3D Pho onic C ys als om highly monodispe se FRET-
based ed luminescen PMMA sphe es

J. F. Galis eo-López,*† M. Ibisa e, A. Muñoz and C. López
Ins i u o de Ciencia de Ma e iales de Mad id (CSIC), c/ So Juana Inés de la C uz 3,
28049 Mad id (Spain)
Red-luminescen PMMA sphe es con aining a Fö s e esonance ene gy
ans e (FRET) pai we e syn hesized ia a wo-s ep polyme iza ion
me hod. Two eac ion pa ame e s, ime and monome olume, a e scanned
in o de o une he sphe e diame e in he 250-500 nm ange. Fu he he
polydispe si y o he sphe es is kep low, a ca. 3%, ega dless o sphe e
diame e o dye concen a ion. A ho ough op ical cha ac e iza ion ia
spec oscopy and ime esol ed measu emen s shows a FRET e iciency o
o e 40% be o e concen a ion quenching e ec s ake place, allowing o
a p ecise uning o hei emission in he ed egion o he isible spec um.
The high quali y o hese sphe es makes hem sui able o ab ica e sel -
assembled 3D pho onic c ys als which ac as pho onic en i onmen o
modi y he spec al p ope ies o he FRET pai ia B agg di ac ion
.

† P esen add ess: Ins i u o de Ciencia de Ma e iales de Se illa (CSIC), c/Amé ico Vespucio
49, 41092 Se illa (Spain)
* e-mail add ess: [email p o ec ed]


In oduc ion
Encapsula ion o ligh sou ces wi hin a polyme ic o ino ganic nano-pa icle (NP)
p o ides a means o shield hem om he en i onmen imp o ing hei pho ophysical
p ope ies and, o ce ain emi e s such as quan um do s, a oiding oxici y issues in hei
use o bio-imaging pu poses. Recen ly he inco po a ion o wo o mo e ypes o
emi e s wi hin he nanopa icle has been eage ly explo ed as a way o imp o e i s
emission p ope ies. In his app oach ene gy om an exci ed emi e can be non-
adia i ely ans e ed ia Fö s e esonance ene gy ans e (FRET) o a nea by (2-10
nm) g ound-s a e accep o gi en a spec al o e lap be ween hei abso p ion and
emission spec a exis s and hei ansi ion dipole momen s a e no o hogonal. [1] The
e iciency o he p ocess is hen dic a ed by he emi e dis ibu ion wi hin he NP which
in many cases can be con olled h ough hei ela i e concen a ion. In his way, a ying
he a io be ween he wo ch omopho es allows p oducing sphe es wi h a common
exci a ion wa eleng h bu di e en pho oluminescence spec a, pa ing he way o
mul iplexed biological de ec ion. Some examples include dye-doped nanosphe es o
di e en na u e including bo h ino ganic, [2,3] o ganic [4,5,6,7] and hyb id ma ices.
[8,9]
Beyond he ield o bio-imaging, applica ions bene i ing om FRET a e g owing and span om
enhanced ene gy ha es ing in pho o ol aics [10,11] o sensing [12] o pho onics (whe e FRET
can be used as a mechanism o imp o e he pe o mance o de ices such as OLEDs [13]).
Rega ding he use o FRET NP, dye doped polyme beads ha e been ecen ly used as gain
media o e icien pho os able ed-emi ing dye-lase s. [14] Fu he i a p ope ly designed
pho onic en i onmen is p o ided, he e iciency o he FRET p ocess may be con olled ia he
local densi y o pho onic s a es (LDOS) a he dye’s loca ion. [15] I he sphe es sa is y a
numbe o equi emen s such as low polydispe si y, diame e in he submic on ange and
colloidal s abili y, hey can be a anged in a pe iodic ashion o cons i u e hemsel es he
pho onic en i onmen in he shape o sel -assembled pho onic c ys als (PhC) o a i icial opals.
