em. In mic oemulsions wi h a bicon inuous o d ople phase, his di e ence is mainly caused by he oil and wa e componen . The e o e, he ob ained di usion coe icien s ep esen he collec i e b ea hing mo ion o he domains o he mo emen o oil-in-wa e o wa e -in-oil s uc u es, espec i ely. R / nm 0 5 10 15 20 25 oil/wa e a io Φ 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Rh, app (DLS) Rh, app (FCS) RSAXS Figu e 59: Appa en hyd odynamic DLS and FCS adius and adius o he SAXS measu emen s de e mined by he GIFT analysis o he C12G2mic oemulsion sys em a di e en oil/wa e a ios Φ. All h ee me hods show an inc ease o he d ople adius om Φ=0.1 o 0.5, a 0.6 he adius dec eases apidly o smalle alues. Compa ed o he SAXS measu emen s, he DLS esul s show he same end. Rhexhibi s bigge alues because o he c owded d ople s which mo e slowe in compa ison wi h a dilu e solu ion. Fu he mo e, he hyd odynamic adius can no be di ec ly compa ed o a adius o gy a ion Rg om he small angle measu emen s. Fo ha d sphe es he a io Rg/Rhis expec ed o be 0.778 [100], he esul s in ig. 59 yield a a io o app oxima ely 0.6, which is an indica ion 86 6 RESULTS AND DISCUSSION 6.3 The C12G2based mic oemulsion sys em o sphe ical d ople s. The FCS measu emen s a e in good ag eemen wi h he DLS and SAXS esul s. A e Φ=0.5 he e is also a simila d op in he adius. 87 6.4 O he mic oemulsion sys ems 6 RESULTS AND DISCUSSION 6.4 O he mic oemulsion sys ems Fu he wo k was done aiming a he s udy o o he sys ems wi h su ac an s ha ing sho e alkyl chain leng hs. Namely he su ac an s n-hexyl-β-d-glucoside (C6G1) and n-hep yl-β-d-glucoside (C7G1) we e s udied using again wa e , cyclohexane, and 1-pen anol as he o he componen s. I u ns ou ha he phase beha io exhibi s no lamella phase in he γ ange, whe e he sys ems we e sc u inized. The phase diag ams a e gi en in ig. 60. While o C6G1 he bicon inuous phase s a s a γ=0.16, in he C7G1sys em he X-poin is loca ed γ=0.22, bu less alcohol con en is needed o o m a single-phase mic oemulsion. In gene al, he CiG2mic oemulsions end mo e o o m a lamella phase compa ed o he CiG1sys ems. 0.05 0.10 0.15 0.20 0.10 0.15 0.20 0.25 0.30 (γ) (δ) 3Φ 1Φ 2Φ 2Φ 0 0.05 0.10 0.15 0.20 0.10 0.15 0.20 0.25 0.30 0.35 (γ) (δ) 3Φ 1Φ 2Φ 2Φ C6G1C7G1 Figu e 60: Phase diag ams o C6G1and C7G1mic oemulsion sys ems (wa e , cyclohexane, 1- pen anol), no lamella phase shows up in he s udied γ ange. SANS measu emen s we e ca ied ou o C7G1samples in Saclay/F ance a he PAXY ins u- men . Th ee samples wi h di e en γ(0.25, 0.30, 0.35) and cons an Φand δ alues we e composed. The in ensi y cu es a e plo ed in ig. 61. The sca e ing cu es exhibi he ypical shape ob ained o bicon inuous mic oemulsions. Hence, he Teubne -S ey app oxima ion was applied o ob ain leng h scales. 88 6 RESULTS AND DISCUSSION 6.4 O he mic oemulsion sys ems I (q) 1 10 100 1,000 1 10 100 1,000 q / Å-1 0.1 11 0.1 11 γ 0.35 0.30 0.275 Figu e 61: SANS measu emen s o C7G1mic oemulsion samples a di e en γ,Φ0.5 and δ0.09. The da a was i ed wi h a Teubne -S ey app oxima ion (equa ion 43). ξ, d / nm 20 40 60 80 γ 0.28 0.3 0.32 0.34 0.36 ξ d Figu e 62: SANS s uc u e sizes de i ed om he Teubne S ey app oxima ion, ξ: co ela ion leng h, d: domain size. The C7G1based sys em has a co ela ion leng h o 3nm, he domain size dec eases om 8nm o 6.5nm o ising γ(compa e igu e 62). This is due o he ising amoun o su ac an in he sys em which leads o an inc ease o he in e acial a ea be ween wa e and oil phase, he e o e 89 7 CONCLUSION AND FUTURE PROSPECTS 7 Conclusion and u u e p ospec s P o eins usually do hei wo k in c owded en i onmen s inside he cell and he espec i e o - ganelles. Hence, an unde s anding o hei di usion is o undamen al impo ance o he un- de s anding o he p ocesses o li e. Mo eo e , also in echnical p ocesses p o eins a e o en used in con ined si ua ions. One example is he use o mic oemulsions as eac ion media o decon amina ion o in chemical p oduc ion. Hence, obse ing he di usion o a p o ein inside he wa e domain