Bicolour fluorescent molecular sensor for cations: design and experimental validation

This jou nal is © he Owne Socie ies 2021 Phys. Chem. Chem. Phys.
Ci e his: DOI: 10.1039/d1cp01203g
Bicolou luo escen molecula senso s o
ca ions: design and expe imen al alida ion
Zo aida F eixa, *
ab
I a
´n Ri illa,
ac
F ancesc Mon abal,
ac
Juan J. Go
´mez-Cadenas
ac
and Fe nando P. Cossı
´o *
cd
Molecula en i ies whose luo escence spec a a e diffe en when hey bind me al ca ions a e e med
bicolou luo escen molecula senso s. The basic design c i e ia o his kind o compound a e p esen ed
and he diffe en luo escen esponses a e discussed in e ms o hei chemical beha iou and elec onic
ea u es. These la e elemen s include in amolecula cha ge ans e (ICT), o ma ion o in amolecula
and in e molecula excime /exciplex complexes and Fo
¨ s e esonance ene gy ans e (FRET). Changes in
he elec onic p ope ies o he luo opho e based on he decoupling be ween i s cons i u i e uni s upon
me al binding a e also discussed. The possibili y o gene a ing luo escen bicolou indica o s ha can
cap u e me al ca ions in he gas phase and a solid–gas in e aces is also discussed.
1. In oduc ion
De ec ion o me al ca ions by means o molecula senso s has
become a e y ac i e esea ch ield, wi h impo an applica ions
in analy ical chemis y,
1
ma e ials
2
and en i onmen al
sciences,
3
and biological chemis y,
4
among o he scien i ic
a eas. Ve y ecen ly, his echnique has eme ged as a
p omising ool in pa icle physics since luminescence
a
Ike basque, Basque Founda ion o Science, 48009 Bilbao, Spain.
E-mail: zo aida_ [email protected]
b
Depa men o Applied Chemis y, Facul y o Chemis y,
Uni e si y o he Basque Coun y (UPV/EHU), 20018 San Sebas ia
´n/Donos ia, Spain
c
Donos ia In e na ional Physics Cen e (DIPC), 20018 San Sebas ia
´n/Donos ia, Spain
d
Depa men o O ganic Chemis y I, Facul y o Chemis y, Uni e si y o he Basque
Coun y (UPV/EHU), 20018 San Sebas ia
´n/Donos ia, Spain
Zo aida F eixa
Zo aida F eixa comple ed he
PhD in he a ea o homogeneous
ca alysis a he Au onomous
Uni e si y o Ba celona. A e a
pos -doc o al s age a P o . an
Leeuwen’s g oup in Ams e dam,
she en olled as Ramo
´n y Cajal
Resea che a he Ins i u e o
Chemical Resea ch o Ca alonia
(ICIQ) as a g oup manage o
P o . an Leeuwen’s g oup. Since
2010, and a e a sho pe iod as
a Lec u e a he Uni e si y o
Ba celona, she has been holding
an Ike basque Resea ch P o esso posi ion a he Uni e si y o he
Basque Coun y (UPV/EHU) in San Sebas ia
´n. She leads a esea ch
g oup ocused on he de elopmen o pho oswi chable and/o chi al
o ganome allic compounds wi h special emphasis on he s udy o
hei ca aly ic and luminescence p ope ies. Since 2019, she has
been in ol ed in he de elopmen o bicolou senso s o ba ium
and adium agging in bb0n eac ions.
I a
´n Ri illa
I a
´n Ri illa g adua ed in
Chemis y a he Uni e si y o
Cas illa-La Mancha. He
ob ained his PhD deg ee in
Chemical Sciences in 2008. He
did his i s pos -doc o al
in e nship unde he supe ision
o P o . P. J. Pe
´ ez a he
Uni e si y o Huel a in 2009. In
2012, he mo ed o he Uni e si y
o he Basque Coun y (UPV/
EHU). Since 2018, he has also
been a isi ing esea che a he
La Sapienza Uni e si y o Rome.
Since 2020 he has been an Ike basque Resea ch Associa e a he
Donos ia In e na ional Physics Cen e (DIPC). His main esea ch
a eas ange om cycloaddi ion eac ions o he design and
syn hesis o new chemical en i ies wi h biological ac i i y. In
addi ion, he is in e es ed in he design and syn hesis o new
chemical senso s, o applica ions in pa icle physics o in he
de ec ion o pa hogens. Mo e ecen ly, he has also begun o s udy
eac ion mechanisms by means o DFT calcula ions.
Recei ed 17 h Ma ch 2021,
Accep ed 17 h June 2021
DOI: 10.1039/d1cp01203g
sc.li/pccp
PCCP
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sensing o ca ions is e y well-sui ed o he de ec ion o special
e en s such as neu inoless double be a decay nuclea
p ocesses.
5
Diffe en echniques de eloped so a o he de ec ion
o me allic ions include a omic abso p ion spec oscopy,
ol amme y, elec ochemical analysis, induc i ely coupled
plasma spec ome y, lase induced b eakdown spec oscopy,
neu on ac i a ion analysis and UV-Vis spec oscopy, among
o he s.
6,7
Howe e , luo escence
8
is pe haps he mos equen ly
used because o he ela i e simplici y o he associa ed de ices,
i s e y high sensi i i y and i s excellen spa ial and empo al
esolu ion.
