Synthesis of Single-Chain Nanoparticles for Catalysis and Sensing

Syn hesis o Single-Chain Nanopa icles
o Ca alysis and Sensing
Au ho : Jokin Pinacho Olaci egui
Supe iso s: P o . José A. Pomposo and P o . Daniel Ta on
Donos ia – San Sebas ián, 2024
(cc) 2025 Jokin Pinacho Olaci egui (cc by-nc-nd 4.0)
CONTENTS
ABSTRACT .............................................................................................................................................. 7
RESUMEN ............................................................................................................................................. 11
RÉSUMÉ ............................................................................................................................................... 16
1. INTRODUCTION ................................................................................................................................ 21
1.1. NANOSCIENCE AND SOFT MATTER ........................................................................................................... 23
1.2. SINGLE-CHAIN NANOPARTICLES .............................................................................................................. 25
1.2.1. Design and syn hesis o SCNPs ................................................................................................ 26
1.2.1.1. P ecu so syn hesis .......................................................................................................................... 27
1.2.1.2. P ecu so unc ionaliza ion .............................................................................................................. 29
1.2.1.3. In achain Folding/Collapse ............................................................................................................. 30
1.2.1.4. Mo phology o Single-Chain Nanopa icles (SCNPs) in Solu ion ...................................................... 36
1.3. APPLICATIONS OF SCNPS ...................................................................................................................... 38
1.3.1. Ca alysis .................................................................................................................................. 39
1.3.2. Sensing .................................................................................................................................... 46
1.3.3. Nanomedicine ......................................................................................................................... 48
1.3.4. O he applica ions ................................................................................................................... 51
1.5. REFERENCES ....................................................................................................................................... 52
2. OUTLINE AND OBJECTIVES OF THE THESIS ........................................................................................ 61
3. LANTHANIDE-BASED SINGLE-CHAIN NANOPARTICLES AS “VISUAL” PASS/FAIL SENSORS OF
MAXIMUM PERMISSIBLE CONCENTRATION OF CU2+ IONS IN DRINKING WATER .................................. 65
3.1 MOTIVATION ........................................................................................................................................ 67
3.2 INTRODUCTION ..................................................................................................................................... 67
3.3. MATERIALS, TECHNIQUES AND METHODS .................................................................................................. 69
3.3.1. Ma e ials ................................................................................................................................. 69
3.3.2. Techniques ............................................................................................................................... 70
3.3.3.Me hods ................................................................................................................................... 71
3.4. RESULTS AND DISCUSSION ...................................................................................................................... 72
2.4.1. Eu-SCNPs as m.p.c.(Cu2+) “ isual” pass/ ail senso s ................................................................ 72
3.4.2. Tb-SCNPs as m.p.c.(Cu2+) “ isual” pass/ ail senso s ................................................................ 84
3.4.3. Dy-SCNPs as m.p.c.(Cu2+) “ isual” pass/ ail senso s .............................................................. 93
3.5. CONCLUSIONS ................................................................................................................................... 100
3.6. REFERENCES ...................................................................................................................................... 101
4. GOLD NANOCLUSTERS SYNTHESIZED WITHIN SINGLE-CHAIN NANOPARTICLES AS CATALYTIC
NANOREACTORS IN WATER ................................................................................................................ 105
4.1. MOTIVATION ..................................................................................................................................... 107
4.2. INTRODUCTION .................................................................................................................................. 107
4.3. MATERIALS AND METHODS .................................................................................................................. 109
4.3.1. Ma e ials ............................................................................................................................... 109
4.3.2. Techniques ............................................................................................................................. 109
4.4. PROCEDURES ..................................................................................................................................... 110
4.4.1. P ocedu e o he Syn hesis o Poly(OEGMA-co-AEMA) ........................................................ 110
4.4.2. P ocedu e o he Syn hesis o Gold Nanoclus e s (Au-NCs) wi hin Poly(OEGMA-co-AEMA)
Single-Chain Nanopa icles (SCNPs) ................................................................................................ 111
4.4.3. P ocedu e o he Reduc ion o 4-Ni ophenol Ca alyzed by AuNCs/SCNPs .......................... 111
4.4.4. P ocedu e o he Reduc ion o Ni obenzene Ca alyzed by AuNCs/SCNPs ........................... 112
4.4.5. P ocedu e o he Reduc ion o 3-(4-Ni ophenyl)-1,3-oxazolidin-2one Ca alyzed by Au-
NCs/SCNPs ....................................................................................................................................... 112
4.5. RESULTS AND DISCUSSION .................................................................................................................... 113
4.5.1. Syn hesis o Gold Nanoclus e s wi hin Single-Chain Nanopa icles (Au-NCs/SCNPs) ............ 113
4.5.2. Gold Nanoclus e s wi hin Single-Chain Nanopa icles (AuNCs/SCNPs) as Ca aly ic
Nano eac o s .................................................................................................................................. 118
4.5.2.1. Reduc ion o 4-Ni ophenol o 4-Aminophenol Ca alyzed by Au-NCs/SCNPs ................................ 118
4.5.2.2. Reduc ion o Ni obenzene o Aniline Ca alyzed by Au-NCs/SCNPs .............................................. 125
4.5.2.3. Reduc ion o 3-(4-Ni ophenyl)-1,3-oxazolidin-2-one o 3-(4-Aminophenyl)-1,3-oxazolidin-2-one
Ca alyzed by Au-NCs/SCNPs ....................................................................................................................... 132
4.6. CONCLUSIONS ................................................................................................................................... 138
4.7. REFERENCES ...................................................................................................................................... 140
5. CONSECUTIVE ONE-POT ALKYNE SEMIHYDROGENATION/ ALKENE DIOXYGENATION REACTIONS BY
PT(II)/CU(II) SINGLE-CHAIN NANOPARTICLES IN GREEN SOLVENT ...................................................... 143
5.1. MOTIVATION ..................................................................................................................................... 145
5.2. INTRODUCTION .................................................................................................................................. 145
5.3. MATERIALS AND METHODS ................................................................................................................... 147
5.3.1. Ma e ials ............................................................................................................................... 147
5.3.2. Techniques ............................................................................................................................. 148
5.4. PROCEDURES ..................................................................................................................................... 149
5.4.1. Syn hesis o P0 ....................................................................................................................... 149
5.4.2. Syn hesis o P1 ....................................................................................................................... 149
5.4.3. Syn hesis o P1-SCNPs (as a con ol) ..................................................................................... 150
5.4.4. Syn hesis o P (II)-SCNPs ........................................................................................................ 150
5.4.5. Syn hesis o P (II)/Cu(II)-SCNPs .............................................................................................. 150
5.4.6. Syn hesis o Cu(II)-SCNPs (as a con ol). ................................................................................ 151
5.4.7. Gene al p ocedu e o he consecu i e one-po alkyne semihyd ogena ion/alkene
dioxygena ion eac ions ca alysed by ............................................................................................. 151
P (II)/Cu(II)-SCNPs in NBP a . . ...................................................................................................... 151
5.5. RESULTS AND DISCUSSION .................................................................................................................... 152
5.6. CONCLUSIONS ................................................................................................................................... 169
5.7. REFERENCES ...................................................................................................................................... 170
6. CONCLUSIONS ................................................................................................................................ 173
7. PUBLICATIONS ................................................................................................................................ 177
8. EXPERIMENTAL TECHNIQUES .......................................................................................................... 181
SIZE EXCLUSION CHROMATOGRAPHY (SEC) .................................................................................................... 183
DYNAMIC LIGHT SCATTERING (DLS) ............................................................................................................. 186
NUCLEAR MAGNETIC RESONANCE (NMR) ...................................................................................................... 187
FOURIER TRANSFORM INFRARED SPECTROSCOPY (FTIR) .................................................................................... 188
ULTRAVIOLET−VISIBLE SPECTROSCOPY (UV-VIS) .............................................................................................. 189
FLUORESCENCE SPECTROSCOPY .................................................................................................................... 190
ELEMENTAL ANALYSIS (EA) .......................................................................................................................... 191
INDUCTIVELY COUPLED PLASMA MASS SPECTROMETRY (ICP-MS) ....................................................................... 192
UV IRRADIATION ....................................................................................................................................... 192
TRANSMISSION ELECTRON MICROSCOPY (TEM) .............................................................................................. 193
REFERENCES............................................................................................................................................. 194
AGRADECIMIENTOS/ACKNOWLEDGMENTS ........................................................................................ 197

7
Abs ac
This hesis di es in o he wo ld o Single-Chain Nanopa icles (SCNPs), om design o
syn hesis, by combining di e en me allic elemen s designed o applica ions in sensing
and ca alysis. Single-Chain Polyme Nanopa icles (SCNPs) a e nanoscale pa icles
o med by he olding and in amolecula c oss-linking o single polyme chains. They
exhibi unique p ope ies such as high su ace a ea, uneable size, and he abili y o
encapsula e a ious subs ances, making hem p omising o applica ions in
nanomedicine, ca alysis, and ma e ial science. Chap e 1 o his Thesis se es as an
in oduc ion o he opic o SCNPs, de ailing hei o ma ion, p ope ies, and po en ial
applica ions. This chap e aims o p o ide a comp ehensi e o e iew o he s a e o he
a in SCNP esea ch, allowing he p og ess made in his Thesis o be unde s ood in he
con ex o exis ing knowledge and echnological ad ancemen s a he ou se .
Agains his backg ound, his wo k ies o u he ailo he capabili ies o SCNPs o e
and abo e he con en ional unc ionali ies wi h he help o a e iew ha a emp ed o
explo e he mos unique chemical and physical p ope ies displayed by some me als,
such as lan hanides (Eu, Tb and Dy), gold, pla inum, and coppe . Indeed, in eg a ion o
me als wi hin he SCNP a chi ec u e enhances hei ca aly ic pe o mance no only
h ough hei elec onic and su ace p ope ies bu also conside s speci ic ea u es o
hese me als' in e ac ion wi h ligh and molecules o enhance hei sensing p ope ies.
Fo example, gold's ou s anding elec on ans e cha ac e is ics o pla inum's esis ance
o a my iad o condi ions, ha e been used as a ca alys owa d a a ie y o chemical
ans o ma ions wi h a unique p eciseness and uggedness. On he o he hand, he
luminescence lan hanides a e employed in he p epa a ion o SCNPs o use as highly
sensi i e and selec i e senso s o en i onmen al analy es. In he conjunc ion o
knowledge comp ising chemis y, ma e ial science, and nano echnology, his is an
in e disciplina y app oach gi ing a comp ehensi e s udy o e he syn hesis o me al
inco po a ed SCNPs. This hesis b ings a se ies o well-designed expe imen s ha no
only con ibu e o he basic knowledge o SCNP assembly and s a egies o me al
in eg a ion bu also open he doo o new possibili ies in applica ions in ca alysis and
8
sensing echnologies, highligh ing he po en ial o SCNPs as a e sa ile and powe ul ool
in nano echnological and ma e ials scien i ic applica ions.
The p esen s udy in Chap e 2 is aimed a syn hesis and cha ac e iza ion o a new class
o wa e -soluble lan hanide-con aining SCNPs ha ha e po en ial o isual sensing o
Cu2+ ions o up o hei maximum pe missible concen a ion (m.p.c.) in d inking wa e .
This no el sensing mechanism explo es he cha ac e is ic p ope ies o lan hanides and
he s uc u al bene i s o SCNPs o design an easy, sensi i e, and selec i e ool o
moni o ing coppe ions, an impo an en i onmen al and heal h conce n. E en i coppe
is an impo an mic o-elemen , i has he po en ial o u n in o a oxic elemen a high
concen a ions, posing ala ming si ua ions bo h o he en i onmen and human heal h.
In his wo k, we de eloped eu opium (Eu), e bium (Tb), and dysp osium (Dy) in eg a ed
SCNPs ha exhibi dis inc luo escence colo changes unde ul a iole ligh upon
eac ing wi h Cu2+ ions, acili a ing a s aigh o wa d " isual" pass/ ail es .
The app oach o his p ocess lies in he de elopmen o an amphiphilic andom
copolyme deco a ed wi h be a-ke oes e unc ional g oups, able o complex lan hanide
ions, so as o ob ain wa e -soluble lan hanide-con aining SCNPs. This me hod does
in achain complexa ion o β-ke oes e /lan hanide, which is signi icance o he
o ma ion and unc ion o hese nanopa icles. The cha ac e iza ion o SCNPs has been
made by di e en app oaches: size-exclusion ch oma og aphy (SEC), dynamic ligh
sca e ing (DLS), induc i ely coupled plasma mass spec ome y (ICP-MS), and
luo escence spec oscopy. SCNPs ha e been success ully o med and a e able o
sensi ize coppe ions (Cu2+).
The esul s seem o clea ly indica e ha all h ee Eu-, Tb- and Dy-based SCNPs can be
e y use ul o a di ec , isual de ec ion o an excess Cu2+ concen a ion in wa e by he
naked eye, due o he espec i e ed- o- anspa en , g een- o- anspa en and yellow o-
anspa en colo changes unde UV ligh . In summa y, his wo k p o ides an impo an
ad ance in en i onmen al moni o ing echnology wi h he p esen a ion o lan hanide-
based SCNPs as e ec i e, easy- o-use senso s o an impo an ask: p o iding sa e
9
d inking wa e . This app oach will no only gi e help owa ds public heal h and
en i onmen al p o ec ion bu opens he a enue o new de elopmen in e ms o simila
senso s o o he con aminan s.
Chap e 3 was based on de eloping a no el idea comp ising he syn hesis o gold
nanoclus e s (Au-NCs) wi hin single-chain nanopa icles (SCNPs) and o apply he o me
as a nano eac o in aqueous ca alysis. The e iciency and selec i i y o me alloenzymes
in cellula en i onmen s ha e p o oked in e es in he p esen s udy, which needs o be
mimicked by na u al ca alys s using simpli ied me alloenzyme-mime ic nano-objec s. As
opposed o mos me al-con aining SCNPs epo ed so a , whe ein he me al ions a e
complexed, in his wo k, he objec i e was he encapsula ion o me al nanoclus e s,
mo e p ecisely Au-NCs wi h sizes smalle han 5 nm, wi hin he SCNPs, in o de o exploi
hei eme gen ca aly ic p ope ies.
Fo he syn hesis o Au-NCs/SCNPs, an amphiphilic andom copolyme o poly
(OEGMAco-AEMA) was used o sel -assemble in wa e and o m SCNPs, educing agen
Au(III) ions o Au(0). The be a-ke oes e g oups loca ed in he AEMA uni s along he
copolyme chain ac ed as e y e ec i e educ an s and s abilizing agen s o he Au-NCs.
On he o he hand, cha ac e iza ions wi h TEM, DLS, and UV-Vis spec oscopy did
es ablish a good con o ma ion o o med and s able Au-NCs/SCNPs.