o a mic oemulsion is o majo impo ance o imp o emen o e.g. decon- amina ion applica ions ealized wi h a p o ein like DFPase [18,21,34]. Howe e , his is no an easy ask because he s uc u e o he mic oemulsion is no much bigge han he pa icles which shall be aced and he mic oemulsion ma ix gi es a kind o “backg ound dynamics” in mos o he possible expe imen s. The e o e me hods ha e o be used, whe e he s uc u e and espec- i ely he dynamics o he mic oemulsion is aded ou and only he desi ed pa icle mo ion can be ollowed. Me hods like dynamic ligh sca e ing a e no usable, because he e ac i e index o he sys em canno be changed easily. Thus he me hod o choice was luo escence co ela ion spec oscopy whe e a luo escen pa icle can be aced. Howe e , in his echnique o he di - icul ies occu ed, and e.g. luo escen impu i ies dis u b he signal o he pa icle o in e es . E en wi h componen s o a high pu i y >99[%]and in 1-pen anol luo escen impu i ies we e ound, in a e y low concen a ion indeed bu enough o being de ec able by he sensible FCS echnique. Wi h echnical g ade su ac an s he luo escence ac i i y is e y high and FCS mea- su emen s a e no possible. Wi h pu e su ac an s and a concen a ion o he luo escen p o ein high enough, he de ec ed luo escen is mainly caused by he p o ein. Besides he mic oemul- sion dynamics can be aken in o accoun by using a wo componen model. Wi h a simila size and he e o e compa able hyd odynamic beha io , GFP+is a sui able model p o ein o s udy dynamics wi h luo escence co ela ion spec oscopy. In his hesis he s uc- u e o wo di e en suga su ac an mic oemulsion sys ems was s udied sys ema ically wi h small angle x- ay and neu on sca e ing measu emen s. I was shown ha he dimensions o his mic oemulsion sys em allow di usion o p o eins inside he wa e domain. The FCS esul s ha e yielded ha GFP+is mobile depending on he co ela ion leng hs o he wa e domains. The con inemen inc eases o highe oil olume ac ion Φ, meaning smalle wa e domains. In he p esen wo k i could be shown ha he di usion o GFP+is hinde ed in he di e en 97 7 CONCLUSION AND FUTURE PROSPECTS mic oemulsions. This beha io was quan i ied in e ms o he sub-di usion exponen αand i u ns ou ha αis in luenced by he composi ion o he mic oemulsion. Fo smalle wa e domains αgoes down sys ema ically. Hence, mic oemulsions a e well sui ed as model sys ems o he s udy o sub-di usion in a con olled way. Fo oo high oil/wa e a ios he p o ein ge s s uck and e lec s he dynamics known as he ”collec i e b ea hing mo ion" o he sponge-like s uc u e. Fu he mo e, he used sys em is ela ed o applica ions. The pu e suga su ac an can be easily eplaced by a echnical g ade su ac an , which leads o cheap mic oemulsions o comme cial use. S udies in he ield o skin iendly and en i onmen ally compa ible componen s a e al- eady ongoing o p o ide a sys em wi h simila dimensions o wide applica ion possibili ies in he decon amina ion a ea. I has o be obse ed in decon amina ion expe imen s which mic oemulsion sys ems show he bes decon amina ion abili y. In some sys ems he p o ein is mobile due o bigge wa e do- mains, in o he cases a high Φo wi h sho suga su ac an sys ems he p o ein is s uck in he wa e phase bu nea o he oil phase whe e he oxic agen is loca ed. Thus i has o be obse ed which in luence he p o ein mobili y and he dis ance o he oxic compound ha e on he decon amina ion e iciency and kine ics. The esul s ela ed o he sub-di usion o GFP+inside he di e en s udies a e unique and i emains o be cla i ied whe he he sub-di usi e beha io pe sis s on all leng h scales o no . Wo ks o Sch eibe e al. [104]sugges ha on e y sho leng h scale Fickian di usion migh be eco e ed. hence, his issue should be add essed using expe imen s wi h an app op ia e esolu ion in leng h. A good choice would be a CiEjmic oemulsion sys em wi h a deu e a ed su ac an in an NSE expe imen . Then he phase beha io could be uned wi h he empe a u e. Fu he mo e, one deu e a ed componen less would be needed and would simpli y he con