[16] These s uc u es ha e been long used as pla o ms o con ol he luminescen p ope ies o
emi e s. [17] Such con ol can be achie ed ei he by elying on he abo e men ioned LDOS
con ol a he emi e posi ion o by modi ying he di ec ionali y o he emission h ough B agg
di ac ion, in oducing a il e e ec . Recen ly, a ew examples explo ing sel -assembled PhC
o modi y FRET ha e been p esen ed using he a i icial opal as a passi e sca old. [18,19,20]
This has been done imp egna ing he opaline ma ix wi h well con olled dono -accep o
con igu a ions [18] o wi h a andom suspension o wo dyes, [19] and also employing an
in e se opal con igu a ion wi h a a e-ea h based ma ix. [20] Fu he , he combined use o
a i icial opals and FRET has been demons a ed as a means o de elop DNA senso s [21] o
sys ems o op ical s o age. [22]
Bu o da e no e idence o opal based PhC wi h FRET pai s wi hin he sphe es has been shown.
Among he ad an ages o his con igu a ion lies he possibili y o achie ing a la ge load o
emi e s since he sphe es ep esen 74% o he o al PhC olume and a p o ec ed en i onmen
whe e in ima e con ac o p oximi y is g an ed o he ac i e species. I one wishes o modi y he
ene gy ans e wi hin he NPs by o ganizing hem in o a pe iodic a ay one mus ab ica e
highly monodispe se NP wi h a diame e o he o de o he emission o he dono species
which, o he case o he isible, ansla es in o a ew hund ed nanome e s. To da e mos o he
app oaches o NP ab ica ion ha e ocused on diame e s in he ange o ens o nm (app op ia e
o hei use in bio-imaging and o he applica ions whe e emission is no mean o be modula ed
by he en i onmen ) p esen ing a la ge polydispe si y, owing o he di icul y in syn hesizing
hem wi h due size con ol, and monodispe se sphe es ha e only appea ed wi h diame e s close
o 2 µm, oo la ge o a p ope con ol o he emission in he isible ange. I one wishes o exe
a con ol on he ene gy ans e wi hin he nanosphe es using he PhC en i onmen , pho onic
ea u es mus be p esen which demand a low polydispe si y o he sphe es. [23] The c i ical
ole o monodispe si y is e iden by he ac ha o alues abo e 5% c ys alliza ion o a i icial
opals is no expec ed o ake place. [24]
In his wo k we demons a e a me hod o ab ica e highly monodispe se polyme ic sphe es
doped wi h wo dyes o he hodamine amily o hei use as FRET-based luminescen media.
Sphe es wi h di e en dye loads a e p epa ed wi h a ine con ol on hei diame e s in he 200-
500 nm ange and ha ing a polydispe si y o ca. 3%. Thei op ical esponse is s udied by means
o pho oluminescence as well as ime esol ed measu emen s demons a ing a FRET e iciency
o up o 50%. Thei high quali y ende s hem app op ia e o hei use as building blocks o
ab ica e h ee dimensional (3D) PhC in he shape o a i icial opals wi h di e en la ice
pa ame e . We show how he luminescen p ope ies o he sphe es can be u he modi ied in
hese s uc u es by means o B agg di ac ion. These esul s pa e he way o u u e
uncon en ional ligh sou ces such as lase s combining he ca i y-less lasing mechanisms
cha ac e is ic o 3D PhC [25] wi h he ad an ages o FRET-based lase s. [26]
Expe imen al
Sphe e syn hesis:
To emo e he inhibi o , Me hylme hac yla e (MMA) (Sigma-Ald ich) was washed wi h
aqueous sodium hyd oxide solu ion (1M), d ied o e anhyd ous magnesium sul a e and inally
passaged downwa d h ough a glass column con aining silica gel. Wa e was pu i ied h ough a
MilliQ pu i ica ion sys em. The es o eagen s we e used wi hou u he pu i ica ion. Sodium
hyd oxide, magnesium sul a e, silica gel 60, po assium pe sul a e (KPS) and hodamine B
(RhB) we e p o ided by Sigma-Ald ich. LD-700 pe chlo a e (Rh700) was acqui ed om
Exci on and e hanol om Pan eac.