The s udy e ealed new u ili ies o hese nanos uc u es as ca aly ic nano eac o s o
he educ ion o 4-ni ophenol, ni obenzene, and 3-(4-ni ophenyl)-1,3-oxazolidin2one
by bo ohyd ide (BH4−) in wa e a . . These eac ions well ep esen benchma ks o he
assessmen o he ca aly ic ac i i y o he syn hesized Au-NCs/SCNPs in wa e and,
he e o e, open up an en i ely new scope o po en ial applica ions ega ding use in
ca alysis, biomedicine, ene gy, and elec onics.
These esul s, he e o e, open new a enues o he syn hesis and inco po a ion o me al
nanoclus e s wi hin SCNPs o ad anced ca alysis in aqueous media. The ac ha
polyme and me al nanoclus e -based sys ems ac ually manage o emula e
16
Résumé
Ce e hèse explo e le monde des nanopa icules à chaîne unique (SCNPs), de leu
concep ion à leu syn hèse, en combinan di é en s élémen s mé alliques des inés à des
applica ions dans la dé ec ion e la ca alyse. Les nanopa icules polymè es à chaîne
unique (SCNPs) son des pa icules à l'échelle nanomé ique o mées pa le epliemen
e la é icula ion in amoléculai e de chaînes polymè es uniques. Elles p ésen en des
p op ié és uniques elles qu'une g ande su ace, une aille modulable e la capaci é
d'encapsule di e ses subs ances, ce qui les end p ome euses pou des applica ions en
nanomédecine, ca alyse e science des ma é iaux. Le chapi e 1 de ce e hèse se
d'in oduc ion au suje des SCNPs, dé aillan leu o ma ion, leu s p op ié és e leu s
applica ions po en ielles. Ce chapi e ise à ou ni un ape çu comple de l'é a de l'a
dans la eche che su les SCNPs, pe me an de comp end e les p og ès éalisés dans
ce e hèse dans le con ex e des connaissances exis an es e des a ancées
echnologiques.
Dans ce con ex e, ce a ail en e de dé eloppe da an age les capaci és des SCNPs au-
delà des onc ionnali és con en ionnelles en s'appuyan su une e ue qui a en é
d'explo e les p op ié és chimiques e physiques les plus uniques a ichées pa ce ains
mé aux, els que les lan hanides (Eu, Tb e Dy), l'o , le pla ine e le cui e. En e e ,
l'in ég a ion des mé aux dans l'a chi ec u e des SCNPs amélio e leu s pe o mances
ca aly iques non seulemen g âce à leu s p op ié és élec oniques e de su ace, mais
p end égalemen en comp e les ca ac é is iques spéci iques de l'in e ac ion de ces
mé aux a ec la lumiè e e les molécules pou amélio e leu s p op ié és de dé ec ion.
Pa exemple, les ca ac é is iques excep ionnelles de ans e d'élec ons de l'o ou la
ésis ance du pla ine à une my iade de condi ions on é é u ilisées comme ca alyseu
pou di e ses ans o ma ions chimiques a ec une p écision e une obus esse uniques.
D'au e pa , les lan hanides luminescen s son u ilisés dans la p épa a ion des SCNPs
pou se i de cap eu s hau emen sensibles e sélec i s pou les analy es
en i onnemen aux. En combinan des connaissances en chimie, science des ma é iaux
e nano echnologie, il s'agi d'une app oche in e disciplinai e o an une é ude

17
complè e su la syn hèse des SCNPs inco po an des mé aux. Ce e hèse p ésen e une
sé ie d'expé iences bien conçues qui non seulemen con ibuen aux connaissances de
base su l'assemblage des SCNPs e les s a égies d'in ég a ion des mé aux, mais ou en
égalemen la oie à de nou elles possibili és d'applica ions dans les echnologies de
ca alyse e de dé ec ion, me an en lumiè e le po en iel des SCNPs comme ou il
poly alen e puissan dans les applica ions nano echnologiques e scien i iques des
ma é iaux.
L'é ude p ésen ée dans le chapi e 2 ise la syn hèse e la ca ac é isa ion d'une nou elle
classe de SCNPs con enan des lan hanides solubles dans l'eau, ayan le po en iel de
dé ec e isuellemen les ions Cu2+ jusqu'à leu concen a ion maximale admissible
(m.p.c.) dans l'eau po able. Ce nou eau mécanisme de dé ec ion explo e les p op ié és
ca ac é is iques des lan hanides e les a an ages s uc u els des SCNPs pou conce oi
un ou il acile, sensible e sélec i pou la su eillance des ions cui e, une p éoccupa ion
impo an e pou l'en i onnemen e la san é. Bien que le cui e soi un mic o-élémen
impo an , il peu de eni un élémen oxique à des concen a ions éle ées, posan des
si ua ions ala man es an pou l'en i onnemen que pou la san é humaine. Dans ce
a ail, nous a ons dé eloppé des SCNPs in ég an de l'eu opium (Eu), du e bium (Tb)
e du dysp osium (Dy) qui p ésen en des changemen s de couleu dis inc s sous lumiè e
ul a iole e lo s de la éac ion a ec les ions Cu2+, acili an un es isuel simple
"pass/ ail".
L'app oche de ce p ocessus éside dans le dé eloppemen d'un copolymè e aléa oi e
amphiphile déco é de g oupes onc ionnels bê a-cé oes e , capable de complexe les
ions lan hanides, a in d'ob eni des SCNPs con enan des lan hanides solubles dans l'eau.
Ce e mé hode éalise une complexa ion in achaîne du complexe β-
cé oes e /lan hanide, ce qui es signi ica i pou la o ma ion e la onc ion de ces
nanopa icules. La ca ac é isa ion des SCNPs a é é éalisée pa di é en es app oches :
ch oma og aphie d'exclusion de aille (SEC), di usion dynamique de la lumiè e (DLS),
spec omé ie de masse à plasma à couplage induc i (ICP-MS) e spec oscopie de
luo escence. Les SCNPs on é é o més a ec succès e son capables de sensibilise les
ions cui e (Cu2+).
18
Les ésul a s semblen indique clai emen que les SCNPs à base de Eu, Tb e Dy peu en
ê e ès u iles pou une dé ec ion di ec e e isuelle d'un excès de concen a ion de Cu2+
dans l'eau à l'œil nu, en aison des changemen s de couleu espec i s de ouge à
anspa en , e à anspa en e jaune à anspa en sous lumiè e UV. En ésumé, ce
a ail appo e une a ancée impo an e dans la echnologie de su eillance
en i onnemen ale a ec la p ésen a ion de SCNPs à base de lan hanides comme cap eu s
e icaces e aciles à u ilise pou une âche impo an e : ou ni de l'eau po able sû e.
Ce e app oche con ibue a non seulemen à la san é publique e à la p o ec ion de
l'en i onnemen , mais ou i a égalemen la oie à de nou eaux dé eloppemen s en
e mes de cap eu s similai es pou d'au es con aminan s.
Le chapi e 3 es basé su le dé eloppemen d'une idée no a ice comp enan la
syn hèse de nanoclus e s d'o (Au-NCs) au sein de nanopa icules à chaîne unique
(SCNPs) e l'applica ion de ces de nie s comme nano éac eu en ca alyse aqueuse.
L'e icaci é e la sélec i i é des mé alloenzymes dans les en i onnemen s cellulai es on
susci é l'in é ê de la p ésen e é ude, qui doi ê e imi ée pa des ca alyseu s na u els
u ilisan des nano-obje s mimé iques de mé alloenzymes simpli iés. Con ai emen à la
plupa des SCNPs con enan des mé aux appo és jusqu'à p ésen , où les ions
mé alliques son complexés, dans ce a ail, l'objec i é ai l'encapsula ion de
nanoclus e s mé alliques, plus p écisémen des Au-NCs de aille in é ieu e à 5 nm, au
sein des SCNPs, a in d'exploi e leu s p op ié és ca aly iques éme gen es.
Pou la syn hèse des Au-NCs/SCNPs, un copolymè e aléa oi e amphiphile de poly
(OEGMA-co-AEMA) a é é u ilisé pou s'au o-assemble dans l'eau e o me des SCNPs,
éduisan les ions Au(III) en Au(0). Les g oupes bê a-cé oes e si ués dans les uni és
AEMA le long de la chaîne du copolymè e on agi comme éduc eu s e agen s
s abilisan s ès e icaces pou les Au-NCs. D'au e pa , des ca ac é isa ions pa TEM,
DLS e spec oscopie UV-Vis on é abli une bonne con o ma ion des Au-NCs/SCNPs
o més e s ables.
L'é ude a é élé de nou elles u ili és de ces nanos uc u es en an que nano éac eu s
ca aly iques pou la éduc ion de 4-ni ophénol, ni obenzène e 3-(4-ni ophényl)-1,3-
19
oxazolidin-2-one pa bo ohyd u e (BH4−) dans l'eau à empé a u e ambian e. Ces
éac ions ep ésen en bien des poin s de é é ence pou l'é alua ion de l'ac i i é
ca aly ique des Au-NCs/SCNPs syn hé isés dans l'eau e ou en donc une nou elle
pe spec i e d'applica ions po en ielles en ca alyse, biomédecine, éne gie e
élec onique.
Ces ésul a s ou en ainsi de nou elles oies pou la syn hèse e l'inco po a ion de
nanoclus e s mé alliques au sein des SCNPs pou une ca alyse a ancée en milieu aqueux.
Le ai que les sys èmes à base de polymè es e de nanoclus e s mé alliques pa iennen
e ec i emen à imi e l'ac i i é des mé alloenzymes ou e donc des p omesses de
dé eloppemen de sys èmes ca aly iques e icaces, s ables e sélec i s dans un la ge
champ d'applica ions.
Le chapi e 4 in odui une nou elle app oche de la ca alyse a ec des nanopa icules
polymè es à chaîne unique hé é obimé alliques P (II)/Cu(II) (SCNPs), in oduisan
s a égiquemen des g oupes onc ionnels α-diazo-β-cé oes e e β-cé oes e nus dans
la s uc u e des SCNPs. Cela implique la géné a ion de ca bène pho oac i ée a ec une
lumiè e de longueu d'onde de 365 nm e des ions P (II), en sélec ionnan le dichlo o(1,5-
cyclooc adiène)P (II) pou son a ini é a ec les g oupemen s ca bène géné és. Pa la
sui e, le Cu(II) es in odui pa des éac ions a ec l'acé a e de Cu(II), ésul an en une
s uc u e polymé ique de complexes Cu(II)-(β-cé oes e )2. Ce e inco po a ion duale de
mé aux es essen ielle pou le epliemen du p écu seu polymé ique en la s uc u e
nanoscopique inale, comme en émoignen la spec oscopie in a ouge, la
ch oma og aphie d'exclusion de aille e la di usion dynamique de la lumiè e, indiquan
la o ma ion de SCNPs P (II)/Cu(II) bien dé inis. La nou eau é de ce e in es iga ion
éside dans le dé eloppemen e l'applica ion de ces nou eaux SCNPs.
1. In oduc ion

1. In oduc ion
23
1.1. Nanoscience and So Ma e
Since he ad en o nanoscience and nano echnology, as pionee ed by Nobel lau ea e
Richa d P. Feynman in his seminal 1959 lec u e, "The e's Plen y o Room a he
Bo om”,1 he scien i ic communi y has wi nessed a ple ho a o ans o ma i e
de elopmen s ac oss he disciplines o physics, chemis y, and biology. These
ad ancemen s ha e ma e ialized Feynman's isiona y concep s o manipula ing ma e
a he mos diminu i e scale, speci ically a he molecula and a omic le els, he ein
e e ed o as he nanoscale.
The e m "nano echnology," albei subjec o a ied in e p e a ions ac oss di e en
scien i ic domains and geog aphical egions, is equen ly employed as an umb ella e m
o encapsula e echnologies ha ope a e on an exceedingly minu e scale. None heless,
nano echnology is p incipally delinea ed as he scien i ic endea o encompassing he
comp ehensi e unde s anding, p ecise manipula ion, and s a egic es uc u ing o
ma e a dimensions on he o de o nanome e s (no ably, less han 100 nm).2
Whi esides (2004) highligh s ha a his scale, he p ope ies o ma e ials unde go a
undamen al shi , dis inguishing hem ma kedly om hei bulk coun e pa s.3 This
ield has wi nessed an exponen ial g ow h o e ecen decades, leading o he
eme gence o g oundb eaking disciplines such as nanomedicine, as elucida ed by Mish a
(2013),4 nanoelec onics, desc ibed by Pue s e al. (2017),5 and nanoca alysis, as
discussed by Bahadu -Singh and Kuma -Tandon (2014).6 The su ge in nanoscience
esea ch and in es men is well-documen ed by Velmu ugan and Radhak ishnan
(2016).7
Solid-s a e physics and elec onics ha e en u ed in o he ealm o nanos uc u es
h ough he employmen o li hog aphy and e ching p ocesses, deno ed as he
" opdown" app oach. This me hodology acili a es he c ea ion o s uc u es wi h
dimensions no less han app oxima ely 20 nanome e s. Con e sely, na u e exempli ies
an al e na i e pa adigm o he assembly o diminu i e s uc u es. In his con ex ,
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indi idual molecules a e amalgama ed in o la ge unc ional en i ies and in ica e
s uc u al hie a chies h ough a p ocess o sel -o ganiza ion, a s a egy e e ed o as he
"bo om-up" app oach.8
In na u e, dynamic indi idual biomac omolecules sel -assemble in o a a ie y o
unc ional nanoen i ies on a ious scales,9 gi ing ise o complex ma e ials like i uses
o DNA. Seen om his angle, he na u al wo ld se es as a model o he design o small
buildings. Fundamen al o he c ea ion o nanos uc u es is he nanopa icle, a
undamen al uni ha is la ge han a oms o simple molecules, which a e subjec o
quan um mechanics, bu much smalle han mac oscopic objec s go e ned by
New onian mechanics.10
Nanopa icles a e classi ied in o wo a ie ies based on composi ion: "ha d
nanopa icles", which a e usually made o ino ganic ma e ials, and "so nanopa icles",
which a e made o o ganic componen s. Excep ional con ol o e size and shape has
been achie ed in he c ea ion o ha d nanopa icles, such as me al oxide nanopa icles,
quan um do s, and gold nanoclus e s.
Mac omolecula a chi ec u e modi ica ion has made emendous s ides in he las
se e al decades in he ield o a i icial so nano-objec s. The de elopmen o e ec i e
Con olled Radical Polyme iza ion (CRP) me hods is p ima ily esponsible o hese
imp o emen s. Many complica ed mac omolecula s uc u es, including s a polyme s,
comb-like copolyme s, and hype b anched mac omolecules, ha e been de eloped by
using CRP me hodologies.11, 12 The ma e ials p oduced as a esul o hese p ocesses
di e signi ican ly om hei linea analogs wi h simila molecula weigh s in e ms o
hei cha ac e is ics (Figu e 1). Wi hin his spec um o a i icial so nano-objec s, a
no able ca ego y is ep esen ed by Single-Chain Nanopa icles (SCNPs).