as ma ching p ocedu e. 98 8 DANKSAGUNG 8 Danksagung In e s e Linie bedanke ich mich bei meinem Dok o a e , P o . D . Thomas Hellweg, ü die Übe lassung des spannenden Themas, das Ve auen und die iel äl igen Fo chungsmöglichkei en. G oße Dank gil auch meinen Kollegen Sebas ian Höhn, Ch is oph Ange mann, Bas ian Wedel, Ch is oph Schul eich, Michael Zeise , Ka ja on Nessen, Susanne Seib , Y onne He le, Simone Wagne und Ral S ehle ü in e essan e Diskussionen, di e se Ra schläge, jegliche Un e s ü zung und die seh gu e Zusammena bei . He n P o . D . S ephan Fö s e danke ich da ü , dass ich meine A bei in Bay eu h e igs ellen du e. Allen “neuen” Kollegen aus de A bei sg uppe Fö s e , insbesonde e meinen Bü okam- e aden Sebas ian Wi h und Jan Sch öde danke ich ü die posi i e A mosphä e und die seh gu e Zusammena bei . Einen besonde en Dank auch an Ka lheinz Lau e bach, de imme ein o enes Oh ha e und sich unablässig um die einwand eie Funk ion de Leh s uhlaus a ung kümme e. Mein Dank gil auch He n P o . D . Ma hias Weiss und den Mi a bei e n aus de Expe imen al- physik I, ü die gu e Zusammena bei und Möglichkei de Nu zung des Con oco 2 auch nach dem Umzug des Ge ä es. Vielen Dank an Uwe Gü h ü He s ellung und Lie e ung de P o einlösungen. Bei Ma in Dulle möch e ich mich ü die Un e s ü zung bei den SAXS-Messungen, Mi eia Subinyà ü die PFG- NMR Messungen und F ank Lüdel ü SANS-Messungen bedanken . Elisabe h Düng elde und Sand a Ge icke will ich ü die Hil e bei allen Themen auße halb de Fo schung bedanken. Beim weh wissenscha lichen Ins i u Muns e möch e ich mich ü die Finanzie ung des P o- jek es bedanken. Vielen Dank den Local Con ac s Alain Lapp in Saclay/LLB und Pe e Falus in G enoble/ILL ü die Un e sü zung wäh end de Messzei en. Von ganzen He zen möch e ich mich auch bei meine Familie und bei meine F eundin Ma ia bedanken ü die Geduld, das Ve s ändnis und dass sie imme an mich geglaub haben. Meinen El e n ein he zliche Dank ü die Un e s ü zung und dass sie mi das S udium übe haup e - möglich haben. 99 A APPENDIX A Appendix A.1 Al e na i e mic oemulsion sys ems Due o he p oblem ha 1-pen anol and simila alcohols wi h o he chain leng hs con ain luo- escen impu i ies which may dis u b FCS measu emen s, i was sea ched o al e na i e luo- escen ee co-su ac an . One candida e is 1-p opanol, his alcohol is a ailable in high pu i y and shows nea ly no luo escence ac i i y. Using he componen s n-dodecyl-β-d-mal oside, cyclohexane and wa e , he o ma ion o a mi- c oemulsion is possible. Bu he main p oblem is he high amoun o 1-p opanol which is needed o each he 1-phase egion, he X-poin is loca ed a γ=0.1, δ=0.39. 0.14 0.19 0.24 0.29 0.34 0.39 0 0.05 0.10 0.15 0.20 0.25 (γ) (δ) 1Φ 2Φ 3Φ Figu e 68: Phase diag am o he sys em C12G2-p opanol-cyclohexane-wa e , he 1-phase bo de s a e loca ed a high alcohol amoun s. The uppe bo de s o he 1-phase a ea could no be de e mined. A such high δ alues, he e is mo e p opanol p esen in he sys em han wa e and oil, he e o e i is unce ain, wha kind o s uc u e is ob ained. Ano he possible explana ion o he ac ha no uppe phase bounda y could be o med would be e ac i e index ma ching wi h wo phases, hen he phases canno be sepa a ed by isible inspec ion. Two ge mo e in o ma ion abou he s uc u e, SANS mea- su emen s we e pe o med a he ILL in G enoble/F ance, igu e 69. The samples we e made a cons an δwi h a ying γs a ing close o he X-poin o a cons an oil/wa e a io. 101 A.1 Al e na i e mic oemulsion sys ems A APPENDIX I(q)/cm-1 1 10 q / nm-1 0.01 0.1 γ 0.15 0.20 0.25 Figu e 69: C12G2-p opanol mic oemulsion SANS measu emen s o di e en γ alues a Φ0.5 and δ0.38. The ed lines a e i s acco ding o he Teubne -S ey app oxima ion. The sca e ing cu es show s uc u e peaks o highe γ alues mo ing o highe q o ising γ meaning dec easing s uc u e sizes. This is common o bicon inuous mic oemulsion sys ems, because wi h inc easing su ac an amoun mo e molecules s ay a he in e ace and a e able o o m smalle wa e /oil domains. The SANS da