9–15
Since in many samples di e en me al ca ions
can coexis , he co esponding senso s mus exhibi high
selec i i y. In his espec , luo escence sensing a ays ha e been
de eloped as a use ul al e na i e based on ensembles o di e en
less selec i e senso s ha exhibi cha ac e is ic esponses o
di e en analy es.
16
Many diffe en ypes o luo escen senso s able o de ec
me al ca ions ha e been desc ibed, including nanopa icles,
17
ca bon-based quan um do s,
18,19
and dend ime s,
20
as well
as me al–o ganic amewo ks (MOFs),
21
and biomolecules
such as DNA
22
and p o eins.
23
These clus e ed and mac o-
molecula senso s a e no included in he p esen Pe spec i e
A icle, which is ocused on disc e e luo escen molecula
senso s.
The luo escence emission
24
spec um o a gi en
luo opho e is p oduced a e exci a ion a a ce ain wa eleng h
(no mally in he UV egion), ollowed by emission o pho ons a
la ge wa eleng hs. The S okes shi exp esses he diffe ence
be ween he maximum abso p ion wa eleng h l
max
abs
and he
maximum emi ed wa eleng h l
max
em
. I his diffe ence is
exp essed in wa enumbe s, he co esponding D
n
S okes
is
gi en by
D
nS okes ¼1
lmax
abs
1
lmax
em
Since l
max
em
4l
max
abs
,D
n
S okes
40. The exci a ion p ocess
gene a es a ansi ion om he g ound s a e S
0
o molecule A o
he i s single exci ed s a e S
1
(A*). The decay om S
1
(A*) o
S
0
(A) is de e mined by he adia i e kine ic cons an k
S
A!
kS
Aþpho onðlemÞ
Juan J. Go
´mez-Cadenas
Juan Jose
´Go
´mez Cadenas
ecei ed bo h his MSc deg ee
and his PhD om he Uni e si y
o Valencia. He has been a S aff
membe a he Eu opean
Labo a o y o Pa icle physics
(CERN), a ull p o esso a he
Uni e si y o Valencia and a ull
p o esso a CSIC. Since 2017, he
has been an Ike basque P o esso
a DIPC. He is he co-
spokespe son o NEXT, a leading
expe imen sea ching o
neu inoless double be a decay
p ocesses (bb0n). Toge he wi h he o he au ho s o his pape
and he membe s o he NEXT Collabo a ion, he is leading an
in e disciplina y effo o de elop a backg ound- ee bb0n
expe imen in xenon based on he agging o he xenon daugh e
(Ba
2+
) in he double be a decay h ough he use o molecula
senso s.
Fe nando P. Cossı
´o
Fe nando P. Cossı
´o ecei ed his
MSc deg ee in chemis y om he
Uni e si y o Za agoza and his
PhD in o ganic chemis y om
he Uni e si y o he Basque
Coun y (UPV/EHU). Since 2002,
he has been a ull p o esso in he
UPV/EHU, whe e he se ed as he
Dean o he Facul y o Chemis y
and he ice chancello o
esea ch and in e na ional
ela ions. Since 2009, he has
been he scien i ic di ec o o
Ike basque, he Basque
ounda ion o he ad ance o science. His esea ch in e es s a e
ocused on expe imen al and compu a ional aspec s o
cycloaddi ion chemis y and C–C bond o ming eac ions, wi h
special emphasis in he design and chemical syn hesis o new
molecules wi h use ul biological and pho ophysical p ope ies in
medicinal chemis y, molecula imaging and neu inoless double
be a decay eac ions leading o Ba
2+
ca ions.
F ancesc Mon abal
F ancesc Mon abal g adua ed in
Physics a he Uni e si y o
Valencia. He ob ained his PhD
a he same Uni e si y
de eloping he echnology o
gaseous xenon TPC o double
be a decay sea ch. In 2015 he
s a ed wo king wi h P o . Da id
R. Nyg en a he Uni e si y o
Texas a A ling on whe e he was
in cha ge o he design and
cons uc ion o he Time
P ojec ion Chambe (TPC) o
he NEXT (Neu io Expe imen
wi h a Xenon Time Chambe )-Whi e de ec o becoming a
echnical coo dina o o he NEXT Collabo a ion he same yea .
Since 2019 he has been an Ike basque Resea ch Fellow a he
Donos ia In e na ional Physics Cen e (DIPC), whe e he is leading
he effo o he cons uc ion o he NEXT-100 de ec o .
Pe spec i e PCCP
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and ollows a usual i s -o de kine ics in he o m
½1A ¼½
1A0exp 
S

In he la e equa ion, [
1
A*]
0
is he concen a ion o exci ed
molecules a = 0, which co esponds o he ins an o he pulse
ligh exci a ion, and
S
is he li e ime o he exci ed s a e S
1
:
S¼1
kS
þks
n
whe e k
S
n
is he o al kine ic cons an associa ed wi h non-
adia i e p ocesses ha do no esul in luo escence emission.
The ime-dependen in ensi y o he luo escence emission
i
F
( ) a e exci a ion a = 0 is p opo ional o he concen a ion
o molecule A in he single exci ed s a e and o he kine ic
cons an associa ed wi h he luo escence emission:
iFð Þ¼kS
½1A ¼kS
½1A0exp 
S

I is impo an o no e ha he measu ed luo escence
in ensi y I
F
is ob ained om he o al decay a diffe en wa e-
leng hs and is exp essed in a bi a y uni s (a.u., no o be
con used wi h a omic uni s) ha depend on he expe imen al
condi ions.