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Figu e 1. Di e en examples o ha d-nanopa icles and so -nanopa icles classi ied by ype and
mo phology.
1.2. Single-Chain Nanopa icles
The de elopmen o Single-Chain Polyme Nanopa icles (SCNPs) cons i u es a ib an
a ea wi hin he ealm o mac omolecula chemis y, aimed a eplica ing he p ecision
o na u al polyme ic models. Analogous o he con igu a ion o p o eins, SCNPs a e
c a ed h ough he in amolecula olding o syn he ic linea polyme p ecu so s (Figu e
2). This inno a i e app oach posi ions SCNPs as iable candida es o a wide a ay o
applica ions ac oss di e se ields such as ca alysis,13,14mic oelec onics,15
nanomedicine,16 DNA deli e y mechanisms,17 senso y echnologies16 o imaging
agen s.18
Figu e 2. Schema ic illus a ion o a linea polyme p ecu so and a single-chain nanopa icle (SCNP)
ob ained h ough in a-chain olding/collapse o indi idual polyme chains a e y dilu e condi ions.
1. In oduc ion
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Figu e 7. Illus a ion o c oss-linke induced collapse echnique employed o he cons uc ion o
SCNPs.
Rega ding he bonding in e ac ions u ilized, single-chain nanopa icles a e syn hesized
h ough in achain co alen bonds (yielding i e e sible SCNPs) o ia in achain
nonco alen and dynamic-co alen bonds (yielding e e sible SCNPs).
A) I e e sible Single-Chain Polyme Nanopa icle Sys ems
Co alen bonding
The syn hesis o i e e sible single-chain nanopa icles (SCNPs) is acili a ed h ough
co alen bonding, u ilizing bo h con en ional o ganic and "click" chemis y eac ions o
he e ec i e c ea ion o obus unimolecula so nanopa icles. I 's impe a i e o no e
ha he co alen s abiliza ion o polyme geome y nega es he dynamic na u e o
single-chain en i ies, hus es ic ing hei applica ion in dynamic biomime ic sys ems,
such as p o ein olding/un olding mechanisms.
Ini ial disclosu es o i e e sible single-chain nanopa icle syn hesis ia he in achain
homocoupling me hod unde highly dilu ed condi ions by Mece eyes e al. u ilized
poly(s y ene)-, poly(alkyl me hac yla e)-, and poly(ε-cap olac one)-based p ecu so s
wi h inyl eac i e g oups.19 Jiang e al. and Che ian e al. espec i ely u ilized
unsa u a ed g oups o poly(4-N-Boc-aminos y ene)- and poly(ca bona e)-based single-
chain nanopa icles.35 Subsequen ly, Ha h e al. syn hesized benzosul one-deco a ed
p ecu so s o indi idual unimolecula nanopa icle c ea ion ia quinodime hane

1. In oduc ion
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o ma ion unde simila condi ions.36 Poly(me hyl me hac yla e)-based SCNPs we e
ob ained by Zhu e al. h ough in amolecula Be gman cycliza ion a lowe
empe a u es (150 ºC).37 The syn hesis o poly(s y ene) and poly(alkyl
me hac yla e)based unimolecula nanopa icles ia in amolecula c oss-linking o
sul onyl azide-38 and benzoxazine-39 unc ionalized polyme s equi ed ele a ed
empe a u es (190–200 ºC). Addi ionally, Di lam e al. ecen ly p epa ed poly(s y ene)-
based unimolecula nanopa icles ia in achain c osslinking h ough oxida i e
polyme iza ion o 3,4p opylenedioxy hiophene unc ional g oups a 50 ºC.40
Figu e 8. Scheme o he Michael syn hesis ou e ollowed by Sanchez-Sanchez e al.41 o he
o ma ion o he SCNPs.
Colmene o e al. p esen ed a s udy using small-angle neu on sca e ing (SANS) and
neu on spin echo (NSE) echniques o explo e he s uc u e and dynamics o i e e sible
single-chain nanopa icles (SCNPs) in a dilu e solu ion. These SCNPs we e ob ained
h ough Michael addi ion-media ed mul idi ec ional sel -assembly (Figu e 8).
T ime hylolp opane iac yla e ac ed as he in achain c oss-linking agen . P ecu so s
we e andom copolyme s o me hyl me hac yla e (MMA) and (2-ace oace oxy)e hyl
me hac yla e (AEMA).41
The in achain he e ocoupling echnique has been employed o he ab ica ion o
i e esible single-chain nanopa icles using e icien azide-alkyne "click" chemis y (i.e.,
coppe -ca alyzed [3+2] cycloaddi ion o alkynes and azides (CuAAC)).42 Ruiz de Luzu iaga
e al. syn hesized bio unc ionalized poly(me hyl me hac yla e) unimolecula
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nanopa icles a oom empe a u e and high yield u ilizing azide- and p o ec ed alkyne-
deco a ed polyme p ecu so s.43 Pomposo e al. simpli ied his echnique, employing
copolyme s wi h p o ec ed alkyne and chlo ome hyl g oups, con e ing hem o
azidome hyl g oups ia a s aigh o wa d subs i u ion eac ion wi h sodium azide.44 The
in achain he e o-coupling me hod has also acili a ed he c ea ion o he mo esponsi e
single-chain nanopa icles.45 Ni ile imine media ed e azole-ene cycloaddi ion (NITEC)
has been applied o he syn hesis o well-de ined luo escen single-chain
nanopa icles.18
The c osslinke -media ed collapse o polyme chains ep esen s ano he e icien
me hodology o ob aining SCNPs. Poly(γ-glu amic acid)-based single-chain
nanopa icles we e p oduced h ough he c osslinke -induced collapse me hod using a
biosyn he ic poly(γ-glu amic acid) p ecu so and 2,2´-(e hylenedioxy)die hylamine as
he bi unc ional c osslinke in he p esence o ca bodiimide.46
B) Re e sible Single-Chain Polyme Nanopa icle Sys ems
Two p incipal ypes o e e sible in e ac ions, i.e., non-co alen in e ac ions and
dynamic co alen bonds, enable he syn hesis o esponsi e, s uc u ally dynamic single-
chain nanopa icles (SCNPs).
Non-co alen In e ac ions
The concep o non-co alen bonds has cap i a ed esea che s ac oss a ious ields since
Linus Pauling's seminal wo k on hyd ogen bonding in he 1930s.47 Jean Ma ie Lehn's
in oduc ion o “sup amolecula chemis y”48 has acili a ed he explo a ion o non-co alen
bonds in cons uc ing ad anced a i icial a chi ec u es, including sup amolecula polyme s.
The suscep ibili y o main-chain sup amolecula polyme s o en i onmen al a ia ions,
mani es ing in sol en pola i y and concen a ion-dependen molecula weigh s, unde sco es
he in luence o ac o s such as empe a u e, p essu e, and concen a ion on non-co alen
bond s eng hs and he equilib ium be ween bonded and unbonded species.
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Non-co alen , o sup amolecula , in e ac ions a e ca ego ized in o h ee classes based
on bond s eng h: i) weak in e ac ions (0-15 kcal/mol), including an de Waals o ces,
hyd ophobic in e ac ions, π-π s acking in e ac ions, and hyd ogen bonds; ii) medium
s eng h in e ac ions (15-60 kcal/mol), such as mul iple hyd ogen bonds and weake
me al coo dina ion complexes; and iii) s ong in e ac ions (>60 kcal/mol), encompassing
ionic in e ac ions, hos -gues in e ac ions, and obus me al coo dina ion o chela e
complexa ion (Figu e 9).
The s eng h o non-co alen bonds is con ingen on ex e nal ac o s like sol en ype,
empe a u e, and concen a ion, p ecluding a s ic ca ego iza ion.49 Non-co alen o
sup amolecula in e ac ions ha e acili a ed he c ea ion o complex a chi ec u es
in ol ing side-chain sup amolecula polyme s,50 mul i-block sup amolecula polyme s,51
and mul i-a m sel -assembled s a s.52
Figu e 9. O ganiza ion o non-co alen in e ac ions by bond s eng h.49
Dynamic Co alen Bonds
Dynamic co alen chemis y encompasses e e sible co alen eac ions ha enable he
exchange o molecula componen s o each he sys em's he modynamic equilib ium.53
Dynamic co alen bonds, exhibi ing he obus ness o co alen bonds, a e only
dis up able and e o mable unde speci ic ex e nal condi ions (e.g., ca alys p esence).
Fu he mo e, dynamic co alen chemis y has success ully con ibu ed o dynamic
combina o ial lib a ies,54 d ug disco e y,55 and con olled ag ance deli e y.56
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Hawke , Kim, and hei colleagues' pionee ing e o s in 2008 demons a ed an e icien
app oach o sup amolecula single-chain nanopa icles ia benzamide dime iza ion,
o ming quad uple hyd ogen bonds be ween benzamide mo i s.57 Subsequen ex ensi e
esea ch by Meije , Palmans, and hei eams ad anced he ield o me as able
nonco alen bonded single-chain nanopa icles h ough he in oduc ion o o hogonal
echniques based on u eidopy imidinone (UPy)58 and benzene-1,3,5- ica boxamide
(BTA)59 hyd ogen bonding mo i s. Meije 's g oup ecen ly showcased sequen ial
sup amolecula nanopa icle syn hesis h ough complemen a y sel -assembly o UPy
and BTA uni s.60 Hos -gues in e ac ions in ol ing cucu bi [n]u il complexa ion61 and
hyd ophobic L-phenylalanine Phe-Phe in e ac ions62 ha e been le e aged o ab ica e
wa e -bo ne sup amolecula single-chain nanopa icles.
1.2.1.4. Mo phology o Single-Chain Nanopa icles (SCNPs) in Solu ion
Unde s anding and con olling he mo phology o SCNPs o e all is a necessi y o p ope ly
u ilize hei p ope ies in a wide a ie y o applica ions including d ug deli e y, sensing,
and he de elopmen o new nanoma e ials. Being able o ex ac he p ecise SCNPs ou
o p ecu so polyme syn hesis will be c i ical in he u u e o how we can e ec i ely
p edic he inal mo phology ype o a well-designed SCNPs o a desi ed applica ion.
As illus a ed in Figu e 10, i all begins in he syn he ic selec ion o he p ecu so :
choosing block copolyme s and enabling mos ly Janus SCNPs wi h gene ally sel -
assembly beha io s enabling complex mul i-molecula s uc u es such as micelles and
lamellae, o andom copolyme s wi h mos ly isola ed/spa se o globula mo phologies
wi hou sel -assembly beha io s in gene al.
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Figu e 10. Scheme o he di e en mo phologies o SCNPs om block copolyme p ecu so and
om andom copolyme p ecu so . I showed some sel -assembly s uc u es o med by Janus
SCNPs and he simila i y o mo phology be ween spa se SCNPs and in insically diso de ed
p o eins (IDPs), and globula SCNPs and enzymes.
Amphiphilic block copolyme s sel -assemble in o nanos uc u es due o block
incompa ibili y. Janus Single-Chain Nanopa icles (SCNPs) 63 ha e bi acial p ope ies,
leading o win o adpole mo phologies, and can o m micelles, lamellae, o esicles. 64

1. In oduc ion
38
Random copolyme s, in con as , a e o med om wo o mo e monome s polyme ized
in a andom sequence, esul ing in a s a is ical dis ibu ion o monome s along he
polyme chain. These andom copolyme s can gi e ise o ei he spa se o globula
SCNPs. Spa se SCNPs a e mo phologically analogous o In insically Diso de ed P o eins
(IDPs), while globula SCNPs esemble enzymes.
In good sol en s, spa se SCNPs exhibi a mo phology wi h ex ended linea sec ions and
compac egions, d i en by he in e play be ween in a-chain c oss-linking and a
p e e ence o sho - ange loop o ma ion, as demons a ed by Molecula Dynamics
(MD) simula ions.65, 66 Globula SCNPs, on he o he hand, possess a homogeneous co e-
shell s uc u e akin o ha o na i e p o eins, such as enzymes. The syn hesis o globula
SCNPs o en in ol es complex syn he ic ou es. Techniques such as he u iliza ion o long
c oss-linke s and bi unc ional g oups ha e been employed o enhance loop o ma ion
and compac ion wi hin hese s uc u es. 67, 68
1.3. Applica ions o SCNPs
The e sa ili y o single-chain nanopa icles (SCNPs) encompasses a b oad spec um o
applica ions, including ca alysis, sensing, and nanomedicine, as depic ed in Figu e 11.
The e icacy o SCNP echnology ac oss hese domains s ems p ima ily om he
nanopa icles' minu e dimensions and hei adap abili y ailo ed o speci ic
unc ionali ies. Al hough esea ch endea o s pe aining o SCNPs ha e p edominan ly
del ed in o undamen al in es iga ions, hese ma e ials ha e mani es ed p ac ical u ili y
in a ious con ex s. In insically, he in e io composi ion o he nanopa icle u nishes a
conduci e chemical milieu, cha ac e ized by unable hyd ophobic o hyd ophilic
p ope ies, alongside con olled size modula ion achie able h ough manipula ion o
in a-chain c oss-linking densi ies and he molecula weigh o he p ecu so polyme .
Mo eo e , he in eg a ion o selec i e unc ional si es wi hin he nanopa icle s uc u e
is acili a ed by con olled polyme iza ion me hodologies and pos -polyme iza ion
modi ica ion echniques. Subsequen sec ions encapsula e ecen applied esea ch
endea o s elucida ing he di e se applica ions o SCNPs.
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Figu e 11. Illus a ion o some examples o po en ial applica ions o SCNPs.
1.3.1. Ca alysis
The de elopmen o ca aly ically ac i e single-chain nanopa icles is conside ed an
essen ial app oach in polyme chemis y, c ea ing new ways o he design o ca alys s
seamlessly in eg a ing he ad an ages o bo h homogeneous and he e ogeneous
ca alysis sys ems. The PhD hesis ega ds he concep o polyme chains se ing as
spa ially demanding suppo s o me al-ions in ca aly ic sys ems and he e ec s o such
con igu a ions on he ca aly ic e iciency. Subs i u ion o molecula ligands by polyme
chains in o ganome allic ca alys s is an e en ha in lic s la ge changes which may
in luence ca aly ic ac i i y ei he by di usional limi a ions o because o dense ca aly ic
pocke s, which hinde subs a e access. This phenomenon no ed by he g oup led by
Pomposo unde sco es he challenge o ensu ing he ee di usion wi hin he SCNP
nano eac o s in o de o main ain ac i i y bu , a he same ime, ecognizing ha
selec i e subs a e access should sha pen eac ion speci ici y.