a can be i ed wi h he Teubne -S ey app oxi- ma ion, he esul s a e shown in able 7. Table 7: S uc u e sizes o C12G2-p opanol-mic oemulsions de e mined by SANS a Φ0.5 and δ 0.38 γ ξ /nm d/nm 0.15 2.1 4.9 0.2 1.8 5.8 0.25 1.4 4.6 The s uc u es a e e y small compa ed o o he sys ems and a leas o γ=0.15 i has o be cla i ied, i he s uc u e is eally bicon inuous o d ople -like and whe e he 1-p opanol molecules a e loca ed, which a e he majo componen in he sys em. 102 A APPENDIX A.2 Sol en sensi i e luo escen dyes A.2 Sol en sensi i e luo escen dyes Fluo escen dyes can be in luenced by he sol en , he consequence is o example a quenched luo escence o a shi in he emission spec a. nile- ed [105,106]is a lipophilic molecule which is sensi i e o he pola i y o he used sol en . Figu e 70 shows luo escence emission o nile- ed in cyclohexane and 1-pen anol. no malized in ensi y 0 0.2 0.4 0.6 0.8 1 emission wa eleng h / nm 500 550 600 650 700 1-pen anol cyclohexane Figu e 70: Fluo escence o nile- ed in di e en sol en s, cyclohexane and 1-pen anol. In wa e , whe e nile- ed is nea ly no soluble, he emission maximum is loca ed a 660nm [105]. The emission maximum is a 535nm o he unpola sol en cyclohexane and a 630nm o 1-pen anol. This is a big sol och omic shi o 95nm. Nile- ed can also be dissol ed in a mi- c oemulsion, which is plo ed in igu es 71 (δ a ia ion), 72 (Φ a ia ion) and 73 (γ a ia ion). The in luence o a ising con en o pen anol in he δ a ia ion shi s he maximum o highe wa eleng hs. 103 A.2 Sol en sensi i e luo escen dyes A APPENDIX no malized in ensi y 0 0.2 0.4 0.6 0.8 1 emission wa eleng h / nm 500 550 600 650 700 δ 0.06 0.08 0.10 Figu e 71: Nile- ed luo escence in mic oemulsions wi h di e en δ((componen s: wa e , Glucopon 220, cyclohexane, 1-pen anol). no malized in ensi y 0 0.2 0.4 0.6 0.8 1 emission wa eleng h / nm 500 550 600 650 700 Φ 0.1 0.3 0.5 0.7 0.9 Φ inc eases Figu e 72: Nile- ed luo escence in mic oemulsion wi h di e en Φ(componen s: wa e , Glucopon 220, cyclohexane, 1-pen anol). When Φis a ied, he emission maximum should inc ease wi h sh inking Φdue o he inc easing wa e amoun . Bu his is no he case, wi h ising Φ he maximum mo es o highe wa eleng h. Mos likely, he necessa y highe amoun o 1-pen anol in luences he nile- ed emission in he mic oemulsion. This is an indica ion, ha he pen anol molecules a e loca ed in he su ac an in e ace and dilu ed in he cyclohexane phase. The e o e, nile- ed could be used as a ma ke 104 A APPENDIX A.2 Sol en sensi i e luo escen dyes o he dis ibu ion o 1-pen anol. A change o he su ac an con en γha dly a ec s he emission wa eleng h, only in a ange o 3 nm. Fo inc easing γ, he mic oemulsion s uc u e mo phs om a bicon inuous phase o a d ople s uc u e. Fu he γinc easing esul s in smalle d ople s. Thus nile- ed is loca ed in he oil domains o d ople s, whe e i is se pe a ed om he wa e phase by he su ac an in e ace. Also he dis ibu ion o pen anol is no changing when γis inc eased. no malized in ensi y 0 0.2 0.4 0.6 0.8 1 emission wa eleng h / nm 500 550 600 650 700 γ 0.14 0.17 0.20 0.23 Figu e 73: Nile- ed luo escence in mic oemulsion wi h di e en γ(componen s: wa e , Glucopon 220, cyclohexane, 1-pen anol). 105 REFERENCES REFERENCES Re e ences [1]P. Winso , “Hyd o opy, solubilisa ion and ela ed emulsi ica ion p ocesses,” T ansac ions o he Fa aday Socie y, ol. 44, pp. 376–398, 1948. [2]J. Schulman, W. S oeckenius, and L. P ince, “Mechanism o o ma ion and s uc u e o mi- c o emulsions by elec on mic oscopy,” The Jou nal o Physical Chemis y, ol. 63, no. 10, pp. 1677–1680, 1959. [3]T. Hoa and J. Schulman, “T anspa en Wa e -in-Oil Dispe sions: he Oleopa hic Hyd o- Micelle,” Na u e, ol. 152, pp. 102–103, 1943. [4]S. F ibe g, Micelliza ion, solubiliza ion, and mic oemulsions (Mi al, K.L. and Ame ican Chemical Socie y). Micelliza ion, Solubiliza ion, and Mic oemulsions, Band 1, Plenum P ess, 1977. [5]S. F ibe g, Mic oemulsions: Theo y and p ac ice (P ince, L.M.). Academic P ess, 1977. [6]G. Gillbe