The luo escence quan um yield o a gi en A* -A ansi ion
is he ac ion o exci ed molecules S
1
(A*) ha decay o he
g ound s a e S
0
(A) wi h emission o pho ons ( ide sup a):
F¼kS
S¼kS
kS
þkS
n
Thus, he quan um yield gi es he ac ion o emi ed
pho ons du ing he o al decay wi h espec o he abso bed
pho ons. This magni ude gi es c ucial in o ma ion o measu e
he efficiency o luo escence emission. Ano he ele an
pa ame e is he b igh ness
4,23
o he luo escence emission
B
F
, which is he p oduc o he quan um yield and he mola
ex inc ion coefficien e
abs
a he exci a ion ligh wa eleng h.
B
F
=
F
e
abs
All hese combined magni udes cha ac e ize he pho ophysical
beha iou o a gi en luo opho e.
The key poin in luo escence sensing o diffe en me allic
ca ions is he change o hese magni udes upon coo dina ion
o binding o he senso wi h he analy e. In a luo escen
monocolou senso he emi ed pho ons a e o iden ical wa e-
leng hs l
em
in he ee s a e and upon coo dina ion o binding
wi h he me al ca ion. This can occu ia pho oinduced elec on
ans e
1,25
(PET) phenomena ia an elec onic dona ion om
one he e oa om o he senso ha quenches he luo escence
elaxa ion o he luo opho e in he ee S
1
s a e. Upon coo dina ion,
his dona ion is di ec ed o he me al and he luo escence decay
akes place efficien ly hus esul ing in an off–on ansi ion
( u n-on enhancemen ) on going om he ee o he me al-
coo dina ed s a e. Ano he less equen ly obse ed PET
consis s o he e e se on–o p ocess ( u n-o quenching). In
mos cases, he PET pe u ba ions o he elaxa ion p ocesses
esul in p e e en ial emission o pho ons wi h l
em
alues
simila o ee and bound s a es, he only di e ence being
he e y low and e y high in ensi ies in he o and on s a es,
espec i ely. The e o e, he la ge he di e ence be ween he
in ensi ies o he o and on s a es, he highe he sensi i i y o
he senso . These monocolo p obes equi e e y high in ensi y-
based sepa a ion ac o s be ween hei ‘o ’ and ‘on’ s a es;
such sepa a ion ac o s a e challenging o a ain, gi en a ious
analy e-independen ac o s which a ec he p obe in ensi y
(e.g. luc ua ions in he exci a ion sou ce, he e ec o he
mic oen i onmen , a ia ions in he p obe concen a ion,
e c.).
26,27
In his Pe spec i e A icle we a he ocus on bicolo
p obes which ely on wa eleng h sepa a ion and a e no
a ec ed by such ac o s.
Molecula bicolou senso s emi pho ons wi h diffe en
wa eleng hs in he ee (l
em
) and bound l0
em

s a es. They
can also be desc ibed as dual emission
28
a iome ic p obes
wi h signal changes associa ed wi h wo independen o co e-
la ed emi e s.
27
Depending on he magni ude o l0
em wi h
espec o l
em
, blue (hypsoch omic) o ed (ba hoch omic)
shi s will be obse ed. Thus, bo h ee and bound bicolou
senso s can be ac i e luo escen emi e s wi hin an on–on0
scheme. In his case, he sensi i i y will be de e mined by he
espec i e in ensi ies and he co esponding pho ophysical
pa ame e s. Two ele an pa ame e s a e he wa eleng h
di e ence DlF¼l0
em lem and he peak disc imina ion
ac o
l
, de ined as
l¼I0
lIl
Il
whe e I0
land I
l
a e he in ensi ies o he signals cha ac e ized
by a wa eleng h la he bound and ee s a es, espec i ely.
This ac o is an indica o o he expec ed signal cleaning o he
me al-bound senso wi h espec o i s ee unbound s a e,
especially when he concen a ion o he me allic ca ion is e y
low ( ide in a). Thus, he g ea e he sepa a ion dic a ed by
l
,
he lesse he o e lap be ween bo h signals. Aside he in insic
alues o quan um yield, b igh ness and in ensi y, hese las
wo ac o s will de e mine he sensi i i y o he molecula
senso wi h espec o he backg ound. This is pa icula ly
impo an in ca ion- agging expe imen s wi h ex emely low
concen a ion o he me allic ca ion (e en ually, only one!), in
which he concen a ion o he ee molecula bicolou senso
is he e o e o de s o magni ude mo e highe han ha o he
bound senso .
Ano he impo an aspec associa ed wi h he design o
molecula luo escen bicolou senso s is he necessi y o
de eloping senso s able o cap u e ca ions in he gas phase.
This in u n implies he explo a ion o sup amolecula
chemis y on solid–gas in e aces, a esea ch ield much less
PCCP Pe spec i e
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de eloped han i s in-solu ion analogues. The use o single
ca ion iden i ica ion has been p oposed as a way o d ama ically
educe he adioac i e backg ound
29
in xenon de ec o s looking
o neu inoless double be a decay. The
136
Xe iso ope decays in o
ba ium, emi ing wo elec ons (and wo an i-neu inos in he
s anda d p ocess). Iden i ica ion o a coincidence signal o he wo
elec ons wi h he app op ia e ene gy wi h he appea ance in he
ac i e olume o a Ba
2+
ionwillbeanunequi ocalsigna u eo he
neu inoless p ocess and, consequen ly, a demons a ion ha
neu inos a e hei own an i-pa icles (Majo ana
30
pa icles).