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The ini ial de elopmen o SCNPs ocused on s uc u al models, using in amolecula
c osslinking o induce chain collapse in o nanopa icle o m. Ea lie s udies on such
sys ems we e aimed a unde s anding changes in in insic iscosi y and he adius o
gy a ion upon chain collapse.69 This e a o disco e y o e ed us c i ical knowledge o he
physical beha iou o SCNPs, bu a ha ime, did no ye le e age hei ca aly ic
po en ial. Signi ican ad ancemen s came in he ea ly 2010s wi h new polyme syn hesis
and c osslinking me hodologies, e i ing in e es in SCNPs and pa ing he way o hei
applica ion in ca alysis.70
The concep o SCNPs as unc ional nano eac o s began o ake shape, le e aging he
idea o in amolecula c osslinking o old polyme chains in o disc e e, s able
nanos uc u es. These nanos uc u es could be ailo ed o inco po a e ca aly ic si es,
mimicking he ac i e si es ound in na u al enzymes. This app oach allowed o he
de elopmen o SCNPs wi h highly speci ic subs a e binding and ca aly ic e iciency,
simila o ha o enzymes.
One pionee ing s udy in his a ea was conduc ed by Pomposo e al., who explo ed he
ca aly ic si e dis ibu ion wi hin 'clickase' SCNPs. They show ha SCNPs wi h clus e ed
ca aly ic si es had signi ican ly highe ac i i y han hose wi h homogeneous ca aly ic
si e dis ibu ion, which sugges ed ha p ecise in e nal mo phology con ol can gi e ise
o enhancemen s in ca aly ic pe o mance o a much g ea e ex en han was ealized a
he beginning o he disco e ies abou SCNPs.71 This disco e y unde sco ed he po en ial
o SCNPs o achie e enzyme-like e iciency h ough syn he ic means.
The inco po a ion o me al ions in o SCNPs was pa icula ly ui ul. Ba ne -Kowollik and
Roesky eams epo ed bime allic con igu a ions in SCNPs. Pla inum and eu opium
me als we e combined, leading o he de elopmen o mul i unc ional nano eac o s.
Such SCNPs o e ed high ca aly ic ac i i y, ease o eco e abili y, and eusabili y— he
wo limi a ions highly ele an o con en ional homogeneous ca alys s.72 In an
illus a i e example, Knö el e al. epo ed he syn hesis o phosphine- unc ionalized
polyme chain-bea ing SCNPs and hei use as homogeneous ca alys s in he amina ion
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41
o allyl alcohol. P (II)-SCNPs, depending on he pola i y o he sol en , could be
sepa a ed om he eac ion mix u e and ecycled in addi ional cycles wi h good
po en ial in e ms o high ca aly ic e iciency and ease o eco e abili y. They also wo ked
on he syn hesis and he ca aly ic applica ions o SCNPs ha con ain bo h Au and Y. The
esea che s syn hesized SCNPs chemically by apping Au(I) and Y(III) ions in o he
polyme ma ix. Such NPs we e obse ed o exhibi ecyclable and e y high ca aly ic
beha io . Resea ch ga e much a en ion o he eco e y and eusing o SCNPs o e
se e al ca aly ic cycles by keeping he e iciency and s abili y o he same. This alluded
o he possible uses o he new he e obime allic SCNPs in sus ainable ca alysis,
oughness, and applica ion o chemical eac ions.73
Fu he ad ancemen s we e made adding addi ional unc ionali ies in o SCNPs. Maag e
al. de eloped SCNPs ha exhibi luo escence, acili a ing easy acking and ecycling o
he ca alys s. This no el y highligh ed he possibili y o combine ca aly ic unc ion wi h
op ical p ope ies in SCNPs, meaning a b oade po en ial applica ion ield.74 A lib a y o
zinc po phy in co es was p epa ed based on poly (me hyl me hac yla e-co-an h acene
me hac yla e) polyme s by Pa enaude, Be da, and Pazicni. These SCNPs we e equipped
wi h di e en unc ional g oups, and showed enhanced eac i i y and subs a e
speci ici y, emphasizing he impo ance o seconda y coo dina ion sphe es in SCNP
ca alysis.75
The s uc u al design o SCNPs was inspi ed by na u al enzymes, pa icula ly hei abili y
o c ea e a speci ied mic oen i onmen o he ca aly ic eac ion. Resea che s ha e
imi a ed hose s uc u al a ibu es o na u al enzymes du ing he design o SCNPs o
a ain high ca aly ic e iciency and subs a e speci ici y. Fo example, Palmans and Meije
de eloped a concep o u henium-based ca alys s, dispe sed in amphiphilic block
copolyme s, o syn hesize SCNPs wi h cen al hyd ophobic co es simila o enzyme ac i e
si es. Such s a egy p o ided an imp o emen no only in e ms o subs a e binding bu
also in he p o ec ion o he ca aly ic si es om deac i a ion. As a esul , ope a ional li e
o he ca alys was ex ended in his design.76
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changes in luo escence changed SCNPs po en ial ac i i y, making hem applicable o
high-sensi i i y and s able luo escen de ec ion o ace le els o H2O2.90
SCNPs ha e been de eloped wi h ou s anding sensi i i y, selec i i y, and e sa ili y as
sensing sys ems. The de elopmen o SCNPs o me al ion de ec ion, p o ein sensing,
and hyd ogen pe oxide de ec ion symbolizes he b oad applicabili y and inno a i e
po en ial o hese nanos uc u es. Wi h ine i able p og ess in esea ch, SCNPs will likely
ind hei applica ions b oadened e en u he and i mly claim hei place in ad anced
sensing echnologies.
1.3.3. Nanomedicine
Nanomedicine is a discipline le e aging he pa icula cha ac e is ics o nanopa icles o
pu poses o diagnosis, moni o ing, he apy, and con ol o biological sys ems. The use o
SCNPs in con eying all such pu poses ou plays hose played by adi ional small
molecules and o he nanopa icle sys ems due o mo e p olonged ci cula ion imes,
lowe deg ada ion, and acili y o a ge ing o speci ic issues o cellula ma ke s o
enhance deli e y and he apeu ic agen e icacy.91
Cy o oxici y S udies
Biocompa ibili y is one o he mos c i ical aspec s a nanoma e ial should possess when
i has been designed o biomedical applica ions SCNPs. Biocompa ible SCNPs we e
syn hesized using he s a egy o o gano-ca alyzed ing-opening polyme iza ion by Qiao
e al.92 The syn hesized SCNPs showed low oxici y o human emb yonic kidney cells
(HEK293T). Thei s udy e ealed ha hese SCNPs, co alen ly c oss-linked by
biodeg adable polyes e linkages, we e non oxic a concen a ions up o 100 µg/mL,
showing p omise o sa e biomedical applica ions.
Fu he in es iga ions by Lemco and Zimme man explo ed he in luence o molecula
weigh on cell iabili y using luo escein-con aining SCNPs. They ound ha SCNPs wi h
molecula weigh s anging om 50 o 100 kDa exhibi ed high HeLa cell iabili y (≥80%)
a a concen a ion o 10 µM. This s udy highligh ed he po en ial o SCNPs o se e as

1. In oduc ion
49
non- oxic ca ie s o imaging agen s o he apeu ic d ugs.93 Addi ional esea ch by
Loinaz e al. (2016), combined in i o and in i o oxici y esea ch owa d SCNPs om
poly(me hac ylic acid) and hey obse ed minimal oxici y in panc ea ic adenoca cinoma
cell lines a concen a ions up o 50 µg/mL and no acu e oxici y in mice a a dose o 100
mg/kg, unde sco ing he sa e y o SCNPs o po en ial clinical use.94
Con olled D ug Deli e y Sys ems
SCNPs ha e been ex ensi ely s udied as nanoca ie s o he con olled deli e y o
he apeu ic agen s, including chi al amino acid de i a i es, pep ides, i amins, and small
molecule d ugs. The p ecise con ol o e SCNP mo phology and unc ionali y enables
ailo ed d ug elease p o iles, enhancing he apeu ic e icacy while minimizing side
e ec s.
Fo ins ance, Hamil on and Ha d de eloped SCNP-based nanoca ie s o he
in acellula deli e y o pep ide he apeu ics. These SCNPs we e deco a ed wi h
dend i ic molecula anspo e s and luo escen p obes, acili a ing e icien cellula
up ake and con olled pep ide elease while p ese ing he biological ac i i y o he
pep ides.95 This app oach has signi ican po en ial o deli e ing he apeu ic pep ides o
in acellula a ge s, which a e o en challenging o each wi h con en ional deli e y
sys ems.
Pomposo e al. syn hesized SCNPs designed o he de mal deli e y o i amin B9. These
SCNPs showed a p og essi e and con olled elease o he i amin ollowing a Fickian
di usion mechanism, wi h comple e elease obse ed wi hin six hou s.96 In a
subsequen s udy, hey explo ed he simul aneous deli e y o hinoki iol and i amin B9,
achie ing e icien elease p o iles a di e en pH le els. This dual-deli e y sys em
demons a ed he e sa ili y o SCNPs in adminis e ing mul iple he apeu ic agen s wi h
dis inc elease kine ics.
Cheng and Zimme man de eloped s imuli- esponsi e SCNPs capable o encapsula ing
he an icance d ug 5- luo ou acil (5-FU). These SCNPs exhibi ed excellen he mo/pH-
1. In oduc ion
50
esponsi e beha io , wi h he highes d ug elease obse ed unde condi ions mimicking
he umo mic oen i onmen (lowe pH and highe empe a u e). This speci ici y
educes he isk o p ema u e d ug elease and enhances he he apeu ic a ge ing o
cance cells.97
Image Con as Agen s
SCNPs ha e also been e alua ed as po en ial agen s o a ious imaging echniques,
including magne ic esonance imaging (MRI), single pho on emission compu e ized
omog aphy (SPECT), and luo escence imaging. These applica ions le e age he unique
op ical and magne ic p ope ies o SCNPs, enhancing he sensi i i y and speci ici y o
imaging modali ies.
Fo example, Loinaz and Od iozola syn hesized gadolinium (Gd3+)-deco a ed SCNPs ha
demons a ed enhanced elaxi i y compa ed o adi ional gadolinium chela es. These
SCNPs, wi h an a e age elaxi i y alue o 6.8 mM-1s-1, o e ed imp o ed con as in MRI
scans, making hem p omising candida es o ad anced diagnos ic imaging.98 Simila ly,
Ha h e al. de eloped wa e -soluble SCNPs wi h dual MRI and luo escence imaging
capabili ies, enhancing hei mul i unc ionali y o diagnos ic applica ions.99 Fluo escen
SCNPs ha e also been de eloped wi h educed pho obleaching and enhanced emission
p ope ies. Zimme man e al. demons a ed he use o luo escein-loaded SCNPs o
long- e m bioimaging, showing s ong luo escence signals inside HeLa cells.100
As a conclusion, The po en ial o SCNPs in nanomedicine is as , wi h p omising
applica ions in d ug deli e y, imaging, and sensing. Thei unique p ope ies, such as
con olled mo phology, biocompa ibili y, and he abili y o unc ionalize wi h a ious
he apeu ic and diagnos ic agen s, posi ion SCNPs as e sa ile ools in he ad ancemen
o nanomedicine. Fu u e esea ch is expec ed o u he explo e and op imize hese
applica ions, pa ing he way o inno a i e he apeu ic and diagnos ic solu ions. The
de elopmen o SCNPs o e s a glimpse in o he u u e o pe sonalized medicine, whe e
ea men s a e ailo ed o indi idual pa ien s' needs, enhancing e icacy and educing
side e ec s.
1. In oduc ion
51
1.3.4. O he applica ions
Beyond he p ima y applica ions p e iously delinea ed, Single-Chain Nanopa icles
(SCNPs) a e poised o e olu ionize he c ea ion o new ma e ials endowed wi h ailo ed
p ope ies. By s a egically enginee ing polyme chain a chi ec u es, i is now possible
o syn hesize ma e ials demons a ing cus omized mechanical, he mal, o elec ical
cha ac e is ics. These ma e ials a e des ined o ind applica ion in e e y hing om
s anda d coa ings o ad anced composi e ma e ials. While hese ep esen mo e
specialized applica ions cu en ly unde in es iga ion, SCNPs ha e been used o enginee
nanos uc u es,101enhance oil eco e y p ocesses,102 and syn hesize plasmonic
nanopa icles.103 The use o hese ma e ials has also been expanded o design
hyd ophobic coa ings104 and ealize hyd ophobic co on wi h g ea ly imp o ed
esis ance,105 ,106 unde sco ing he pe asi e u ili y o SCNPs in he ield o ma e ial
science and enginee ing.
1. In oduc ion
52
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3. Lan hanide-based Single-Chain Nanopa icles as
“Visual” Pass/Fail Senso s o Maximum Pe missible
Concen a ion o Cu2+ Ions in D inking Wa e
3. Lan hanide-based Single-Chain Nanopa icles as “Visual” Pass/Fail Senso s o Maximum Pe missible
Concen a ion o Cu2+ Ions in D inking Wa e
67
3.1 Mo i a ion
The maximum pe missible concen a ion (m.p.c.) o Cu2+ ions in d inking wa e , as se by
he Wo ld Heal h O ganiza ion (WHO) is m.p.c.(Cu2+)WHO = 30 µM, whe eas he US
En i onmen al P o ec ion Agency (EPA) es ablishes a mo e es ic i e alue o
m.p.c.(Cu2+)EPA = 20 µM. He ein, we de elop - o he i s ime e e - a amily o
m.p.c.(Cu2+) “ isual” pass/ ail senso s based on wa e -soluble lan hanide-con aining
single-chain nanopa icles (SCNPs) exhibi ing an a e age hyd odynamic diame e below
10 nm. Bo h eu opium (Eu)- and e bium (Tb)-based SCNPs allow excessi e Cu2+
concen a ion o be eadily de ec ed in wa e , as can be naked-eye moni o ed by ed- o-
anspa en and g een- o- anspa en colo changes unde ul a- iole (UV) ligh
i adia ion, espec i ely, aking place a ca. 30 µM o Cu2+ ions in bo h cases.
Complemen a y, dysp osium (Dy)-based SCNPs show a yellow- o- anspa en colo
ansi ion unde UV ligh i adia ion a ca.15 µM o Cu2+ ions. Eu-, Tb- and Dy-con aining
SCNPs p o e o be selec i e o Cu2+ ions as hey do no espond agains o he me al
ions, such as Fe. These new m.p.c.(Cu2+) “ isual” pass/ ail senso s a e ho oughly
cha ac e ized by a combina ion o echniques including size exclusion ch oma og aphy
(SEC), dynamic ligh sca e ing (DLS), induc i ely coupled plasma mass spec ome y
(ICP-MS), in a- ed (IR), UV and luo escence spec oscopy.