g, H. Leh inen, and S. F ibe g, “NMR and IR in es iga ion o he condi ions de e mining he s abili y o mic oemulsions,” Jou nal o Colloid and In e ace Science, ol. 33, no. 1, pp. 40 – 53, 1970. [7]K. Shinoda and T. Ogawa, “The solubiliza ion o wa e in nonaqueous solu ions o non- ionic su ac an s,” Jou nal o Colloid and In e ace Science, ol. 24, no. 1, pp. 56 – 60, 1967. [8]K. Shinoda and H. Sai o, “The e ec o empe a u e on he phase equilib ia and he ypes o dispe sions o he e na y sys em composed o wa e , cyclohexane, and nonionic su ac an ,” Jou nal o Colloid and In e ace Science, ol. 26, no. 1, pp. 70 – 74, 1968. [9]K. Shinoda and H. Sai o, “The s abili y o o/w ype emulsions as unc ions o empe a u e and he HLB o emulsi ie s: The emulsi ica ion by PIT-me hod,” Jou nal o Colloid and In e ace Science, ol. 30, no. 2, pp. 258 – 263, 1969. [10]D. Shah, Su ace phenomena in enhanced oil eco e y. Plenum P ess, 1981. [11]C. Solans and H. Kunieda, Indus ial Applica ions o Mic oemulsions. Su ac an Science Se ies, M. Dekke , 1997. 113 REFERENCES REFERENCES [12]M.-J. Schwuge , K. S ickdo n, and R. Schomaecke , “Mic oemulsions in echnical p o- cesses,” Chemical Re iews, ol. 95, no. 4, pp. 849–864, 1995. [13]R. Schomäcke and Baye -AG, “Mik oemulsionen als Medium ü chemische Reak ionen,” Nach ich en aus Chemie, Technik und Labo a o ium, ol. 40, no. 12, pp. 1344–1352, 1992. [14]M. Pileni, “Re e se micelles as mic o eac o s,” The Jou nal o Physical Chemis y, ol. 97, no. 27, pp. 6961–6973, 1993. [15]J. Sjöblom, R. Lindbe g, and S. E. F ibe g, “Mic oemulsions - phase equilib ia cha ac e i- za ion, s uc u es, applica ions and chemical eac ions,” Ad ances in Colloid and In e ace Science, ol. 65, no. 0, pp. 125 – 287, 1996. [16]M. Hö e , “Chemische Kamp s o e: Ein Übe blick,” Chemie in unse e Zei , ol. 36, no. 3, pp. 148–155, 2002. [17]H. Mu akami, A. Bi nbaum, and P. Gab iel, Unde g ound. Vin age In e na ional, 2001. [18]M.-M. Blum and A. Richa d , Decon amina ion o Wa a e Agen s: Enzyma ic Me hods o he Remo al o B/C Weapons. Wiley-VCH Ve lag GmbH & Co. KGaA, 2008. [19]M. Kahlwei , G. Busse, B. Faulhabe , and H. Eibl, “P epa ing non oxic mic oemulsions,” Langmui , ol. 11, no. 11, pp. 4185–4187, 1995. [20]S. Welle , H. Al mann, A. Richa d , A. Lapp, P. Falus, B. Fa ago, and T. Hellweg, “Dy- namics o he in e acial ilm in bicon inuous mic oemulsions based on a pa ly ionic su ac an mix u e: A neu on spin-echo s udy,” The Eu opean Physical Jou nal E: So Ma e and Biological Physics, ol. 33, pp. 243–250, 2010. 10.1140/epje/i2010-10668-1. [21]A. Richa d , M.-M. Blum, and S. Mi chell, “Was wissen Calama i übe Sa in? Enzyma- ische Dekon amina ion on Ne enkamp s o en,” Chemie in unse e Zei , ol. 40, no. 4, pp. 252–259, 2006. [22]C. S uben auch, Mic oemulsions: Backg ound, New Concep s, Applica ions, Pe spec i es. Ox o d: WILEY-VCH Ve lag, 2009. [23]W. P i zkow, “Die Tenside, He ausgegeben on K. Kosswig und H. S ache München,” Jou nal ü P ak ische Chemie/Chemike -Zei ung, ol. 336, no. 5, pp. 473–473, 1994. 114 REFERENCES REFERENCES [24]H. Ro h and H. Fenne , A zneis o e. S u ga : Thieme, 1998. [25]B ockhaus ABC Chemie. VEB F. A. B ockhaus Ve lag Leipzig, 1965. [26]K. Kosswig and H. S ache, Die Tenside. Hanse , 1993. [27]T. Hellweg, “Phase s uc u es o mic oemulsions,” Cu en Opinion in Colloid and In e ace Science, ol. 7, no. 1 - 2, pp. 50 – 56, 2002. [28]A. Biasio, C. Came i, P. Codas e ano, P. Ta aglia, J. Rouch, and S. Chen, “Phase beha io o dense h ee-componen ionic mic oemulsions and elec ical conduc i i y in he lamel- la phase,” Physical Re iew E, ol. 47, pp. 4258–4264, Jun 1993. [29]A. Biasio, C. Came i, P. Codas e ano, P. Ta aglia, J. Rouch, and S. Chen, “Elec ical conduc i i y in he lamella phase o a wa e -in-oil mic oemulsion,” in T ends in Colloid and In e ace Science VII, ol. 93 o P og ess in Colloid & Polyme Science, pp. 191–193, S einkop , 1993. [30]S. Bu aue , L. Belkou a, C. S uben auch, and R. S ey, “Bicon inuous mic oemulsions e isi ed: a new