Demons a ing his hypo hesis would ha e deep consequences
in pa icle physics and in cosmology.
31,32
A p omising ‘‘we ’’
ba ium agging expe imen has been epo ed as a p oo o
concep .
33
In o de o ep oduce he equi ed ‘‘d y’’ condi ions
imposed by hese expe imen s, se e al in e es ing ba ium- agging
senso s ha e been epo ed so a ,
34,35
all o hem based on PET
o –on luo escence emission. Ou app oach o ba ium- agging is
ins ead based on he de elopmen o sui able on–on’ bicolou
luo escen senso s, which bene i om a wa eleng h-shi ing
mechanism.
The many con ibu ions o he de elopmen o a iome ic
molecula luo escen senso s o ca ions ha e been comp e-
hensi ely desc ibed in leading e iews and book chap e s in he
a ea.
9,26,27,36–42
In his Pe spec i e A icle, we shall p esen and
discuss he main ea u es and design c i e ia o molecula
bicolou luo escen senso s o me allic ca ions, in ended o
he de ec ion o ex emely low ca ion concen a ions (e en ually
only one) on a su ace co e ed by unbounded senso molecules,
in d y media. In he ollowing sec ions, we will desc ibe he
main modes o senso –ca ion in e ac ions and he elec onic
phenomena esponsible o he change in he luo escence
spec a upon me al coo dina ion o a e in e ac ion wi h he
ca ionic cen e. We will also epo on ecen esul s ob ained
in ou labo a o ies on ba ium agging in d y media in ol ing
solid–gas phases.
Bicolou luo escen p obes can be conside ed a special ype
o he so-called single- luo opho e a iome ic p obes wi h wo
analy e-sensi i e emission signals, as hey change he emission
in ensi y and wa eleng h upon in e ac ion wi h he analy e.
They can also be desc ibed as dual emission ma e ials
composed o wo independen emi e s ( ee and bound) in
equilib ium.
28,43
In gene al, hese molecules inco po a e diffe en
componen s ha include a syn he ic ionopho e ( he ca ion
ecogni ion-binding domain), he luo opho e and, op ionally, a
space and a linke o ancho he senso wi h sui able su aces
o biomolecules. The co alen connec ions among hese
componen s can be diffe en , hus gi ing ise o diffe en
senso a chi ec u es, he mos usual ones being hose shown in
Fig. 1A.
The binding g oups (ionopho es) a e esponsible o he
efficien and p e e ably highly selec i e cap u e o he me al
ca ion and can consis o chela ing g oups
4
such as poly-
ca boxylic acids B1, podands B2, co onands such as c own
e he s
44–46
B3 and c yp ands B4. O he ionopho es
47,48
such as
calixa enes, cyclodex ins and pilla a enes, no shown in
Fig. 1B, ha e been desc ibed.
The s uc u es o luo opho e componen s (Fig. 1C) a e e y
a ied
4
and comp ise a oma ic hyd oca bons such as py enes
C1, and polyhe e ocyclic a oma ic moie ies such as naph hyli-
mides C2, e acyclic s uc u es based on imidazo[1,2(5)a]
py idines
49,50
C3, luo escein de i a i es C4 and BIODIPY-
based s uc u es C5, among o he s.
When equi ed, he space s can consis o alipha ic linea
chains o polye hylene glycol (PEG) de i a i es, which can in e -
cala e wi h a oma ic hyd oca bons o he e ocycles. Finally, he
linke s can be ialkoxysilyl de i a i es D1 ( e y use ul o ancho ing
he luo escen molecula senso s o solid su aces) o maleimides
D2, which can bind – wi h low selec i i y – cys eine-con aining
Fig. 1 Gene al design pa e ns o luo escen molecula senso s o
ca ions (A), showing hei main componen s: binding g oups (ionopho es, B),
luo opho es (C) and linke s (D).
Pe spec i e PCCP
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p o eins o bio hiols, hus gi ing ise o he co esponding
conjuga ed de i a i es.
51
Molecula luo escen bicolou senso s can change he
in ensi y and emission wa eleng h o hei luo escence signals
in diffe en ways a e in e ac ing wi h me al ca ions (Fig. 2).
When an ionopho e is p esen , me al binding can modi y he
pho ophysical p ope ies o he senso ia o ma ion o monome ic
and dime ic complexes (Fig. 2, mode A.1). In some cases, when he
luo opho e i sel is able o bind di ec ly he me allic ca ion, he
pho ophysical p ope ies o he chela e can be diffe en o hose o
he ee luo opho e (Fig. 2, mode A.2), hus yielding a bicolou
senso . In many cases, a he e oa om X possessing a sui able lone-
pai p esen in he luo opho e can also bind he me allic cen e
hus o ming a complex in which bo h he syn he ic ionopho e and
he he e oa om a e bound o he ca ionic analy e (Fig. 2, no e he
dashed line in monome ic complex A.1). Finally, eac i e unc ional
g oups deco a ing he luo opho e can eac selec i ely wi h he
me al o be ans o med in o a diffe en luo opho e, hus gi ing
ise o an ionopho e- ee luo escen bicolou senso (Fig. 2B).
These conside a ions apply o no only single emi e s om one
exci ed elec onic s a e, bu also o dual emi e s wi h a sole
luo opho e possessing wo emi ing s a es and dual emi e s wi h
wo independen o co ela ed emission cen es.