Dye doped sphe es wi h a 250-380 nm diame e we e ab ica ed using a modi ied e sion o he
p ocedu e gi en in e e ence [5]. In a 250 mL 3-neck ound-bo om lask equipped wi h a
condense and a gas inle , 140 mL o wa e and he co esponding MMA olume ( om 10 o 25
mL) we e added. The mix u e was deoxygena ed by bubbling ni ogen gas a oom empe a u e
o 45 min and hen empe ed o 60 min a 80 ºC. To s a he polyme iza ion 5 mL o a
deoxygena ed KPS wa e solu ion (0.1 g/mL) we e added unde magne ic s i ing. 5 min a e
he polyme iza ion s a s, 9 mL o a dye solu ion we e added a a cons an a e o 0.67 mL/min.
The dye solu ion con ained 6.2 mg o RhB wi h di e en amoun s o Rh700 ( o ob ain mola
a ios om 1:1 o 1:4 RhB:Rh700) in a 1:1 ( / ) e hanol:wa e mix u e. The eac ion ime was
ixed o 40 min, excep o he sample made om 25 mL o MMA whose eac ion ime was 2 h.
In o de o inc ease he NPs size beyond 380 nm a wo-s age dispe sion polyme iza ion was
conduc ed. In his p ocedu e he p e ious p o ocol was ollowed ixing he ini ial olume o
MMA a 25 mL and adding an ex a MMA olume (10, 15 o 20 mL a a a e o 0.67 mL/min)
immedia ely a e he dyes solu ion addi ion. The eac ion was allowed o p oceed o 2 h,
longe imes leading o agg ega ion o he sphe es. As dyes we e added once he eac ion was
ini ia ed, he g own PMMA sphe es will ha e a ew nm sized co e wi hou any dye in i . In
addi ion an inc easing g adien in he dye concen a ion will ake place adially ac oss he
PMMA NP due o he slow dye addi ion o he eac ion lask. T ansmission Elec on
Mic oscopy (TEM) images we e aken in o de o es ima e he diame e o he g own sphe es
using a JEOL 2000 FX II model. S a is ics we e de i ed om 100 sphe es o each o sample.
Op ical cha ac e iza ion:
Op ical cha ac e iza ion was ca ied ou by means o pho oluminescence (PL) spec oscopy and
ime esol ed measu emen s. The samples we e all measu ed unde he same condi ions. Qua z
cu e es wi h a 1 mm op ical pa h con aining a NP aqueous solu ion wi h ixed sphe e
concen a ion (1 mg/mL) we e used o all accep o o dono a ios QAD. The concen a ion was
kep low in o de o a oid sca e ing om he sphe es in luencing he measu emen s. Op ical
pumping was pe o med wi h a unable pulsed lase (OPe A-Solo om Cohe en ) ha ing a
epe i ion a e o 1 kHz and deli e ing 150 s long pulses. The pump wa eleng h (515 nm) was
chosen in o de o maximize op ical exci a ion o he dono while minimizing ha o he
accep o . PL spec a we e collec ed wi h a ibe coupled spec ome e USB2000 (Ocean Op ics).
Time esol ed measu emen s we e pe o med (wi h a esolu ion o 80 ps) wi h a Time
Co ela ed Single Pho on Coun ing ca d (SPC-300 om Becke & Hickl) a a ixed wa eleng h
selec ed wi h a monoch oma o .
A i icial opal ab ica ion and cha ac e iza ion:
Thin ilm a i icial opals we e ab ica ed by e ically placing a clean glass subs a e in o a 20
mL ial con aining a 0.1% ol. aqueous dispe sion o sphe es. The dispe sion was le in a
humidi y (60%) and empe a u e (45ºC) con olled chambe o 24 hou s. No mal incidence
e lec ance spec a we e collec ed using a FTIR spec ome e (B uke IFS-66/S) coupled o an
op ical mic oscope wi h a 10× objec i e (NA=0.12). PL spec a om he opals we e collec ed in
an in e ed mic oscope using a high NA objec i e (NA=0.75) as ocusing/collec ion op ics.
Fig. 1 Chemical s uc u e o dono (a) and accep o (b) molecules. (c) Abso p ion
(dashed) and PL (solid line) spec a o dono ( ed) and accep o (black line).

Resul s
The dyes selec ed as dono and accep o we e RhB and Rh700 (see Figu e 1) which sa is y a
numbe o condi ions: hey could be indi idually in oduced in o PMMA sphe es (up o a
maximum concen a ion o 0.8 mg/mL o MMA) be o e concen a ion quenching ook place,
he dono can be op ically pumped using wa eleng hs o which he polyme ic ma ix is
anspa en and he e is a good spec al o e lap be ween dono emission and accep o
abso p ion. Fu he , he spec al o e lap be ween he abso p ion bands o he species is small
enough as o g an a selec i e exci a ion o he dono alone ( ide in a).