app oach o eeze ac u e elec on mic oscopy (FFEM),” Colloids and Su aces A: Physicochemical and Enginee ing Aspec s, ol. 228, no. 1-3, pp. 159 – 170, 2003. Fes sch i o P o esso Bjo n Lindman. [31]N. F eibe ge , C. Moi zi, L. de Campo, and O. Gla e , “An a emp o de ec bicon inui y om sans da a,” Jou nal o Colloid and In e ace Science, ol. 312, no. 1, pp. 59 – 67, 2007. [32]M. Kahlwei , G. Busse, and J. Winkle , “Elec ic conduc i i y in mic oemulsions,” The Jou nal o Chemical Physics, ol. 99, no. 7, pp. 5605–5614, 1993. [33]A. Zilman and R. G anek, “Undula ions and dynamic s uc u e ac o o memb anes,” Physical Re iew Le e s, ol. 77, pp. 4788–4791, Dec 1996. [34]S. Welle , B. Tie sch, J. Koe z, A. Richa d , A. Lapp, O. Holde e , J. Gäb, M.-M. Blum, C. Schul eich, R. S ehle, and T. Hellweg, “The DFPase om loligo ulga is in suga su ac an -based bicon inuous mic oemulsions: S uc u e, dynamics, and enzyme ac i - i y,” Eu opean Biophysics Jou nal, ol. 40, pp. 761–774, 2011. 115 REFERENCES REFERENCES [35]R. S ey, “Mic oemulsion mic os uc u e and in e acial cu a u e,” Colloid and Polyme Science, ol. 272, pp. 1005–1019, 1994. [36]M. Teubne and R. S ey, “O igin o he sca e ing peak in mic oemulsions,” The Jou nal o Chemical Physics, ol. 87, pp. 3195–3200, 1987. [37]S. Welle , M. Ka g, H. Imho , A. S eppin, H.-J. Al mann, M. Dolle, A. Richa d , B. Tie sch, J. Koe z, A. Lapp, and T. Hellweg, “S uc u e o biodiesel based bicon inuous mic oemul- sions o en i onmen ally compa ible decon amina ion: A small angle neu on sca e ing and eeze ac u e elec on mic oscopy s udy,” Jou nal o Colloid and In e ace Science, ol. 325, no. 1, pp. 250 – 258, 2008. [38]T. So mann, “Solubiliza ion e iciency boos ing by amphiphilic block co-polyme s in mi- c oemulsions,” Cu en Opinion in Colloid and In e ace Science, ol. 7, no. 1, pp. 57–65, 2002. [39]B. Jakobs, T. So mann, R. S ey, J. Allgaie , , and D. Rich e , “Amphiphilic block copoly- me s as e iciency boos e s o mic oemulsions,” Langmui , ol. 15, no. 20, pp. 6707– 6711, 1999. [40]H. Endo, J. Allgaie , M. Mihailescu, M. Monkenbusch, G. Gomppe , D. Rich e , B. Jakobs, T. So mann, and R. S ey, “Amphiphilic block copolyme s as e iciency boos e s in mi- c oemulsions: a SANS in es iga ion o he ole o polyme s,” Applied Physics A, ol. 74, pp. 392–395, 2002. [41]S. Welle , H. Imho , M. Dolle, H.-J. Al mann, A. Richa d , and T. Hellweg, “Decon am- ina ion o chemical wa a e agen s using pe chlo oe hylene - ma lowe IHF-H2O-based mic oemulsions: we ing and ex ac ion p ope ies on ealis ic su aces,” Colloid & Poly- me Science, ol. 286, pp. 417–426, 2008. [42]E. Scha , J. Koepke, G. F i zsch, C. Luecke, and H. Rue e jans, “RCSB p o ein da a bank: 1E1A, c ys al s uc u e o diisop opyl luo ophospha ase om Loligo ulga is.” h p://www. csb.o g/pdb/explo e/explo e.do?s uc u eId=1E1A. (01/2013). 116 REFERENCES REFERENCES [43]E. Scha , J. Koepke, G. F i zsch, C. Lücke, and H. Rü e jans, “C ys al s uc u e o di- isop opyl luo ophospha ase om loligo ulga is.,” S uc u e, ol. 9, no. 6, pp. 493–502, 2001. [44]C. Schul eich, C. Ange mann, S. Hoehn, R. Neubaue , S. Seib , R. S ehle, A. Lapp, A. Richa d , A. Diekmann, and T. Hellweg, “Bicon inuous mic oemulsions wi h ex emely high empe a u e s abili y based on skin iendly oil and suga su ac an ,” Colloids and Su aces A: Physicochemical and Enginee ing Aspec s, ol. 418, pp. 39–46, 2013. [45]S. Welle , M. Ka g, O. Holde e , A. Richa d , and T. Hellweg, “Tempe a u e dependence o he su ac an ilm bending elas ici y in a bicon inuous suga su ac an based mi- c oemulsion: A quasielas ic sca e ing s udy,” Phys. Chem. Chem. Phys., ol. 13, pp. 3092– 3099, 2011. [46]E. Elson and D. Magde, “Fluo escence co ela ion spec oscopy. I. Concep ual basis and heo y,” Biopolyme s, ol. 13, no. 1, pp. 1–27, 1974. [47]J. Lakowicz, P inciples o Fluo escence Spec oscopy. Sp inge London, 2009. [48]P. P ingsheim, Fluo escence and phospho escence. In e science Publishe s New Yo k and London, 1963. [49]R. Kellogg and R. Benne , “Radia ionless In e molecula Ene gy T ans e . III. De e mi- na ion o Phospho escence E iciencies,” The Jou nal o Chemical Physics, ol. 41, no. 10, pp. 3042–3045, 1964. [50]A. Follenius-Wund, M. Bou o e, M. Schmi , F. Iyice, H. Lami, J.