28
2. Mechanisms o bicolou sensing
2.1. In amolecula cha ge ans e (ICT)
I changes in he elec onic s uc u e o he luo escen bicolou
senso a e conside ed, one possibili y consis s o modi ying he
pola iza ion pa e ns be ween subs i uen s o he luo opho e
in he ee and bound s a es. This p ocess is named Pho o-
induced Cha ge T ans e (PCT), which almos in all cases akes
place in amolecula ly. The e o e, he la e p ocess will be
e e ed o as In amolecula Cha ge T ans e (ICT). In hese
sys ems, an elec on- eleasing g oup (ERG), which ac s as a
dona ing g oup, ans e s a ce ain elec on densi y o an
elec on-wi hd awing g oup (EWG), which is he accep o o
his cha ge ans e . This p ocess in ol es complex elec onic
in e ac ions. Wi hin a simpli ied scheme in ol ing delocalized
on ie molecula o bi als, wo si ua ions can be dis inguished,
depending on he p oximi y o ERG and EWG moie ies o he
me allic ca ion. O cou se, speci ic elec onic in e ac ions can be
impo an , especially when he e is a di ec union be ween he
luo opho e and he me al ion. When he ERG is close o he M
n+
cen e bound o he ionopho e, he wo-elec on ERG -M
n+
in e ac ion will compe e wi h he ERG -EWG dona ion hus
inc easing he n0
em equency associa ed wi h he luo escence
S
1
-S
0
ansi ion (Fig. 3A). The e o e, a blue shi will be
obse ed, in many cases wi h a ce ain dec ease in b igh ness.
Al e na i ely, when he EWG is close o he ca ion, he ERG -
EWG dona ion will inc ease in magni ude and he EWG-M
n+
in e ac ion will esul in he lowe ing o he lowes unoccupied
molecula o bi al (LUMO) ene gy o he luo opho e, hus
esul ing in a lowe n0
em equency and he co esponding ed
shi , usually associa ed wi h a b igh e signal (Fig. 3B). In bo h
cases, he sensi i i y o he ICT senso co ela es wi h he
espec i e diffe ences in b igh ness, as well as wi h he peak
disc imina ion ac o s ( ide sup a).
2.2. Excime s
Ano he way o ob ain adiome ic bicolou senso s consis s o
designing luo opho es ha , upon binding he me allic ca ion
h ough he adequa e ionopho e, o m dime s (homo- o
he e odime s) ha a e mo e s able in he S
1
exci ed s a e. Thus,
an excime (exci ed dime ) o igina es om he in e ac ion o an
exci ed luo opho e wi h a nea by congene in he g ound s a e.
I he wo in e ac ing uni s a e non-iden ical, he whole
chemical en i y is named an exciplex (exci ed complex). The
wo uni s can be connec ed by a space and inco po a e hei
espec i e incomple e ionopho e componen s ha , upon
coo dina ion wi h he me allic ca ion, gene a e he op imal
binding g oup (Fig. 4A). An analysis o he on ie molecula
o bi als (FMOs) o he monome ic uni s o he senso a he S
0
and S
1
(Fig. 4B) s a es shows ha he ene gy le els o he FMOs
o he excime (o he exciplex) a e modi ied upon dime iza ion
Fig. 2 Main ypes o bicolou senso s classi ied acco ding o hei
s uc u e and in e ac ion wi h he me al. Diffe en chela ion modes
(A, monome ic and dime ic chela ing g oups, as well as chela ing
luo opho es) a e shown. Bicolou senso s in which he change in he
luo opho e is gene a ed by a chemical eac ion wi h he me allic ion a e
also shown (mode B). Op ional space and linke componen s a e omi ed.
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and usually esul in a sho e HOMO–LUMO gap (HOMO
s ands o he highes occupied molecula o bi al).
52
In u n,
his sh inkage in ene gy le els gene a es a ed shi ha pe mi s
o disc imina e he ee and bound s a es o he senso . The
binding abili y o he pa ial ionopho es in he ee monome ic
s a e and he s abili y o he excime /exciplex can esul in a
backg ound signal ha , depending upon he espec i e
in ensi ies, pe mi s o es ablish an accu a e measu emen o
he adiome ic esponse (Fig. 4C). In o he cases, he excime /
exciplex is ele an in he absence o he me allic ca ion, whose
coo dina ion esul s in he o al o pa ial loss o luo escen
esponse associa ed wi h excime inhibi ion p omo ed by
ca ion coo dina ion.
2.3. FRET
A ela ed phenomenon in which he emission in ol es an
in e ac ion be ween wo luo opho es (Fig. 5A) is he Fo
¨ s e
esonance ene gy ans e (FRET).
53–55
This p ocess consis s o
a non- adia i e exci a ion ans e
56
om he exci ed dona ing
(D*) g oup o he accep o g oup (A) in i s g ound s a e (Fig. 5B).
The la e g oup elaxes ia an A*(S
1
)-A(S
0
) p ocess hus
emi ing wi h a di e en wa eleng h (Fig. 5C).
57
Any FRET
equi es app op ia e ela i e o bi al ene gies associa ed wi h
he FMOs o D and A. In he case o a iome ic bicolou
senso s, he FRET is usually obse ed when he senso is bound
o he ca ionic cen e. I is no ewo hy ha he dis ance R
equi ed o FRET is signi ican ly la ge han he a e age alues
o he combined an de Waals adii o he D and A g oups. The
Fo
¨ s e dis ance R
0
is de ined as he dis ance a which he FRET
e iciency
FRET
=[1+(R/R
0
)
6
]
1
is 0.5. Fo ins ance, i D =
py ene and A = couma in, R
0
= 3.9 nm.