As men ioned abo e wo pa ame e s we e changed in o de o con ol he sphe e diame e :
monome olume and eac ion ime. Fo a single MMA addi ion and 40 min eac ion ime,
sphe es in he 250-380 nm ange we e ob ained changing he MMA ini ial olume. In o de o
u he inc ease he sphe e diame e up o 500 nm a second addi ion o MMA was in oduced
and eac ion ime inc eased o 2 hou s. Figu e 2a shows he change in sphe e diame e wi h o al
MMA olume o he wo eac ion imes. Figu e 2b shows he size dis ibu ion o sphe es
ab ica ed using 25 mL o MMA in 140 mL o dis illed wa e . Fo he sample con aining only
dono molecules (QAD=0) a sphe e diame e o 383 nm and a polydispe si y o 2.2 % was
ob ained. Upon addi ion o inc easing amoun s o accep o molecules he sphe e quali y was no
a ec ed, he polydispe si y aising o jus 3.7% (Figu e 2c). Fu he , changing he QAD a io did
no a ec he sphe e diame e and only a ia ions o ca. 3% in sphe e diame e we e obse ed
om sample o sample.
Fig. 2 (a) Dependence o sphe e diame e on MMA olume. The wo highligh ed
egions co espond o: (I) 40 minu es and (II) 2 hou s eac ion ime. Dashed lines
a e guides o he eye. (b) Size dis ibu ion o sphe es ab ica ed om 25 mL o
MMA and a a io QAD=0. Inse shows an SEM image o he sphe es (scale ba is 1
µm). (c) E olu ion o he polydispe si y o sphe es ab ica ed wi h 25 mL o MMA
and inc easing QAD a io.
Nex we op ically cha ac e ized he sphe es employing PL spec oscopy as well as ime esol ed
expe imen s. Figu e 3 shows PL measu emen s o samples ha ing a diame e o 380 nm
(co esponding o samples g own om 25 mL o MMA) and a a iable accep o o dono a io.
He e we can see how o he sphe es con aining dono molecules only (QAD = 0) a PL peak
cen e ed a 577 nm, cha ac e is ic o RhB, is p esen . As we inc ease he accep o concen a ion
a PL peak cen e ed a 670 nm appea s associa ed wi h he emission o Rh700.

Fig. 3 (a) Aqueous suspensions and (b) no malized PL spec a o PMMA sphe es
ab ica ed om a o al olume o 25 mL o MMA and ha ing di e en QAD a ios
(indica ed o each sample and PL cu e).

In o de o e alua e he in luence o ene gy ans e and di ec op ical pump in he e olu ion o
he accep o emission, we ca ied ou a se o con ol measu emen s. We pumped, using he
same wa eleng h and powe , a se o con ol samples consis ing o sphe es con aining only
accep o molecules wi h iden ical concen a ion as he es samples bu wi hou dono species.
Figu e 4 shows he in ensi y o PL maxima o dono and accep o emission ( aken a 577 and
670 nm espec i ely). He e i is e iden ha as D emission decays, A emission inc eases and
only abou 20 % o i is due o di ec op ical exci a ion, he es being due o ene gy ans e
om he accep o . Fo alues o QAD ≥ 3 we can see how he ise in A PL sa u a es and hen
dec eases. This beha io poin s o he o ma ion o non-emi ing complexes ei he be ween
dono and accep o o be ween iden ical molecules (sel -quenching) o such la ge dye
concen a ions. As a ma e o ac , quenching is likely aking place be ween dono and accep o
molecules as o he case o he con ol samples (con aining only accep o molecules) he
sa u a ion beha io is less e iden . A simila beha io was obse ed o samples wi h smalle
sphe es (250 nm) a a smalle a io (QAD = 1). Fo la ge sphe es (480 nm) no sa u a ion was
obse ed o all QAD a ios unde conside a ion.
