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