58
The FRET e iciency
also depends on he ela i e o ien a ion o D and A, as well as
Fig. 3 Schema ic ep esen a ion o in amolecula cha ge ans e
p ocesses (ICT) ha modi y he luo escence emission o bicolou senso s
in he p esence o ca ions M
n+
. (A) Blue shi effec o an elec on- eleasing
g oup (ERC) close o he ionopho e. (B) Red shi effec o an elec on-
wi hd awing g oup (EWG) close o he ionopho e.
Fig. 4 (A) Ca oon showing he o ma ion o excime s/exciplexes in he
p esence o a me allic ca ion. (B) Quali a i e diag am showing he dis o ion
o he FMO ene gy le els in he S
1
s a e o he excime wi h espec o hose
o he monome . (C) Red shi o he emission spec a o monome s and
excime s/exciplexes associa ed wi h he diag am shown in (B). Fig. 5 (A) Schema ic ep esen a ion o a luo escen senso inco po a ing
dono and accep o uni s. R
0
is he Fo
¨ s e adius (see he ex ). (B) Fo
¨ s e -
esonance elec on ans e (FRET) associa ed wi h he on ie molecula
o bi als o he dono and he accep o . (C) A ep esen a ion o he
luo escence emission spec a o he FRET senso in he ee and bound
s a es.
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on he o e lap be ween he emission spec um o D and he
abso p ion spec um o A.
2.4. Two-componen luo opho es
In he p e ious sec ions, we ha e p esen ed bicolou senso s based
on luo opho es ha ac as indi isible pho oac i e ensembles.
Ano he possibili y consis s o spli ing a gi en luo opho e in o
wo sepa a e o a able uni s connec ed by a single co alen bond
(Fig. 6). In u n, his wo-uni luo opho e 1is co alen ly a ached o
an ionopho e. When a me allic ca ion is cap u ed by he la e
binding moie y, he na u e o he in e ac ion is ein o ced by
addi ional coo dina ion wi h a he e oa om o luo opho e 1, hus
yielding an ICT-complex (Fig. 2, A.1 pa e n) ha in addi ion
inco po a es a p–ca ion in e ac ion
59
be ween he me allic cen e
M
n+
and he A
2
g oup ga he ed in Fig. 25. This A
2
–M
n+
complex
p omo es a A
2
-M
n+
cha ge ans e , as well as a o a ion o A
2
wi h espec o he polyhe e ocyclic He A
1
uni . The ou come o
hese me al-induced changes is he decoupling be ween he He A
1
and A
2
g oups. The e o e, he new He A
1
luo opho e 2 hus
o med (Fig. 25) is less conjuga ed han i s p edecesso luo opho e
1in he unbound s a e and will emi pho ons wi h sho e
wa eleng h. I an op ical il e is ins alled in he de ec ion sys em
o hide he emission associa ed wi h luo opho e 1, hisblueshi
should pe mi an essen ially backg ound- ee signal upon ca ion
complexa ion i he peak disc imina ion ac o
l
is la ge enough.
3. Design and alida ion o bicolou
p obes
All o he abo e discussed bicolou senso s ha e been
classi ied in e ms o he chemical in e ac ions in ol ed
( luo opho e–ionopho e, di ec chela ion by he luo opho e
and a chemical eac ion wi h he ca ion) o he elec onic
e ec s ha p omo e he change in he wa eleng h o he
emi ed pho ons (ICT, excime /exciplex, FRET and ela ed
phenomena). In he ollowing sec ions, an o e iew o selec ed
examples o hese s a egies o he cons uc ion o bicolou
luo escen p obes o ca ions is p esen ed and discussed.
3.1. ICT-Based senso s
Many e iew a icles and books desc ibe he pho o-induced ICT
phenomenon and he po en ial applica ions o ICT-based
compounds.
24,60
In pola sol en s, ea angemen o he sol a ion
shell a ound he ICT exci ed s a e p oduces i s s abiliza ion and
he concomi an des abiliza ion o he landing g ound s a e,
aising i s ene gy le el.
41
The e o e, hese compounds may show
dual emission: a peak in he mos ene ge ic egion o he emission
spec a ha o igina es a he locally exci ed s a e (LE), and a
ed-shi ed signal om he sol en -s abilized ICT exci ed s a e.
Addi ionally, i he dono and he accep o a e connec ed h ough
a o a ingp-elec on b idge, a elaxa ion o he ICT exci ed s a e
can occu h ough an in amolecula o a ion, decoupling he
o bi als o he dono and accep o , o ming a highly pola exci ed
s a e. This phenomenon is e e ed o as ‘‘ wis ed in amolecula
cha ge ans e ’’ (TICT), and i is cha ac e ized by ex a-la ge
S okes shi s in pola sol en s.
Bo h ICT- and TICT-based luo opho es a e sui able
scaffolds o he cons uc ion o bicolou p obes o ca ions
by in eg a ing a ca ion ecep o ei he on he ERG o on he
EWG si es o he luo opho e (Fig. 3). The simplici y o he
design makes ICT-based senso s he mos widely used a io-
me ic p obes o ca ions. Among he ea lies examples a e
Indo-1 and Fu a-2 (Fig. 7), de eloped in 1985 by Tsien and
collabo a o s.
61
Nowadays, Indo-1 and Fu a-2 a e he leading
compounds o a se ies o comme cially a ailable luo escen
a iome ic p obes o Ca
2+
and o he ca ions.