-J. Bou guignon, J. Haiech, and C. Pigaul , “Fluo escen de i a i es o he GFP ch omopho e gi e a new insigh in o he GFP luo escence p ocess,” Biophysical Jou nal, ol. 85, no. 3, pp. 1839 – 1850, 2003. [51]R. Rigle and E. Elson, Fluo escence Co ela ion Spec oscopy: Theo y and Applica ions. Be lin: Sp inge , 2001. [52]S. Kelle , Fluo eszenz-Ko ela ions-Spek oskopie in Polyme lösungen. PhD hesis, München, 2004. 117 REFERENCES REFERENCES [53]J. Ricka and T. Binke , “Di ec measu emen o a dis inc co ela ion unc ion by luo es- cence c oss co ela ion,” Physical Re iew A, ol. 39, pp. 2646–2652, Ma 1989. [54]J. Wideng en and R. Rigle , “Mechanisms o pho obleaching in es iga ed by luo escence co ela ion spec oscopy,” Bioimaging, ol. 4, no. 3, pp. 149–157, 1996. [55]J. Wideng en, U. Me s, and R. Rigle , “Fluo escence co ela ion spec oscopy o iple s a es in solu ion: A heo e ical and expe imen al s udy,” The Jou nal o Physical Chem- is y, ol. 99, no. 36, pp. 13368–13379, 1995. [56]M. Weiss, M. Elsne , F. Ka be g, and T. Nilsson, “Anomalous subdi usion is a measu e o cy oplasmic c owding in li ing cells.,” Biophys. J., ol. 87, pp. 3518–3524, No . 2004. [57]S. P o enche , “In e se p oblems in polyme cha ac e iza ion: Di ec analysis o poly- dispe si y wi h pho on co ela ion spec oscopy,” Die Mak omolekula e Chemie, ol. 180, no. 1, pp. 201–209, 1979. [58]D. Koppel, “Analysis o mac omolecula polydispe si y in in ensi y co ela ion spec- oscopy: The me hod o cumulan s,” The Jou nal o Chemical Physics, ol. 57, no. 11, pp. 4814–4820, 1972. [59]S. P o enche , “A cons ained egula iza ion me hod o in e ing da a ep esen ed by linea algeb aic o in eg al equa ions,” Compu e Physics Communica ions, ol. 27, no. 3, pp. 213 – 227, 1982. [60]S. P o enche , “CONTIN: A gene al pu pose cons ained egula iza ion p og am o in- e ing noisy linea algeb aic and in eg al equa ions,” Compu e Physics Communica ions, ol. 27, no. 3, pp. 229–242, 1982. [61]A. G. Langen/Ge many, “ALV-5000 DLS ins umen .” h p://www.al gmbh.de. (11/2012). [62]U. Tübingen, “Small-angle sca e ing and da a analysis.” h p://www.so -ma e .uni- uebingen.de/ eaching/SASTu o ial.pd . (12/2012). [63]J. Kohlb eche and I. B essle , “SAS i .” h ps://ku .web.psi.ch/sans1/SANSSo /sas i .h ml. (01/2013). 118 REFERENCES REFERENCES [64]O. Gla e , “GIFT: small angle sca e ing da a e alua ion so wa e.” h p://physchem.k unig az.ac.a /sm/Index.h ml. (01/2013). [65]A. Be gmann, G. F i z, and O. Gla e , “Sol ing he gene alized indi ec Fou ie ans o - ma ion (GIFT) by Bol zmann simplex simula ed annealing (BSSA),” Jou nal o Applied C ys allog aphy, ol. 33, pp. 1212–1216, Oc 2000. [66]F. Mezei, C. Pappas, and T. Gu be le , Neu on Spin Echo Spec oscopy: Basics, T ends and Applica ions. Lec u e No es in Physics, Sp inge , 2003. [67]T. Munich, “RESEDA-Handou .” h p://www2009.ph. um.de/s udium/be ieb/p ak ika/ op a/ e suche/61/. (12/2012). [68]“Ins i u Laue-Lange in, G enoble, F ance - IN15.” h p://www.ill.eu/h ml/ins umen s- suppo /ins umen s-g oups/ins umen s/in15/. (12/2012). [69]C. Ba ge on, “Measu emen o a con inuous dis ibu ion o sphe ical pa icles by in en- si y co ela ion spec oscopy: Analysis by cumulan s,” The Jou nal o Chemical Physics, ol. 61, no. 5, pp. 2134–2138, 1974. [70]T. Hellweg, M. G adzielski, B. Fa ago, and D. Lange in, “Shape luc ua ions o mic oemul- sion d ople s: A neu on spin-echo s udy,” Colloids and Su aces A: Physicochemical and Enginee ing Aspec s, ol. 183-185, no. 0, pp. 159 – 169, 2001. [71]S. Milne and S. Sa an, “Dynamical luc ua ions o d ople mic oemulsions and esicles,” Physical Re iew A, ol. 36, pp. 4371–4379, No 1987. [72]J. Huang, S. Milne , B. Fa ago, and D. Rich e , “S udy o dynamics o mic oemulsion d ople s by neu on spin-echo spec oscopy,” Physical Re iew Le e s, ol. 59, pp. 2600– 2603, No 1987. [73]T. Hellweg and D. Lange in, “The dynamics in dodecane/C10E5/wa e mic oemulsions de e mined by ime esol ed sca e ing echniques,” Physica A: S a is ical Mechanics and i s Applica ions, ol. 264, no. 3-4, pp. 370 – 387, 1999. 