Fig. 4 In ensi y a he maximum o PL o dono (a) and accep o (b) emission
co esponding o λ=577 and 670 nm espec i ely. G ey ci cles co espond o
di ec op ical pumping o e e ence samples con aining only accep o molecules.
To u he e alua e he in luence o quenching due o agg ega ion o he molecules wi hin he
polyme sphe es, he decay dynamics o con ol and FRET samples we e s udied. In a i s s ep
a se o PMMA beads we e ab ica ed wi h an inc easing amoun o dono dye. Fo RhB
concen a ions exceeding ha used in he p esen wo k by a ca. ou - old ac o (see
Expe imen al sec ion) decay dynamics o he sphe es we e collec ed a λ=585 nm. Figu e 5
shows esul s o sphe es ab ica ed om 25 mL o MMA. A beha io close o he single-
exponen ial ideal scena io, whe e all molecules, once encapsula ed wi hin he PMMA sphe e,
a e sensing he same en i onmen was obse ed (see Fig.5 a). Ne e heless, o co ec ly i he
da a a mul iexponen ial i co esponding o a dis ibu ion o decay a es [27] has o be assumed.
In ou case, he dis ibu ion ha bes i ed he da a was a logno mal one, as p e iously obse ed
in dye-doped biopolyme ic ma ices. [28] The need o a mul iexponen ial i o desc ibe he
decay dynamics o dyes inco po a ed in o polyme ic beads is known e en o comme cial
sphe es o di e en size. [29] In ou case, he need o a dis ibu ion o decay a es is likely
ela ed o an inhomogeneous dis ibu ion o dye molecules wi hin he sphe e in insic o he
g ow h me hod whe e he dye concen a ion inc eases du ing he polyme iza ion p ocess (see
abo e). The decay dis ibu ions o he con ol samples wi h di e en dye concen a ions
p esen ed a small ull wid h a hal maximum (FWHM) o (0.11 ± 0.02 ns-1) and a cons an mos
equen alue ΓMF. The la e indica es ha no addi ional non- adia i e decay pa hs associa ed
wi h sel -quenching a e aking place independen o dye concen a ion. The ob ained decay a e
o he dono wi hin he PMMA beads (0.342 ± 0.007 ns-1) was sligh ly la ge han he alue o
molecules in an e hanol solu ion (0.320 ± 0.002 ns-1) as a consequence o he di e en
en i onmen o he dye molecules. This beha io in he dynamics o he dyes was accompanied
by a linea inc ease in i s PL, u he e idence o he absence o agg ega ion.
In a second se o con ol expe imen s, he dynamics o accep o molecules was s udied in
FRET samples con aining bo h dyes as he ones in Figs.3 and 4. The samples we e pumped wi h
λ=633 nm and PL was collec ed a λ=680 nm (see Fig. 5c). Unde hese pump condi ions we a e
ce ain ha accep o molecules a e no being exci ed ia FRET. Again, expe imen al da a could
be i ed wi h logno mal dis ibu ions wi h a small FWHM o 0.03 ± 0.01 ns-1) and a nea ly
cons an mos equen decay a e o 0.31 ± 0.01 ns-1 we e ob ained sa e om he sample ha ing
he highes accep o load (QAD=4) o which a as e dynamics wi h ΓMF = 0.36 ns-1 was
ob ained. Such change in he accep o dynamics is ela ed o agg ega ion in he p esence o
accep o as iden ical samples, only con aining no dono molecules (g ey do in he igu e),
showed a alue simila o ha o he FRET sphe es wi h lowe dono load.
In o de o e alua e whe he ene gy ans e be ween dono and accep o is aking place ia a
esonan p ocess (i.e. FRET) in he sphe es con aining bo h dyes we nex s udied he dynamics
o he emission o he dono in he absence and p esence o accep o . I a dono molecule is
su icien ly close o an accep o one, non- adia i e pa hs o ene gy ans e will be a ailable o
he o me ia long- ange dipole-dipole in e ac ion wi h he la e . The addi ion o non- adia i e
(FRET) de-exci a ion pa hs wi h a decay a e ΓFRET o he adia i e ones ΓD causes an inc ease
o he o al decay a e ΓT measu ed in a ime esol ed expe imen . Thus, measu ing he li e ime
o he dono in he absence (ΓD) and p esence (ΓT = ΓD + ΓFRET) o accep o yields he FRET
decay a e. Once his is known, he e iciency o FRET is gi en by he a io be ween he decay
a e associa ed wi h FRET and he o al a e.