62
They combine a
s ilbene skele al luo opho e ( ein o ced by ex a he e ocyclic
bonds) wi h he well-es ablished BAPTA agmen as an ion-
ecogni ion si e. These molecula p obes a e cha ac e ized by a
s ong luo escen a iome ic esponse o Ca
2+
. Because o he
inhibi ion o he ICT upon coo dina ion o he Ca
2+
o he
Fig. 6 Design c i e ia o wo-componen luo escen bicolou indica o s
(FBI) based on he decoupling o one componen o he s a ing luo opho e
upon me al coo dina ion. The me al-induced hypsoch omic shi and he
eco ding o a backg ound- ee signal by means o an op ical il e a e also
highligh ed. He A and A deno e polyhe e ocyclic a oma ic and a oma ic
g oups, espec i ely. Fig. 7 Fu a-2 and Indo-1 ICT-based senso s o Ca
2+
.
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aniline ni ogen o he BAPTA ecep o , a blue-shi ed
luo escenc emission is obse ed (Fig. 3A). One o he main
limi a ions o hese p obes was he need o UV-ligh exci a ion.
I was ci cum en ed using wo-pho on abso p ion (TPA)
mic oscopy, which pe mi ed he s udy o ca ion dis ibu ion
in li e issues.
63
Mo e ecen ICT-based senso s a e based on luo opho es
ha can be exci ed in he isible egion. In 2019 Wood uff and
O’Hallo an syn hesized a amily o BODIPY-based luo opho es
con aining an appended N,N,N0- is(2-py idylme hyl)-1,2-
e hanediamine ( ispicen) chela o as a Zn
2+
ecep o si e.
64
Among hem, ZincBY-4 (Fig. 8), con aining a pending ace amide
on he 5-posi ion o he BODIPY co e ou pe o med o he
de i a i es due o in amolecula hyd ogen bonding be ween
he amide hyd ogen and he luo ine a oms o he BF
2
cen e,
hus inc easing he Zn
2+
affini y. In pola sol en s, he ICT-based
luo escence o he ee p obe is s ongly quenched, whe eas a
blue-shi ed emission is eco e ed upon Zn
2+
chela ion due o
dis up ion o he ICT p ocess.
In 2017, Han, Zang and colleagues published a iome ic
p obes o Cd
2+
, also based on ca ion-induced ICT deple ion,
cha ac e ized by a long wa eleng h luo escence emission
(Me
4
BOPHY-BPA in Fig. 8).
65
The senso consis ed o a e a-
me hyla ed BOPHY luo opho e conjuga ed o bis(py idine-2-yl)
amine (BPA) podand ac ing as a Cd
2+
selec i e ecep o . Cd
2+
–
Chela ion caused a clea hypsoch omic shi o he luo escence
emission e ealing he cha ac e is ic yellow emission o he
Me
4
BOPHY dye. In 2019, Zeng e al. pushed his concep u he
de eloping a Hg
2+
-p obe (Lin Fig. 8) wi h NIR emission in bo h
he holo and he apo o m.
66
This senso consis s o a ch ome-
nylium–cyanine dye con aining a conjuga ed NS
2
O
2
Hg
2+
–che-
la ing uni . The s ong ICT p ocess be ween he N-a om o he
ca ion ecep o and he luo escen dye, oge he wi h he NIR
emission o he naked luo opho e, pe mi ed he au ho s o
cons uc his a iome ic NIR senso ope a i e unde isible-
ligh exci a ion.
The clea ad an ages o TPA mic oscopy compa ed o
con en ional one-pho on abso p ion (OPA) mic oscopy o he
analysis o ca ion dis ibu ion in li e cells, issues, and
o ganisms (i.e. imp o ed pene a ion, educed oxici y, educed
au o luo escence, and minimal ligh sca e ing) d o e he
de elopmen o molecula p obes op imized o TPA.
67–69
When conside ing ICT-based senso s, his has been pu sued
by binding he ca ion ecogni ion si e o he accep o domain o
an ICT– luo opho e. The e o e, upon me al binding, a he
han ICT deple ion (as obse ed in he examples men ioned
abo e) he pola iza ion o he ICT exci ed s a e will inc ease.
This p obe con igu a ion should no only p oduce a ba hoch omic
shi in he luo escence emission o he ca ion-bound
p obe (Fig. 3B), bu also enhance he TPA c oss-sec ion, and he
b igh ness o he luo escence emission.
69
Based on his p emise, in he las ew yea s a se ies o
a iome ic TPA p obes appea ed in he li e a u e. Se e al Zn
2+
-
selec i e senso s ha e been de eloped based on a BPA podand
bound o diffe en luo opho es. Rep esen a i e examples a e
shown in Fig. 9.
70–73
MPVQ-se ies, ini ially de eloped by Meng’s
g oup,
73
consis ed o BPA-appended quinoline as he
luo escen skele on wi h a conjuga ed dono on he
6-posi ion, whe eas ch omis-1 con ains 2-a yl hiazole as a
push–pull luo opho e. In all cases, a ed-shi ed luo escence
was obse ed in he p esence o Zn
2+
, which was accompanied
by an inc eased TPA c oss-sec ion.
Mo e complex a iome ic senso s o ca ions, op imized o
TPA, ha e been cons uc ed using cen osymme ic dono –p–
accep o –p–dono quad upola con igu a ions, in which he
ca ion ecogni ion si e is in eg a ed in o he cen al accep o
uni , hus cons i u ing a chela ing luo opho e (Fig. 2, pa e n
A.2). The GBC p obe (Fig. 10), de eloped in 2014,
74
was a
g ound-b eaking example o senso s based on his s uc u al
mo i and inspi ed u he de elopmen s.