119 REFERENCES REFERENCES [74]C. Schul eich, “Bikon inuie liche Mik oemulsionen als neua ige Dekon amina ionsmi - el: Phasen e hal en und S uk u un e suchungen in Zucke ensid - Sys emen (Zulas- sungsa bei ),” Bay eu h 2009. [75]R. Wheele , “RCSB p o ein da a bank: 1EMA, GFP s uc u e.” www.wikipedia.o g. (11/2012). [76]X. Shu, N. Shane , C. Ya b ough, R. Tsien, and S. Reming on, “RCSB p o ein da a bank: 2H5Q, c ys al s uc u e o mChe y.” h p://www. csb.o g/pdb/explo e/explo e.do?s uc u eId=2H5Q. (11/2012). [77]O. Shimomu a, F. H. Johnson, and Y. Saiga, “Ex ac ion, pu i ica ion and p ope ies o ae- quo in, a bioluminescen p o ein om he luminous hyd omedusan, aequo ea,” Jou nal o Cellula and Compa a i e Physiology, ol. 59, no. 3, pp. 223–239, 1962. [78]O. Scholz, A. Thiel, W. Hillen, and M. Niede weis, “Quan i a i e analysis o gene ex- p ession wi h an imp o ed g een luo escen p o ein,” Eu opean Jou nal o Biochemis y, ol. 267, no. 6, pp. 1565–1570, 2000. [79]M. Chal ie and S. Kain, Me hods o Biochemical Analysis, G een Fluo escen P o ein: P op- e ies, Applica ions and P o ocols. Me hods o Biochemical Analysis, Wiley, 2005. [80]S. Bokman and W. Wa d, “Rena u a ion o aequo ea g een- luo escen p o ein,” Biochem- ical and Biophysical Resea ch Communica ions, ol. 101, no. 4, pp. 1372 – 1380, 1981. [81]N. Shane , R. Campbell, P. S einbach, B. Giepmans, A. Palme , and R. Tsien, “Imp o ed monome ic ed, o ange and yellow luo escen p o eins de i ed om Discosoma sp. ed luo escen p o ein,” Na u e Bio echnology, ol. 22, pp. 1567–1572, Dec. 2004. [82]H. Ze l, New Applica ions o Fluo escence Co ela ion Spec oscopy in Ma e ials Science. PhD hesis, Bay eu h, 2006. [83]H. Bagge -Jö gensen, L. Coppola, K. Thu esson, U. Olsson, and K. Mo ensen, “Phase beha io , mic os uc u e, and dynamics in a nonionic mic oemulsion on addi ion o hy- d ophobically end-capped poly(e hylene oxide),” Langmui , ol. 13, no. 16, pp. 4204– 4218, 1997. 120 REFERENCES REFERENCES [84]J. Huang, J. Sung, and X.-L. Wu, “The e ec o H2O and D2O on a wa e -in-oil mic oemul- sion,” Jou nal o Colloid and In e ace Science, ol. 132, no. 1, pp. 34 – 42, 1989. [85]I. Vasilie , “Q iplo .” h p://so .p oindependen .com/q iplo .h ml. (10/2012). [86]J. Nelde and R. Mead, “A simplex me hod o unc ion minimiza ion,” The Compu e Jou nal, ol. 7, no. 4, pp. 308–313, 1965. [87]“Ganesha - Small Angle X-Ray Sca e ing ins umen .” SAXS LAB /Denma k. [88]“PAXY ins umen a he LLB Saclay/F ance.” h p://www-llb.cea. /en/ -en/pd /paxy- llb.pd . (11.2012). [89]G. Bu ne , G. Rees, D. S ey le , and B. Robinson, “Fluo escence co ela ion spec oscopy o wa e -in-oil mic oemulsions: An applica ion in speci ic cha ac e isa ion o d ople s con aining biomolecules,” Colloids and Su aces A: Physicochemical and Enginee ing As- pec s, ol. 250, no. 1-3, pp. 171 – 178, 2004. [90]M. G adzielski, “Recen de elopmen s in he cha ac e isa ion o mic oemulsions,” Cu en Opinion in Colloid & In e ace Science, ol. 13, no. 4, pp. 263 – 269, 2008. [91]E. Po ma, W. de Boeij, L. Bosg aa , J. Roelo s, P. an Haas e , and D. Wie sma, “Reduced p o ein di usion a e by cy oskele on in ege a i e and pola ized dic yos elium cells,” Biophysical Jou nal, ol. 81, no. 4, pp. 2010 – 2019, 2001. [92]S. Sun, Physical Chemis y o Mac omolecules: Basic P inciples and Issues. Wiley New Je sey, 2004. [93]T. So mann and R. S ey, “E idence o co esponding s a es in e na y mic oemulsions o wa e - alkane - CiEj,” Jou nal o Physics: Condensed Ma e , ol. 8, no. 25A, p. A39, 1996. [94]M. Summe s, J. Eas oe, S. Da is, Z. Du, R. M. Richa dson, R. K. Heenan, D. S ey le , and I. G illo, “Polyme iza ion o ca ionic su ac an phases,” Langmui , ol. 17, no. 17, pp. 5388–5397, 2001. 121