PL
d
di e
diam
e
b
eco
m
Fu
h
cu
e
dono
wo
o
inco
due
o
(see
E
Fig. 5 (a)
P
(g ey) and
equen
a
PMMA sp
h
associa ed
R
h700 (g
e
concen a
i
d
ecay cu e
s
en QAD a
i
e
e o 380
m
e s eepe
h
e
mo e, si
m
e
s become
/accep o c
o
o
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o
po a ed in o
o
he ac h
a
E
xpe imen
a
P
L decay c
u
i wi h a l
o
a
lue ΓMF o
h
e es ab i
c
wi h he d
e
e
y do co
e
i
on as QAD=
4
s
a he do
n
i
os when pu
m
nm. As acc
e
poin ing o
m
ila o o h
e
s ongly m
u
o
n igu a ion
s
o
n he one h
a
he polyme
a
he amou
n
a
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e o a di
s
o
gno mal di
s
he a e dis
c
a ed using
e
cay o Rh
7
e
sponds o
a
4
only wi h
n
n
o ’s emissi
m
ped wi h
λ
e
p o molec
u
he in od
u
e
polyme ic
u
l iexponen
s
exis . In h
e
a
nd a dis i
b
ic ma ix.
O
n
o dye in
h
s
pe sion o
P
s
ibu ion o
ibu ion as
25ml o
MM
7
00 measu
e
a e e ence
n
o dono ).
i
on (λ=585
λ
=515 nm. F
i
u
les a e incl
u
u
c ion o n
o
c
ma ices s
u
ial which
i
e
p esen ca
s
b
u ion o D/
A
O
n he o he
h
he eac ion
i
P
MMA sphe
decay a es
a unc ion
o
MM
A con ain
d o sphe
e
sample ha
nm) we e
m
i
gu e 6 sho
w
u
ded wi hin
o
n- adia i e
u
ch as DN
A
i
ndica es h
a
s
e he dis ib
u
A
sepa a io
n
h
and a adi
a
i
nc eases as
es con aini
n
(black cu
e
o
dye conce
n
i
ng RhB o
n
e
s con aini
n
ing he sa
m
m
easu ed o
w
s esul s o
he sphe es
(FRET) d
e
A
-CTMA co
a
an inc e
u
ion o FR
E
n
s and o ien
a
l concen a
i
he polyme
i
n
g only RhB
e
). (b) Mo
s
n a ion o
n
ly. (c) ΓMF
n
g RhB and
m
e accep o
samples
h
sphe es ha
s
he decay
c
e
-exci a ion
o
mplexes,
[
2
e
asing num
b
E
T p ocesse
s
a ions as h
e
i
on g adien
i
za ion ake
s
h
a ing
ing a
c
u es
pa hs.
8
]
he
b
e o
s
has a
e
y a e
exis s
s
place
Fig. 6 PL decay cu es measu ed o sphe es wi h a diame e o 380 nm. (a)-(e)
co espond o QAD=0-4. G ey cu e shows he esul o QAD=0 and is shown o
compa ison.
In o de o gain u he insigh in o he FRET p ocess wi hin he PMMA sphe es as he QAD
a io inc eases we will conside he decay dis ibu ion ex ac ed om he i ing o he
expe imen al cu es. Figu e 7 shows logno mal dis ibu ions ρ(Γ) o he di e en alues o QAD
conside ed in he p esen wo k as de i ed om he PL decay cu es. He e we can see how as
he accep o concen a ion inc eases o a ixed amoun o dono molecules he dis ibu ion
shi s o la ge Γ alues, co esponding o an o e all as e decay. Since no agg ega ion be ween
he molecules is aking place as men ioned abo e, his co esponds o he in oduc ion o
addi ional non- adia i e decay (FRET) pa hs. Fu he , as he dis ibu ion shi s o la ge alues i
becomes b oade , e idencing he exis ence o an inc easing numbe o D/A con igu a ions. The
la e likely comp ises bo h sepa a ions and o ien a ions as no p e e ed molecule o ien a ion is
expec ed o ake place wi hin he polyme ic ma ix.
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