3.2 In oduc ion
Coppe is an essen ial ace elemen o animals and plan s.1 In ac , Cu2+ - he 3 h mos
impo an me al ion p esen in biological sys em- is in ol ed as co ac o o nume ous
me alloenzymes.2,3 Cu2+ ions a e also essen ial o he human body and de ec o his
c ucial mic onu ien can impa cy openia (a educ ion in he numbe o ma u e blood
cells) and p o ound neu ological de ici s.4,5 On he con a y, an excessi e Cu2+ in ake
om occupa ional exposu e o con amina ed wa e can cause gas oin es inal p oblems,
li e and kidney damage, hemoly ic anemia and impai ed immune unc ion, among
o he e ec s.6,7 Thus, Cu2+ ions ac as one o he en i onmen pollu an s o con ol due
3. Lan hanide-based Single-Chain Nanopa icles as “Visual” Pass/Fail Senso s o Maximum Pe missible
Concen a ion o Cu2+ Ions in D inking Wa e
68
o hei inc eased use a se e al le els (home, indus ial, and ag icul u al ope a ions), as
well as en i onmen al pe sis ency.8
Di e en egula o y O ganisms ha e es ablished he maximum pe missible
concen a ion (m.p.c.) o Cu2+ ions in d inking wa e . The m.p.c. o Cu2+ ions in d inking
wa e by he Wo ld Heal h O ganiza ion (WHO) is m.p.c.(Cu2+)WHO = 30 µM, whe eas
he US En i onmen al P o ec ion Agency (EPA) es ablishes a mo e es ic i e alue o
m.p.c.(Cu2+)EPA = 20 µM.9,10 Al hough di e en analy ical echniques a e a ailable o
accu a ely de e mine he concen a ion o Cu2+ ions in d inking wa e (e.g., a omic
abso p ion spec oscopy, ion ch oma og aphy, induc i ely coupled plasma mass
spec ome y), hese echniques o en in ol e sophis ica ed ins umen a ion and
equi e skilled use s. Consequen ly, al e na i e me hods based on “ isual” pass/ ail
senso s p o iding ope a ional simplici y, sensi i i y and selec i i y a e highly desi able.
To ackle ha challenge, we en isioned o design single-chain polyme nanopa icles
(SCNPs) inco po a ing lan hanide me al ions as a e sa ile pla o m owa ds a new
gene a ion o m.p.c.(Cu2+) ope a ing as “ isual” pass/ ail senso s. SCNPs a e indi idual
polyme ic chains ha a e in amolecula ly olded h ough in a-chain in e ac ions.11 The
olding o disc e e syn he ic polyme chains in o SCNPs a emp s o mimic he na u al
olding o biomac omolecules, such as p o eins.12 The chain olding p ocess leads o
locally compac domains wi hin SCNPs, which can be o p ac ical use o bind ac i e
species (e.g., me al ions,13 luminopho es,14 d ugs15). Consequen ly, SCNPs o e
in e es ing oppo uni ies o he de elopmen o imp o ed senso s16, inno a i e d ug
deli e y ehicles17 and biomime ic ca alys s,18 among o he p ac ical applica ions. On
he o he hand, a a ie y o complexes based on lan hanide ions19-22 ha e been
in es iga ed as luo escen “ u n-o ” senso s o Cu2+ ions.
Howe e , mos o hese molecula senso s a e no di ec ly ope a i e in wa e , bu
ins ead, in o ganic sol en /wa e mix u es. We hypo hesized ha , by using an
amphiphilic andom copolyme ea u ing be a-ke oes e unc ional g oups able o
complex wi h lan hanide ions, wa e -soluble lan hanide con aining SCNPs could be
3. Lan hanide-based Single-Chain Nanopa icles as “Visual” Pass/Fail Senso s o Maximum Pe missible
Concen a ion o Cu2+ Ions in D inking Wa e
69
achie ed ia in a-chain be a-ke oes e /lan hanide complexa ion. The esul ing
lan hanide based SCNPs we e hus employed as inno a i e m.p.c.(Cu2+) “ isual” pass/ ail
senso s in d inking wa e . He e we epo he a ional design o eu opium (Eu)-, e bium
(Tb)- and dysp osium (Dy)-based SCNPs, as well as hei use - o he i s ime e e - as
e icien “ isual” pass/ ail senso s o maximum pe missible concen a ion o Cu2+ ions in
wa e .
3.3. Ma e ials, echniques and me hods
3.3.1. Ma e ials
Oligo(e hylene glycol) me hyl e he me hac yla e (OEGMA) (a e age Mn 300 g/mol),
(2-ace oace oxy)e hyl me hac yla e (AEMA) (95%), 2,2-azobis(2-me hylp opioni ile)
(AIBN) (≥98%), ie hylamine (E 3N) (>99%), me hyl ace oace a e 99%, 1,4-dioxane
(anhyd ous, 99.8%), hexane (anhyd ous, 95 %), 4-cyano-4-( hiobenzoyl hio)pen anoic
acid (≥ 97%), eu opium ichlo ide hexahyd a e (EuCl3 x 6 H2O) (99.99% ace me als
basis), e bium ichlo ide hexahyd a e (TbCl3 x 6 H2O) (99.99% ace me als basis),
dysp osium ichlo ide hexahyd a e (DyCl3 x 6 H2O) (99.99% ace me als basis), coppe
(II) ace a e (98%), i on (II) ace a e (95%), cobal (II) ace a e e ahyd a e (99%), ba ium
ace a e (99%), nickel (II) ace a e e ahyd a e (99%), me cu y (II) ace a e (≥ 98.0%), lead
(II) ace a e e ahyd a e (99%) zinc (II) ace a e dehyd a e (99%), i on (III) ace ylace ona e
(97%), calcium ace a e monohyd a e (99%), manganese (II) ace ylace one (98%),
magnesium chlo ide (98%) and ch omium(III) ace a e (98%) we e supplied by Me k
(SigmaAld ich). Po assium hyd oxide (KOH) (≥ 85%, pelle s) was supplied by PanReac
AppliChem (ITW Reagen s). Sil e (I) ace a e (99%) was supplied by ITW Reagen s (Ac os
O ganics). Deionized wa e was ob ained om a The mo Scien i ic appa a us (Ba ns ead
TII Pu e Wa e Sys em). Te ahyd o u an (THF) was supplied by Scha lab. Deu e a ed
chlo o o m (CDCl3) (99.8 a om % D) was supplied by Me k.

3. Lan hanide-based Single-Chain Nanopa icles as “Visual” Pass/Fail Senso s o Maximum Pe missible
Concen a ion o Cu2+ Ions in D inking Wa e
70
3.3.2. Techniques
1H nuclea magne ic esonance (NMR) spec a we e ob ained a oom empe a u e ( . .)
using a B uke spec ome e ope a ing a 400 MHz wi h CDCl3 as he sol en . Size
exclusion ch oma og aphy (SEC) measu emen s we e conduc ed a 30 °C in an Agilen
1200 sys em ha was equipped wi h PLgel 5 μm Gua d and PLgel 5 μm MIXED-C
columns. The measu emen s employed a iple de ec ion sys em, which included a
di e en ial e ac i e index de ec o (Op ilab Rex, Wya ), a mul i-angle lase ligh
sca e ing (MALLS) de ec o (MiniDawn T eos, Wya ), and a iscosime ic (VIS) de ec o
(ViscoS a -II, Wya ). SEC da a we e analysed using Wya 's ASTRA So wa e ( e sion 6.1).
Te ahyd o u an (THF) was used as he eluen wi h a low a e o 1 mL/min. Fo bo h he
p ecu so and he single-chain nanopa icles, a alue o dn/dc = 0.1150 was applied.
Dynamic Ligh Sca e ing (DLS) measu emen s we e ca ied ou a . . on a Mal e n
Ze asize Nano ZS appa a us. Me al con en in he single-chain nanopa icles was
de e mined by induc i ely coupled plasma mass spec ome y (ICP-MS). Fou ie
ans o m in a ed (FTIR) spec a we e eco ded a . . on a JASCO 3600 FTIR
spec ome e . UV spec oscopy was ca ied ou in an Agilen 8453A spec ome e .
Pho oluminescence (PL) spec a we e eco ded a . . on an Agilen Ca y Eclipse
spec ome e . Ho iba Laqua win-pH-33 compac pH-me e was used o pH
measu emen s.
Scheme 1. Schema ic illus a ion o he syn hesis o lan hanide (Ln)-based single-chain polyme
nanopa icles (SCNPs) using an amphiphilic andom copolyme deco a ed wi h be a-ke oes e
unc ional g oups, namely, poly(OEGMA-co-AEMA).
3. Lan hanide-based Single-Chain Nanopa icles as “Visual” Pass/Fail Senso s o Maximum Pe missible
Concen a ion o Cu2+ Ions in D inking Wa e
71
3.3.3.Me hods
Syn hesis o amphiphilic andom copolyme ea u ing be a-ke oes e unc ional
g oups
OEGMA (1.54 mL, 5.6 mmol), AEMA (0.26 mL, 1.38 mmol), 4-cyano-4-( hiobenzoyl hio)
pen anoic acid (18.3 mg, 0.065 mmol), and AIBN (2.15 mg, 0.013 mmol) we e dissol ed
a . . in 1,4-dioxane (3 mL). The esul ing mix u e was degassed by pu ging wi h a gon
o 15 min. Then, he mix u e was subjec ed o e e sible addi ion agmen a ion chain-
ans e (RAFT) copolyme iza ion a 70 °C o 24 h. The esul ing poly(OEGMAco-AEMA)
copolyme , a pink oil, was isola ed by p ecipi a ion in hexane. Subsequen ly, he p oduc
was dissol ed in a minimal amoun o THF and added o an excess o hexane ( wice).
A e emo al o ola ile o ganic sol en s, u he d ying was ca ied ou a . . unde
acuum and co i med by 1H NMR (Figu e 1).
Figu e 1. 1H NMR spec um o poly(OEGMA-co-AEMA).
3. Lan hanide-based Single-Chain Nanopa icles as “Visual” Pass/Fail Senso s o Maximum Pe missible
Concen a ion o Cu2+ Ions in D inking Wa e
72
Syn hesis o lan hanide-based single-chain polyme nanopa icles (SCNPs)
Poly(OEGMA-co-AEMA) (15 mg, 0.03 mmol) and he co esponding lan hanide
ichlo ide hexahyd a e (LnCl3 x 6 H2O; Ln = Eu, Tb, Dy) (30 μM) we e dissol ed in wa e
(15 mL) a . . and pH = 7.5 o 24 h. The esul ing lan hanide-con aining SCNPs we e
pu i ied by dialysis agains deionized wa e . ICP-MS da a abou he lan hanide con en in
he SCNPs a e p o ided in Table 1. Success ul o ma ion o SCNPs was con i med by SEC
and DLS measu emen s, acco ding o well-es ablished li e a u e p ocedu es.11 To
mi iga e possible in e e ence om molecula oxygen, all lan hanide-based SCNPs we e
subjec ed o an ex ensi e degassing (N2) p ocess be o e pe o ming he luo escence
measu emen s.
Table 1. SEC and ICP-MS da a o Ln-SCNPs.
Ln-SCNPs
Mw(MALLS)
(kDa)
Ɖ
Ln3+ con en
(µg/mg)a
Ln3+ con en
(µg/mg)b
Eu-SCNPs
85.0
1.12
4.6
2.7
Tb-SCNPs
88.9
1.36
4.8
2.0
Dy-SCNPs
-
-
4.9
-
aTheo e ical alue. bExpe imen al alue de e mined om ICP-MS measu emen s.
3.4. Resul s and discussion
2.4.1. Eu-SCNPs as m.p.c.(Cu2+) “ isual” pass/ ail senso s
Eu opium-based single-chain nanopa icles (Eu-SCNPs) we e p epa ed a a
concen a ion o 1 mg/mL, ollowing he p ocedu e depic ed in Scheme 1. A linea
amphiphilic andom copolyme , namely, poly(OEGMA-co-AEMA) was i s syn hesized by
3. Lan hanide-based Single-Chain Nanopa icles as “Visual” Pass/Fail Senso s o Maximum Pe missible
Concen a ion o Cu2+ Ions in D inking Wa e
73
RAFT copolyme iza ion o he hyd ophilic monome OEGMA and he hyd ophobic
monome AEMA. Poly(OEGMA-co-AEMA) was ound o con ain 35 mol% o
be ake oes e unc ional g oups, as de e mined by 1H NMR spec oscopy (Figu e 1).
Poly(OEGMA-co-AEMA) showed a weigh -a e age molecula weigh o Mw = 80.7 kDa
and ela i ely low dispe si y, Ɖ = 1.11 (Table 1). Eu-SCNPs esul ed om he complexa ion
o Eu3+ ions in solu ion by be a-ke oes e unc ional g oups o poly(OEGMA-co-AEMA)
(see Scheme 1).
7.5 8 8.5 9 9.5
Eu-SCNPs
poly(OEGMA-co-AEMA)
SEC e en ion ime (min)
Figu e 2. SEC aces o he p ecu so , poly(OEGMA-co-AEMA), and he Eu-SCNPs
The o ma ion o Ln3+/be a-ke oes e complexes in ol ing low molecula weigh o ganic
compounds is well documen ed.23-25 To he bes o ou knowledge, howe e , he e a e
no p eceden s o single-chain polyme nanopa icle o ma ion ia in a-chain
lan hanide/be a-ke oes e complex o ma ion. Success ul p epa a ion o Eu-SCNPs was
con i med by a combina ion o s uc u al and size cha ac e iza ion echniques. Hence,
SEC esul s con i med a shi o he SEC elu ion ime a peak maximum owa ds longe
e en ion ime and, hence, smalle hyd odynamic size o Eu-SCNPs when compa ed o
3. Lan hanide-based Single-Chain Nanopa icles as “Visual” Pass/Fail Senso s o Maximum Pe missible
Concen a ion o Cu2+ Ions in D inking Wa e
80
Figu e 11. FTIR spec a o poly(OEGMA-co-AEMA) (blue colou ) and Eu-SCNPs ( ed colou )
Figu e 12 shows he PL spec a o Eu-SCNPs in wa e in he p esence o inc easing
amoun s o Cu2+ ions (λexc = 254 nm). As shown in Figu e 13, Eu-SCNPs can be used as
“ isual” pass/ ail senso s o m.p.c. o Cu2+ ions in wa e , acco ding o he WHO c i e ion
(m.p.c.(Cu2+)WHO = 30 µM).9 A clea ed- o- anspa en colou change unde UV ligh (λexc
= 254 nm) is indeed obse ed also in Figu e 13 on passing om [Cu2+] ≤ 27.5 µM o [Cu2+]
= 30 µM.
100020003000
Poly(OEGMA-co-AEMA)
Eu-SCNPs
Wa enumbe [cm-1]
1632.4 cm-1

3. Lan hanide-based Single-Chain Nanopa icles as “Visual” Pass/Fail Senso s o Maximum Pe missible
Concen a ion o Cu2+ Ions in D inking Wa e
81
Figu e 12. PL spec a o Eu-SCNPs in wa e in he p esence o inc easing amoun s o Cu2+
ions.