75,76
The ca ion
ecogni ion is achie ed h ough a cen al bipy idine uni wi h
wo appended ca bazole dono s linked h ough inylic p-elec on
Fig. 8 Recen examples o me al-induced ICT deple ion p obes.
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b idges. GBC also con ains oxye hylene side chains o inc ease
i s solubili y in aqueous media and i s cell pe meabili y. The
emission ed shi and he enhanced TPA obse ed upon me al
binding we e ini ially a ibu ed o ICT, because o he inc eased
elec on-wi hd awing cha ac e o he Zn-coo dina ed bipy idine
uni . The mechanism esponsible o he la ge TPA cha ac e is ic
o hese sys ems has been u he analysed om a heo e ical
pe spec i e aiming a es ablishing s uc u e–p ope y
ela ionships.
77–79
These s udies e ealed ha luo escence emis-
sion unde wo-pho on exci a ion o igina es om he S
2
exci ed
s a e a he han om S
1
. Addi ionally, coo dina ion o he Zn
2+
ca ion equi es he o a ion o he cen al bipy idine uni om
he ans o he cis coplana o m. This con o ma ional change
esul s in an inc ease o he dipole and quad upole momen s o
he molecule by a loss o he cen e o in e sion p esen in he
mos s able con o ma ion o he unbound p obe.
77
3.1.1. Mul i- a ge senso s. The elec onic o igin o he
shi in he emission obse ed in ICT-based senso s makes i
easible o de elop mul i- a ge senso s, able o disc imina e
be ween simila ca ions due o he diffe en emission colou s o
hei co esponding complexes. The ca ion-dependen poly-
ch omic emission obse ed in some ICT-based senso s has
been a ibu ed o diffe ences in elec onic affini ies,
80
binding
modes,
81
o ion sizes.
81
Rep esen a i e examples a e shown in
Fig. 11. I is wo h poin ing ou ha senso L2 showed an
ex emely low de ec ion limi o Cd
2+
(0.24 nM), and ha bo h L1
and L2 we e es ed on solid suppo s, as he ac i e componen s o
indica o pape .
82
In he case o HQZn, he ca ion-dependen
emission colou o his TPA senso , oge he wi h he highe
affini y o he senso o Cd
2+
compa ed o Zn
2+
, pe mi ed
he au ho s o use he Zn-chela ed ligand as a Cd
2+
senso ,
elimina ing he ecu en p oblem o Zn
2+
in e e ence du ing
Cd
2+
de ec ion in biological sys ems.
83
3.1.2. Reac i i y-based ICT bicolou senso s: chemo-
dosime e s. Highly efficien a iome ic p obes o ca ions
based on ICT can also be cons uc ed elying on ca ion-
induced i e e sible bond-clea age/bond- o ma ion eac ions
on he molecula p obe (Fig. 2, pa e n B). Such ca ion-
media ed chemical changes in he p obe can p oduce an
enhancemen o deple ion o he cha ge- ans e p ocess, which
e en ually affec s he colou o he luo escence emission. The
examples ange om p obes based on he chemical eac i i y o
he ca ion wi h speci ic unc ional g oups o o m a ca ion-
coo dina ed ICT luo opho e,
84,85
o hose based on ca ion-
media ed umpolung eac ions on he p obe (usually p o ec ion/
dep o ec ion o unc ional g oups) eleasing a ca ion- ee ICT
luo opho e.
86–92
Selec ed examples a e shown in Fig. 12. One o
he ea lies examples was de eloped by Lin e al. in 2009.
88
The p obe was based on a phenan h oimidazole dye con aining
an appended elec on- ich 1,3-di hiane. Cu
2+
-P omo ed desul u -
iza ion o he hioace al ende ed a o myl EWG, wi h he
Fig. 9 TPA ICT-based a iome ic p obes using BPA as a selec i e Zn
2+
ecep o .
Fig. 10 GBC a iome ic p obe o Zn
2+
.
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luo opho e can inco po a e di e en o a able uni s ha can
be decoupled in he p esence o a sui able me al ca ion hus
esul ing in di e en luo opho es in he ee and coo dina ed
s a es.
I is expec ed ha in he yea s o come hese designs will
pe mi he de elopmen o mo e sensi i e and selec i e
luo escen bicolou senso s able o de ec speci ically me al
ca ions in e y low concen a ions. This si ua ion will pe mi
u he ad ances in ields anging om cell biology o pa icle
physics. In addi ion, he de elopmen o luo escen senso s
able o de ec me allic ca ions in he gas phase and a solid–gas
in e aces would pe mi he explo a ion o sup amolecula
chemis y in he absence o sol en s, hus expanding he ield
om ‘‘we ’’ o ‘‘d y’’ chemis y.
Con lic s o in e es
The e a e no con lic s o decla e.
Acknowledgemen s
This wo k was suppo ed by he Basque Go e nmen (G an s IT-
1346-19 and IT1180-19), by he Spanish Minis y o Science and
Inno a ion (MICINN-FEDER, G an s PID2019-104772GB-I00,
PID2019-111281GB-I00, RED2018-102387-T, and RED2018-
102471-T), and by he Eu opean Resea ch Council (ERC) unde
he Eu opean’s Union Ho izon 2020 esea ch and inno a ion
p og amme (G an ag eemen ERC-2020-SyG 951281).
Re e ences
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