Figu e 13. (e) demons a ion o he u ili y o Eu- SCNPs as “ isual” pass/ ail senso s o m.p.c. o
Cu2+ ions in wa e acco ding o he WHO c i e ion (i: 5 µM, ii: 10 µM, iii: 15 µM, i : 22.5 µM, :
25 µM, i: 27.5 µM and ii: 30 µM).
Analysis o he da a in Figu e 14 using he S e n-Volme equa ion (I0/I = 1+ KSV[Cu2+])
p o ided a alue o KSV = 1.4 x 105 M-1 and a squa ed coe icien o linea eg ession o
R2 = 0.99.
0
10
20
30
40
50
60
70
80
580 590 600 610 620 630 640 650
Eu-SCNPs
[Cu2+] 5 µM
[Cu2+] 7.5 µM
[Cu2+] 10 µM
[Cu2+] 12.5 µM
[Cu2+] 15 µM
[Cu2+] 17.5 µM
[Cu2+] 20 µM
[Cu2+] 22.5 µM
[Cu2+] 25 µM
[Cu2+] 27.5 µM
[Cu2+] 30 µM
In ensi y (a.u.)
Wa elengh (nm)
3. Lan hanide-based Single-Chain Nanopa icles as “Visual” Pass/Fail Senso s o Maximum Pe missible
Concen a ion o Cu2+ Ions in D inking Wa e
82
Figu e 14. S e n-Volme plo (I0/I =1+ KSV[Cu2+]) o Eu-SCNPs (e o ba s es ima ed om iple
measu emen s)
Rema kably, Eu-SCNPs p o ed highly selec i e o Cu2+ ions agains o he me al ions, such
as Fe2+, Ag+, Co2+, Ba2+, Ni2+, Hg2+, Pb2+, Zn2+, Fe3+, Ca2+, Mn2+, Mg2+ and C 3+. F om he
ba cha (Figu e 15), i 's clea ha he esponse o Eu-SCNPs o Cu2+ is signi ican ly
highe han o o he me al ions, indica ed by he owe ing peak o Cu2+. This sugges s
ha Eu-SCNPs ha e a high selec i i y o Cu2+ ions. The line g aph (Figu e 16) shows he
pho oluminescence (PL) spec a o Eu-SCNPs in he p esence o di e en me al ions,
including Cu2+. The spec a o Cu2+ shows a p onounced quenching o Eu3+ emission,
which is no obse ed wi h o he me al ions. This quenching is a ibu ed o ene gy
ans e om Eu3+ o Cu2+ ions, ollowed by non adia i e elaxa ion o he g ound s a e
o he Cu2+ ions,27,28 con i ming he selec i i y o Eu-SCNPs o Cu2+ ions o e o he s. This
p ope y is pa icula ly aluable o he de elopmen o selec i e senso s o Cu2+ in
a ious applica ions.
1
2
3
4
5
6
0 5 10 15 20 25 30
I0/I
[Cu2+] µM
KSV = 1.4 x 105 M-1
R2 = 0.99
3. Lan hanide-based Single-Chain Nanopa icles as “Visual” Pass/Fail Senso s o Maximum Pe missible
Concen a ion o Cu2+ Ions in D inking Wa e
83
Figu e 15. PL in ensi y o Eu-SCNPs in he p esence o di e en me al ions a a concen a ion o
27.5 µM.
Figu e 16. Selec i i y o Eu-SCNPs o Cu2+ ions agains o he me al ions.
0
1
2
3
4
5
6Eu-SCNPs
Fe2+
Ag+
Co2+
Ba2+
Cu2+
Ni2+
Hg2+
Pb2+
Zn2+
Fe3+
Ca2+
Mn2+
Mg2+
C 3+
I0/I
Me al ions
0
10
20
30
40
50
60
70
80
580 590 600 610 620 630 640 650
Eu-SCNPs
Fe2+
Ag+
Co2+
Ba2+
Cu2+
Ni2+
Hg2+
Pb2+
Zn2+
Fe3+
Ca2+
Mn2+
Mg2+
C 3+
In ensi y (a.u.)
Wa elengh (nm)
3. Lan hanide-based Single-Chain Nanopa icles as “Visual” Pass/Fail Senso s o Maximum Pe missible
Concen a ion o Cu2+ Ions in D inking Wa e
84
3.4.2. Tb-SCNPs as m.p.c.(Cu2+) “ isual” pass/ ail senso s
Tb-SCNPs we e ob ained om he complexa ion o Tb3+ ions in solu ion by be a-
ke oes e unc ional g oups o poly(OEGMA-co-AEMA), as displayed in Scheme 1. The
a e age hyd odynamic diame e o he Tb-SCNPs was 7.2 nm (Figu e 17), a alue smalle
han ha o he poly(OEGMA-co-AEMA) p ecu so (8.3 nm). The SEC ace o Tb-SCNPs
(Mw = 88.9 kDa, Ɖ = 1.36, see ESI) con i med he absence o mul i-chain agg ega es, in
acco dance wi h DLS esul s.
Figu e 17. DLS size dis ibu ion o he p ecu so , poly(OEGMA-co-AEMA), and he Tb-SCNPs.
The Tb-based SCNPs we e isualized as g eenish luo escen nanoma e ials unde UV
ligh i adia ion (λexc = 254 nm) in a ange o pH be ween 7.5 and 9 (see Figu e 18). Unde
s ong acidic condi ions he luo escence disappea ed due o he dis up ion o
Tb3+/be a-ke oes e complexes, whe eas unde high basic pH a dec ease in luo escence
was no ed, as a consequence o agg ega ion o he Tb-SCNPs. In his way, he b igh
0
5
10
15
20
25
30
110 100
Poly(OEGMA-co-AEMA)
Tb-SCNPs
In ensi y (%)
Hyd odynamic diame e (nm)
3. Lan hanide-based Single-Chain Nanopa icles as “Visual” Pass/Fail Senso s o Maximum Pe missible
Concen a ion o Cu2+ Ions in D inking Wa e
85
g een glow o he Tb-SCNPs unde UV i adia ion was clea ly isible in Figu e 19,
indica ing ha hey could be used in sensing wi hin a pH ange o 7.5-9.
0
0.2
0.4
0.6
0.8
1
1.2
2 4 6 8 10 12 14
Rela i e PL in ensi y
pH
Figu e 18. Rela i e PL in ensi y s. pH o Tb-SCNPs.
Figu e 19. Illus a ion o he g eenish luo escen Tb-SCNPs unde UV ligh i adia ion (λexc = 254
nm)

3. Lan hanide-based Single-Chain Nanopa icles as “Visual” Pass/Fail Senso s o Maximum Pe missible
Concen a ion o Cu2+ Ions in D inking Wa e
86
Shown in Figu e 20 is he "S okes shi " (Δλ). This is how he wa eleng h con as
be ween he peaks o he abso bance and emission spec a a e de ined. Fo luo escen
ma e ials, his is an all-impo an shi because he la ge i is, he mo e ene gy is los
om ligh abso p ion o emission and he less emi ed ligh will be e-abso bed. As a
esul , luo escence e iciency is inc eased. Wha you a e looking a is a peak o
abso bance and a sha pe peak o emission. The S okes shi is ob iously he di e ence
in wa eleng h be ween hose wo peaks: 302 nm.
Figu e 20. Abso bance (do ed) s emission (con inuous) and S okes shi (Δλ) o Tb-SCNPs.
Tb-SCNP luo escence in 3D and 2D exci a ion-emission landscapes is shown in Figu e 21
and 22. 3D luo escence analysis (Figu e 21) o e s an in ui i e p esen a ion. Emission
in ensi ies a e shown as peaks and in e p e a ion h ough one heigh is possible a he
di e en exci a ion wa eleng hs. A 2D con ou plo (Figu e 22) p o ides a co ela ed
p ojec ion on a plane and simpli ies he iden i ica ion o he wa eleng hs whe e Tb-
SCNPs display peak luo escence. These isualiza ion ools a e essen ial o he
0
0,2
0,4
0,6
0,8
1
1,2
0
200
400
600
800
1000
1200
240 320 400 480 560 640
Abso bance (a.u.)
Emission (a.u.)
Wa eleng h (nm)
3. Lan hanide-based Single-Chain Nanopa icles as “Visual” Pass/Fail Senso s o Maximum Pe missible
Concen a ion o Cu2+ Ions in D inking Wa e
87
iden i ica ion o he exci a ion-emission pai mos sui able o Tb-SCNPs’ analy ical
applica ions.
Figu e 21. 3D s udy o exci a ion s. emission o Tb-SCNPs.
Figu e 22. 2D s udy o exci a ion s. emission o Tb-SCNPs.
200 250 300 350 400 450
300
400
500
600
700
Emi ion (nm)
Exi a ion (nm)
-0.5000
12.06
24.63
37.19
49.75
62.31
74.88
87.44
100.0
In ensi y
3. Lan hanide-based Single-Chain Nanopa icles as “Visual” Pass/Fail Senso s o Maximum Pe missible
Concen a ion o Cu2+ Ions in D inking Wa e
88
As in he case o Tb-SCNPs, he s abili y esul s closely aligned wi h he p ecious
indings. The s udy ocused on he luo escence in ensi y o Tb-SCNPs a in e als o 10
minu es, 1 hou , 1 day, and 1 mon h o assess he nanopa icles' s abili y. The nea ly
iden ical cu es om bo h se s o expe imen s indica e ha he Tb-SCNPs consis en ly
main ain hei luo escence in ensi y o e he examined pe iod (see Figu e 23).
Figu e 23. S abili y o he luo escence in ensi y o e ime o Tb-SCNPs.
The pH adjus men co al es is shown in Figu e 24, which demons a es he a ia ion o
he pho oluminescence (PL) in ensi y o Tb-SCNPs wi h pH al e a ions be ween neu al
(pH 7.5) and acidic (pH 4) en i onmen s. The ma ked dis inc ions in PL in ensi y as a
unc ion o pH show he pH-dependency o he nanopa icles' luo escence. Thei
luo escence in ensi y is a g ea e a pH 7.5 han a pH 4, whe e a s iking educ ion in
luo escence is e iden . In addi ion, as shown in Figu e 24, Tb-SCNPs endu e 3-4 cycles
o pH al e a ions un il hei luo escence en i ely disappea s.
0
200
400
600
800
1000
1200
520 530 540 550 560 570
10 min
1h
1 day
1 mon h
In ensi y (a.u.)
Wa eleng h (nm)
5.72 %
3. Lan hanide-based Single-Chain Nanopa icles as “Visual” Pass/Fail Senso s o Maximum Pe missible
Concen a ion o Cu2+ Ions in D inking Wa e
89
Figu e 24. E olu ion o he PL in ensi y o Tb-SCNPs upon consecu i e pH changes be ween 7.5
and 4.
Figu e 25. FTIR spec a o poly(OEGMA-co-AEMA) (blue colou ) and Tb-SCNPs (g een
colou ).
0
100
200
300
400
500
0 2 4 6 8 10
pH=7.5
pH=4
In ensi y
Cycles
100020003000
Poly(OEGMA-co-AEMA)
Tb-SCNPs
Wa enumbe [cm-1]
1637.3 cm-1
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Concen a ion o Cu2+ Ions in D inking Wa e
96
unde go signi ican deg ada ion o quenching ha would o he wise a ec hei
luo escence.
Figu e 35. S abili y o he luo escence in ensi y o e ime o Dy-SCNPs.
The pH adjus men co al es is shown in Figu e 36, which demons a es he a ia ion o
he pho oluminescence (PL) in ensi y o Eu-SCNPs wi h pH al e a ions be ween neu al
(pH 7.5) and acidic (pH 4) en i onmen s. The ma ked dis inc ions in PL in ensi y as a
unc ion o pH show he pH-dependency o he nanopa icles' luo escence. Thei
luo escence in ensi y is a g ea e a pH 7.5 han a pH 4, whe e a s iking educ ion in
luo escence is e iden . In addi ion, as shown in Figu e 36, Eu-SCNPs endu e 3-4 cycles
o pH al e a ions un il hei luo escence en i ely disappea s.
0
50
100
150
200
250
550 560 570 580 590 600
10 min
1h
1 day
1 mon h
In ensi y (a.u.)
Wa elengh (nm)
6.71 %

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Concen a ion o Cu2+ Ions in D inking Wa e
97
Figu e 36. E olu ion o he PL in ensi y o Dy-SCNPs upon consecu i e pH changes be ween 7.5
and 4.
In he FTIR spec um o he Dy-based SCNPs, he s e ching ib a ion o he enol
au ome o he be a-ke oes e g oups bonded o Dy3+ was obse ed a 1642.1 cm-1,
which could be assigned o he s e ching ib a ion o he enol au ome o he
be ake oes e g oups bonded o Dy3+ (Figu e 37).
Figu e 37. FTIR spec a o poly(OEGMA-co-AEMA) (blue colou ) and Dy-SCNPs (yellow colou )
0
50
100
150
200
250
300
350
0 2 4 6 8 10
pH=7.5
pH=4
Rela i e PL in ensi y
Cycles
100020003000
Poly(OEGMA-co-AEMA)
Dy-SCNPs
Wa enumbe [cm-1]
1642.1 cm-1
3. Lan hanide-based Single-Chain Nanopa icles as “Visual” Pass/Fail Senso s o Maximum Pe missible
Concen a ion o Cu2+ Ions in D inking Wa e
98
Dy-SCNPs show a yellow- o- anspa en colo ansi ion unde UV ligh i adia ion a ca.
15 µM o Cu2+ ions (Figu e 38) - e y close o he US-EPA c i e ion (m.p.c.(Cu2+)EPA = 20
µM)- making hese Dy-based SCNPs p ac ical “ isual” pass/ ail senso s o m.p.c. o Cu2+
ions in wa e acco ding o he EPA egula ion.
Figu e 38. PL spec a o Dy-SCNPs in wa e in he p esence o inc easing amoun s o Cu2+ ions.
Figu e 39. Demons a ion o he u ili y o Dy-SCNPs as “ isual” pass/ ail senso s o m.p.c. o Cu2+
ions in wa e acco ding o he EPA c i e ion (i: 1 µM, ii: 2.5 µM, iii: 10 µM, i : 15 µM, : 20 µM,
and i: 30 µM),
0
50
100
150
200
250
550 560 570 580 590 600
Dy SCNPs
[Cu2+] 5 µM
[Cu2+] 7.5 µM
[Cu2+] 10 µM
[Cu2+] 12.5 µM
[Cu2+] 15 µM
[Cu2+] 17.5 µM
[Cu2+] 20 µM
[Cu2+] 22.5 µM
[Cu2+] 25 µM
[Cu2+] 27.5 µM
[Cu2+] 30 µM
In ensi y (a.u.)
Wa eleng h (nm)
3. Lan hanide-based Single-Chain Nanopa icles as “Visual” Pass/Fail Senso s o Maximum Pe missible
Concen a ion o Cu2+ Ions in D inking Wa e
99
A S e n-Volme plo o Dy-SCNPs p o ided KSV = 2.9 x 105 M-1 and R2 = 0.99 (see Figu e
40). Simila ly, o Eu-based and Tb-based SCNPs, Dy-based SCNPs p o ed selec i e o
Cu2+ ions agains o he me al ions in solu ion (see Figu e 41 and 42).
Figu e 40. S e n-Volme plo o Dy-SCNPs (e o ba s es ima ed om iple measu emen s)
Figu e 41. Selec i i y o Dy-SCNPs o Cu2+ ions agains o he me al ions.
1
2
3
4
5
6
0 2 4 6 8 10 12 14 16
I0/I
[Cu2+] µM
KSV = 2.9 x 105 M-1
R2 = 0.99
0
1
2
3
4
5
6
Dy-SCNPs
Fe2+
Ag+
Co2+
Ba2+
Cu2+
Ni2+
Hg2+
Pb2+
Zn2+
Fe3+
Ca2+
Mn2+
Mg2+
C 3+
I0/I
Me al ions
3. Lan hanide-based Single-Chain Nanopa icles as “Visual” Pass/Fail Senso s o Maximum Pe missible
Concen a ion o Cu2+ Ions in D inking Wa e
100
Figu e 42. PL in ensi y o Dy-SCNPs in he p esence o di e en me al ions a a concen a ion o
15 µM.
3.5. Conclusions
We in oduced a new gene a ion o m.p.c.(Cu2+) “ isual” pass/ ail senso s based on
wa e -soluble lan hanide (Eu, Tb, o Dy)-con aining SCNPs o sub-10 nm size ange.
Unde UV ligh i adia ion a λexc = 254 nm, Eu-SCNPs and Tb-SCNPs showed a naked-eye
ed- o- anspa en and g een- o- anspa en colou ansi ions, espec i ely, nea [Cu2+]
= 30 µM in wa e a pH = 7.5. As o Dy-based SCNPs, hey displayed a
yellow o anspa en colou change a ca. [Cu2+] = 15 µM. The cha ac e is ic FTIR
s e ching ib a ion o he enol au ome o he be a-ke oes e g oups bonded o Eu3+,
Tb3+ and Dy3+ ions in Eu-SCNPs, Tb-SCNPs and Dy-SCNPs was loca ed a 1632.4, 1637.3
and 1642.1 cm-1, espec i ely. These “ isual” pass/ ail senso s show high selec i i y
owa ds Cu2+ ions agains a a ie y o o he me al ions (Fe2+, Ag+, Co2+, Ba2+, Ni2+, Hg2+,
Pb2+, Zn2+, Fe3+, Ca2+, Mn2+, Mg2+ and C 3+). Consequen ly, Eu-SCNPs and Tb-SCNPs can
be used as “ isual” pass/ ail senso s o m.p.c. o Cu2+ ions in wa e acco ding o he WHO
c i e ion (m.p.c.(Cu2+)WHO = 30 µM). Complemen a y, Dy-SCNPs can be use ul as “ isual”
pass/ ail senso s o m.p.c. o Cu2+ ions in wa e acco ding o he EPA egula ion
(m.p.c.(Cu2+)EPA = 20 µM).
0
50
100
150
200
250
300
550 560 570 580 590 600
Dy-SCNPs
Fe2+
Ag+
Co2+
Ba2+
Cu2+
Ni2+
Hg2+
Pb2+
Zn2+
Fe3+
Ca2+
Mn2+
Mg2+
C 3+
In ensi y (a.u.)
Wa eleng h (nm)
3. Lan hanide-based Single-Chain Nanopa icles as “Visual” Pass/Fail Senso s o Maximum Pe missible
Concen a ion o Cu2+ Ions in D inking Wa e
101
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chain nanopa icles, Polym. In ., 2016, 65, 855-860.
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polyme ic nanopa icles as compa men alised senso s o me al ions, Polym.
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14. J. De-La-Cues a, E. Ve de-Ses o, A. A be and J. A. Pomposo, Sel -Repo ing o
Folding and Agg ega ion by O hogonal Han zsch Luminopho es Wi hin a Single
Polyme Chain, Angew. Chem. In . Ed., 2021, 60, 3534-3539.
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polyme ic nanopa icles owa ds he de elopmen o e icien d ug deli e y
sys ems, Polym. Chem., 2016, 7, 6164-6169.
16. A. La o e-Sánchez and J. A. Pomposo, A simple, as and highly sensi i e
colo ime ic de ec ion o zein in aqueous e hanol ia zein-py idine-gold
in e ac ions, Chem. Commun., 2015, 51, 15736-15738.
17. N. M.Hamelmann, J. W. D. Paa s, Y. A alos-Padilla, E. Lan e o, I. Siden-Kiamos, L.
Spanos, X. Fe nandez-Busque s and J. M. J. Paulusse, Single-Chain Polyme
Nanopa icles Ta ge ing he Ookine e S age o Mala ia Pa asi es, ACS In ec . Dis.,
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18. S. Ga mendia, S. B. Law enson, M. C. A no, R. K. O'Reilly, D. Ta on and A. P. Do e,
Ca aly ically Ac i e N-He e ocyclic Ca bene Release om Single-Chain
Nanopa icles Following a The molysis-D i en Un olding S a egy, Mac omol.
Rapid Commun., 2019, 40, 1900071.
19. T. Gunnlaugsson, J. P. Leona d, K. Sénéchal and A. J. Ha e, Eu(III)–cyclen–phen
conjuga e as a luminescen coppe senso : he o ma ion o mixed polyme allic
mac ocyclic complexes in wa e , Chem. Commun., 2004, 782-783.
20. A. Nona , A. J. Ha e, K. Senechal-Da id, J. P. Leona d and T. Gunnlaugsson,
Luminescen sensing and o ma ion o mixed –d me al ion complexes be ween a
Eu(III)cyclen-phen conjuga e and Cu(II), Fe(II), and Co(II) in bu e ed aqueous
solu ion, Dal on T ans., 2009, 4703-4711.
21. Z. Ekmekci, Highly selec i e luo escence ‘ u n-o ’ senso s o Cu2+ in aqueous
en i onmen s, Te ahed on Le ., 2015, 56, 1878-1881.
22. L. M. A oua, R. Ali, A. E. A. E. Albad i, S. Messaoudi, F. M. Alminde ej and S. M.
Saleh, A New, Ex emely Sensi i e, Tu nO Op ical Senso U ilizing Schi Base o
Fas De ec ion o Cu(II), Biosenso s, 2023, 13, 359.
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Concen a ion o Cu2+ Ions in D inking Wa e
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23. B.S. Sankha and R.N. Kapoo , O ganic compounds o sama ium. II. Reac ions o
sama ium isop opoxide wi h e hyl ace oace a e, Can. J. Chem., 1966, 44, 1369-
1372.
24. N.K. Du and S. Rahu , Chemis y o lan hanons-XXIII. The o ma ion cons an s o
he e hyl ace oace a e complexes o a e ea h, J. Ino g. Nucl. Chem. 1969, 31,
3177-3179.
25. A. M. Mishchenko, E. K. T uno a and A. S. Be ezhny ska, Lan hanide Complexes
wi h Allyl Ace oace a e in Mixed Wa e –O ganic Media: Fo ma ion, S abili y and
Bonding, J. Solu ion Chem., 2015, 44, 2117-2128.
26. O. S e n and M. Volme , Übe die Abklingzei de Fluo eszenz, Zei sch i ü
Physik, 1919, 20, 183-188.
27. M. L. Aulseb ook, B. G aham, M. R. G ace and K. L. Tuck, Lan hanide complexes
o luminescence-based sensing o low molecula weigh analy es, Coo d. Chem.
Re ., 2018, 375, 191220.
28. K. Szyszka, S. Ta gońska, A. Lewińska, A. Wa as and R. J. Wiglusz, Quenching o
he Eu3+ Luminescence by Cu2+ Ions in he Nanosized Hyd oxyapa i e Designed
o Fu u e BioDe ec ion, Nanoma e ials, 2021, 11, 464.
4. Gold Nanoclus e s Syn hesized wi hin Single-
Chain Nanopa icles as Ca aly ic Nano eac o s in
Wa e
4. Gold Nanoclus e Syn hesized wi hin Single-Chain Nanopa icles as Ca aly ic Nano eac o s in Wa e
112
μL o 4-ni ophenol and 30.35 mg o NaBH4 we e sequen ially added unde a gon in o a
ial a 0 C. The 4-ni ophenol educ ion eac ion was hen moni o ed ia UV-Vis
spec opho ome y. Abso bance was eco ded by aking 1 μL o c ude a a gi en eac ion
ime, which was hen dilu ed in 2 mL o deionized wa e . A e eac ion, 4-aminophenol
was pu i ied ia p epa a i e hin-laye ch oma og aphy (TLC) (n-hexane/e hyl ace a e
1:1), wi h a yield o 95%. 1H NMR (400 MHz, DMSO-d6, ppm): δ 4.36 (s, 2H), 6.42–6.46
(m, 4H), 8.34 (s, 1H).
4.4.4. P ocedu e o he Reduc ion o Ni obenzene Ca alyzed by
AuNCs/SCNPs
The Au-NC/SCNPs we e used as ca alys s o he educ ion a . . o ni obenzene (0.2
mmol) o aniline in he p esence o NaBH4 (0.8 mmol). The same p ocedu e desc ibed in
Sec ion 3.4.3. was ollowed. Abso bance was eco ded by aking 2 μL o c ude a a gi en
eac ion ime, which was hen dilu ed in 4 mL o deionized wa e . A e eac ion, aniline
was pu i ied ia p epa a i e TLC (n-hexane/e hyl ace a e 1:1), wi h a yield o 96%. 1H
NMR (400 MHz, DMSO-d6, ppm): δ 3.65 (s, 2H), 6.67–6.70 (m, 2H), 6.73–6.77 (m, 1H),
7.13–7.17 (m, 2H).
4.4.5. P ocedu e o he Reduc ion o 3-(4-Ni ophenyl)-1,3-oxazolidin-
2one Ca alyzed by Au-NCs/SCNPs
The Au-NC/SCNPs we e used as ca alys s o he educ ion a . . o 3-(4-ni ophenyl)-
1,3-oxazolidin-2-one (0.2 mmol) o 3-(4-aminophenyl)-1,3-oxazolidin-2-one in he
p esence o NaBH4 (0.8 mmol). The same p ocedu e desc ibed in Sec ion 3.4.3. was
ollowed. Abso bance was eco ded by aking 2 μL o c ude a a gi en eac ion ime,
which was hen dilu ed in 10 mL o deionized wa e . I is wo h no ing ha due o he
he e ogenous cha ac e o he eac ion and o he igo ous gene a ion o molecula
hyd ogen gas, aliquo s o analysis we e p epa ed ensu ing only he aqueous phase was
aken. The p oduc , 3-(4-aminophenyl)oxazolidin-2-one, was pu i ied ia p epa a i e TCL

4. Gold Nanoclus e Syn hesized wi hin Single-Chain Nanopa icles as Ca aly ic Nano eac o s in Wa e
113
(n-hexane/e hyl ace a e 1:1), wi h a yield o 89%. 1H NMR (400 MHz, CDCl3), ppm): δ
4.63 (s, 2H), 3.97–4.01 (dd, 2H), 4.43–4.47 (dd, 2H), 6.68–6.71 (d, 2H), 7.27–7.30 (d, 2H).
4.5. Resul s and Discussion
4.5.1. Syn hesis o Gold Nanoclus e s wi hin Single-Chain Nanopa icles
(Au-NCs/SCNPs)
We a ge ed an amphiphilic poly(OEGMA-co-AEMA) andom copolyme ea u ing
hyd ophilic oligo(e hyleneglycol) me hyl e he me hac yla e (OEGMA) and hyd ophobic
(2-ace oace oxy)e hyl me hac yla e (AEMA) uni s as a empla e o he syn hesis o
AuNCs. Based on he li e a u e da a o low-molecula -weigh ligands,24 we su mised ha
he be a-ke oes e g oup o AEMA could be used as a educ an o Au(III) ions, as well as
a s abilizing and s uc u e-di ec ing agen , o gene a e he Au-NCs. Poly(OEGMA-
coAEMA) was syn hesized by means o e e sible addi ion agmen a ion chain- ans e
(RAFT) polyme iza ion (see Figu e 44).
Figu e 44. Syn hesis o an amphiphilic poly(OEGMA-co-AEMA) andom copolyme ea u ing
hyd ophilic oligo(e hyleneglycol) me hyl e he me hac yla e (OEGMA) and hyd ophobic (2-
4. Gold Nanoclus e Syn hesized wi hin Single-Chain Nanopa icles as Ca aly ic Nano eac o s in Wa e
114
ace oace oxy)e hyl me hac yla e (AEMA) epea uni s ia e e sible addi ion agmen a ion
chain ans e (RAFT) polyme iza ion. Schema ic illus a ion o he syn hesis o Au-NCs/SCNPs
om poly(OEGMA-co-AEMA) sel -assembled in wa e in he o m o SCNPs.
The copolyme showed a weigh -a e age molecula weigh (Mw) o 80.7 kDa and a low
dispe si y (D) alue o 1.12, as de e mined by SEC. The con en o AEMA uni s in he
copolyme was 35 mol%, as es ima ed om 1H NMR spec oscopy. I is well-known om
p e ious wo ks ha amphiphilic poly(OEGMA-co-AEMA) andom copolyme s wi h his
AEMA con en a e able o sel -assemble in amolecula ly in wa e a high dilu ion (1
mg/mL) in o disc e e co e–shell-like SCNPs.25,26 In ag eemen wi h p e ious esul s, DLS
measu emen s o poly(OEGMA-co-AEMA) in wa e a 1 mg/mL e ealed an a e age
hyd odynamic diame e o Dh = 11.0 nm co esponding o disc e e, indi idual SCNPs
wi hou any sign o he p esence o mul i-chain agg ega es (see Table 2 and Figu e 44).
Table 2. E olu ion o a e age nanopa icle sizes du ing he syn hesis o Au-NCs/SCNPs.
Ma e ial Type
Reac ion Time,
DLS Hyd odynamic
Diame e , Dh (nm) 2
Poly(OEGMA-co-AEMA) 1
0 min.
11.0
Au-NCs/SCNPs
1 min.
11.0
Au-NCs/SCNPs
15 min.
10.7
Au-NCs/SCNPs
1 h
11.3
Au-NCs/SCNPs
1 day
11.2
Au-NCs/SCNPs
1 week
11.6
1 Sel -assembled in wa e in he o m o co e–shell-like SCNPs. 2 S anda d de ia ion ca. ± 0.3
nm.
4. Gold Nanoclus e Syn hesized wi hin Single-Chain Nanopa icles as Ca aly ic Nano eac o s in Wa e
115
Figu e 44. DLS size dis ibu ion o poly(OEGMA-co-AEMA) SCNPs and Au-NCs/SCNPs.
Figu e 45. Compa ison o he UV-Vis spec a o Au-NPs syn hesized in he p esence o
poly(OEGMA-co-AEMA) SCNPs a a (Au(III))/(be a-ke oes e (AEMA)) a io o 1 s Au-NCs
syn hesized wi hin poly(OEGMA-co-AEMA) SCNPs a a (Au(III))/(be a-ke oes e (AEMA)) a io o
0.08.
0
0.1
0.2
0.3
0.4
0.5
200 300 400 500 600 700 800
Poly (OEGMA-co-AEMA)
Au-NPs
Au-NCs
In ensi y (%)
Wa eleng h (nm)
4. Gold Nanoclus e Syn hesized wi hin Single-Chain Nanopa icles as Ca aly ic Nano eac o s in Wa e
116
Fo he syn hesis o Au-NCs wi hin he SCNPs h ough be a-ke oes e -media ed Au(III)
educ ion (Figu e 45), we ound con ol o he (Au(III)/(be a-ke oes e (AEMA)) a io o
be c i ical. Hence, by using a (Au(III))/(be a-ke oes e (AEMA)) a io o 1, we ob ained
gold nanopa icles (Au-NPs) ins ead o Au-NCs, as e ealed by he in ense localized
su ace plasmon esonance (LSPR) UV-Vis abso bance signal cha ac e is ic o Au-NPs
(Figu e 45). In e es ingly, by lowe ing he (Au(III))/(be a-ke oes e (AEMA)) a io o 0.08,
we obse ed ia TEM he p esence o Au-NCs (diame e < 5 nm) wi hin indi idual
poly(OEGMA-coAEMA) SCNPs (Figu e 46), as well as he o al absence o he LSPR band
ypical o la ge Au-NPs (Figu e 45). No signi ican di e ences we e ound be ween he
IR spec a o nea poly(OEGMA-co-AEMA) and he Au-NCs/SCNPs, as illus a ed in Figu e
47, which we a ibu e o he ela i ely low (Au(III))/(be a-ke oes e (AEMA)) a io
employed. No ably, he size o he Au-NCs/SCNPs was ound o be e y s able o e ime,
as illus a ed in Table 2 and Figu e 44, showing he no o ious s abilizing e ec o he
SCNPs agains Au-NCs agg ega ion o e ime. Con e sely, by using a
(Au(III))/(be ake oes e (AEMA)) a io o 1, he diame e o he esul ing Au-NPs was
ound o g ow wi h ime, as illus a ed in Figu e 48 by he associa ed colo changes. A e
1 day, he DLS hyd odynamic diame e o he Au-NPs was ound o be 67.1 nm.
(a) (b)
Figu e 46. (a) TEM pic u e o Au-NCs syn hesized wi hin poly(OEGMA-co-AEMA) SCNPs a a
(Au(III))/(be a-ke oes e (AEMA)) a io o 0.08. (b) Illus a ion o he p esence o (da ke ) AuNCs
wi h a diame e < 5 nm wi hin a ep esen a i e single-chain nanopa icle o ca. 11 nm in
diame e .
4. Gold Nanoclus e Syn hesized wi hin Single-Chain Nanopa icles as Ca aly ic Nano eac o s in Wa e
117
Figu e 47.IR spec a o nea poly(OEGMA-co-AEMA) SCNPs (blue colo ) and Au-NCs/SCNPs ( ed
colo ).
Figu e 48. Au-NPs syn hesized in he p esence o poly(OEGMA-co-AEMA) SCNPs a a
(Au(III))/(be a-ke oes e (AEMA)) a io o 1 s Au-NCs syn hesized a a a io o 0.08.
800100012001400160018002000
Poly(OEGMA-co-AEMA)
Au-NCs
Wa enumbe [cm-1]

4. Gold Nanoclus e Syn hesized wi hin Single-Chain Nanopa icles as Ca aly ic Nano eac o s in Wa e
118
Taken oge he , he abo e esul s demons a e he e icien syn hesis o s abilized Au-
NCs wi h size < 5 nm wi hin disc e e poly(OEGMA-co-AEMA) SCNPs o ca. 11 nm in
diame e . Key o he access o s abilized Au-NCs wi hin SCNPs, ins ead o o la ge AuNPs
ha g ow in size o e ime, is he con ol o he (Au(III))/(be a-ke oes e (AEMA)) a io
employed du ing he syn hesis.
4.5.2. Gold Nanoclus e s wi hin Single-Chain Nanopa icles
(AuNCs/SCNPs) as Ca aly ic Nano eac o s
We epo he ein he esul s o he use o Au-NCs wi hin SCNPs as ca aly ic
nano eac o s o he educ ion o 4-ni ophenol, ni obenzene, and 3-(4-ni ophenyl)-
1,3-oxazolidin2one by bo ohyd ide (BH4−) in wa e a . .
4.5.2.1. Reduc ion o 4-Ni ophenol o 4-Aminophenol Ca alyzed by Au-
NCs/SCNPs
Pa ace amol (a popula analgesic and an ipy e ic agen used o ea e e and mild o
mode a e pain) can be ypically syn hesized om 4-ni ophenol as an in e media e ia
i s educ ion o 4-aminophenol and subsequen ace yla ion wi h ace ic anhyd ide.
4ni ophenol is highly soluble in wa e (11.6 mg/mL a 20 ◦C). We in es iga ed he
educ ion o 4-ni ophenol (4-NP) o 4-aminophenol (4-AP) by BH4− in wa e a . . by
using he Au-NCs/SCNPs as highly e icien ca aly ic nano eac o s. This ans o ma ion
has eme ged as an impo an model eac ion o assessing he ca aly ic ac i i y o
me allic nanopa icles in wa e . Sec ion 3.4.3. de ails he expe imen al p ocedu e ha
ollowed, which is depic ed schema ically in Figu e 49.
4. Gold Nanoclus e Syn hesized wi hin Single-Chain Nanopa icles as Ca aly ic Nano eac o s in Wa e
119
Figu e 49. (a) Reduc ion o 4-ni ophenol o 4-aminophenol in wa e a . . by using he
AuNCs/SCNPs as highly e icien ca aly ic nano eac o s. (b) Colo changes obse ed du ing he
educ ion o 4-ni ophenol (λmax ≈ 400 nm) o 4-aminophenol (λmax ≈ 300 nm) ca alyzed by
AuNCs/SCNPs.
The con e sion o 4-aminophenol o 4-aminophenol was ollowed by UV-Vis
spec ome y due o he di e en , well-sepa a ed abso p ion bands o he eac an and
p oduc , as illus a ed in Figu e 49(b) and Figu e 50. In e es ingly, unde ou eac ion
condi ions, an isosbes ic poin loca ed a ca. λ = 325 nm was obse ed in he UV-Vis
spec a du ing he educ ion o 4-aminophenol (λmax ≈ 400 nm) o 4-aminophenol (λmax
≈ 300 nm). This ac sugges s he majo in ol emen o a di ec ou e mechanisms in he
educ ion o 4ni ophenol o 4-aminophenol in wa e a . . wi h Au-NCs/SCNPs.27 No
eac ion was obse ed in a model expe imen lacking he Au-NCs/SCNPs ca alys .
4. Gold Nanoclus e Syn hesized wi hin Single-Chain Nanopa icles as Ca aly ic Nano eac o s in Wa e
120
Figu e 50. Kine ics o he educ ion o 4-ni ophenol o 4-aminophenol ca alyzed by
AuNCs/SCNPs as de e mined by UV-Vis spec oscopy.
The pu i ied p oduc ’s chemical s uc u e was con i med using 1H nuclea magne ic
esonance spec oscopy (1H NMR). The spec um shows nume ous p o on en i onmen s
wi hin he molecule. A single a δ 4.36 (s, 2H) sugges s he p esence o a pai o
equi alen p o ons, he amino g oup. The mul iple om δ 6.42-6.46 (m, 4H) signi ies an
a oma ic sys em wi h p o ons which expe ience sligh ly di e en elec onic
en i onmen s, likely due o he in luence o an adjacen subs i uen , such as he -NH2
and -OH g oups. Finally, he single a δ 8.34 o he emaining p o on (s, 1H) is ypical
o an a oma ic p o on ha is deshielded, ypically due o an elec onega i e g oup (such
as a ni o o ca bonyl) nea by o , in his case, his single signi ies he in luence o a
hyd oxyl g oup in -pa a o he amino g oup. This spec um indica es a clean p oduc wi h
no signi ican impu i ies, in ha only he expec ed signals a e obse ed.
0
0.2
0.4
0.6
0.8
1
1.2
300 400
0 min
2 min
3min
4min
5min
7min
9min
12min
15min
Wa eleng h (nm)
Abso ion (a.u.)
4-NP
4-AP
4. Gold Nanoclus e Syn hesized wi hin Single-Chain Nanopa icles as Ca aly ic Nano eac o s in Wa e
121
Figu e 51. 1H MNR spec um a e isola ion ia p epa a i e TCL o he 4-aminophenol
p oduc .
Me iculously cons uc ed calib a ion cu es we e done o all compounds ia UV- isible
spec oscopy. The goal o his e o was o de e mine he mola ex inc ion coe icien s
o each compound. This pa ame e is c i ical o quan i a ing he abili y o a compound
o abso b ligh a a pa icula wa eleng h. The p ocess o c ea ing a calib a ion cu e
in ol es making a se ies o s anda d solu ions wi h accu a ely known concen a ions o
he a ge compound. Each se o s anda ds is hen subjec ed o UV- is spec oscopy,
whe e we eco d he abso bance a he wa eleng h whe e he compound exhibi s
maximum abso p ion. The ela ionship be ween abso bance and concen a ion o
hese s anda ds should be linea , acco ding o he Bee -Lambe law, o e he ange o
concen a ions used.
The mola ex inc ion coe icien , ε, e e s o he abso bance o a one mola (1 M) solu ion
o he compound in a cu e e o c oss-sec ion 1 cm. Plo ing he abso bance alues
e sus he concen a ions o he s anda ds gene a es a calib a ion cu e. The slope o
4. Gold Nanoclus e Syn hesized wi hin Single-Chain Nanopa icles as Ca aly ic Nano eac o s in Wa e
128
p oduc .
Figu e 59. Expe imen al da a poin o he in ol emen o bo h he di ec ou e (uppe ) and he
condensa ion ou e (bo om) mechanisms.
I was impe a i e o quan i y he mola ex inc ion coe icien s so ha we could
unde s and how he sys em e ol es in e ms o he mola ac ions o he cons i uen
species as well as he mechanis ic p og ession and he kine ics o he eac ion. Once
again, he mola ex inc ion coe icien s we e deduced h ough UV- isible spec oscopic
analysis (Figu e 60,61 and 62) whe e calib a ion cu es we e cons uc ed. Mola
ex inc ion coe icien s u nished a c ucial ounda ion o ou kine ic analysis, allowing us
o quan i y he mola ac ions o he eac an s, in e media es, and p oduc s h oughou
he cou se o he eac ion, hus p o iding a window in o he in ica e molecula
in e ac ions and ans o ma ions ha dic a e he cou se o he chemis y o he eac ion
unde s udy.

4. Gold Nanoclus e Syn hesized wi hin Single-Chain Nanopa icles as Ca aly ic Nano eac o s in Wa e
129
Figu e 60. Calib a ion cu e o de e mina ion o he UV-Vis mola ex inc ion coe icien o
ni obenzene in wa e a . .: ε (λmax = 265 nm) ≈ 16632 M-1cm-1.
Figu e 61. Calib a ion cu e o de e mina ion o he UV-Vis mola ex inc ion coe icien o
azobenzene in wa e a . .: ε (λmax = 230 nm) ≈ 16566 M-1cm-1.
0
0.1
0.2
0.3
0.4
0.5
0 5 10-6 1 10-5 1.5 10-5 2 10-5 2.5 10-5
y = 0.0096729 + 16632x R2= 0.99641
Abso bance (a.u.)
[C] (mol/L)
0
0.2
0.4
0.6
0.8
1
1.2
1.75 10-5 3.5 10-5 5.25 10-5 7 10-5
y = -0.094268 + 16566x R2= 0.99279
Abso bance (a.u.)
[C] (mol/L)
4. Gold Nanoclus e Syn hesized wi hin Single-Chain Nanopa icles as Ca aly ic Nano eac o s in Wa e
130
Figu e 62. Calib a ion cu e o de e mina ion o he UV-Vis mola ex inc ion coe icien o
aniline in wa e a . .: ε (λmax = 230 nm) ≈ 20239 M-1cm-1.
Figu e 63 shows he e olu ion o he concen a ion o he eac an and he p oduc o e
ime as es ima ed om da a in Figu e 56. A eac ion yield o 96% was achie ed in 90 min.
o eac ion ime, whe e he amoun o ni obenzene was o ally consumed. As illus a ed
in Figu e 55, he appa en kine ic cons an (kapp) o he eac ion was es ima ed o be kapp
= 3.8 × 10−4 s−1.
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0 5 10-6 1 10-5 1.5 10-5 2 10-5 2.5 10-5
y = 0.026859 + 17171x R2= 0.99692
Abso bance (a.u.)
[C] (mol/L)
4. Gold Nanoclus e Syn hesized wi hin Single-Chain Nanopa icles as Ca aly ic Nano eac o s in Wa e
131
Figu e 63. E olu ion o he concen a ion o ni obenzene (blue), cis-azobenzene ( ed) and
aniline (g een) o e ime du ing he educ ion o ni obenzene o aniline ca alyzed by
AuNCs/SCNPs.
Figu e 64. Appa en kine ic cons an (kapp) o he educ ion o ni obenzene o aniline ca alyzed
by Au- NCs/SCNPs.
0
0.2
0.4
0.6
0.8
1
020 40 60 80
Ni obenzene
cis-azobenzene
Aniline
Mola ac ion
(min)
-2.5
-2
-1.5
-1
-0.5
0
0 1000 2000 3000 4000 5000 6000
ln(A/A0)
(s)
kapp = 3.8 x 10-4 s-1
4. Gold Nanoclus e Syn hesized wi hin Single-Chain Nanopa icles as Ca aly ic Nano eac o s in Wa e
132
4.5.2.3. Reduc ion o 3-(4-Ni ophenyl)-1,3-oxazolidin-2-one o 3-(4-
Aminophenyl)-1,3-oxazolidin-2-one Ca alyzed by Au-NCs/SCNPs
Mo i a ed by he high con e sion obse ed du ing he Au-NCs/SCNPs-ca alyzed
educ ion o bo h 4-ni ophenol and ni obenzene, we addi ionally in es iga ed he
educ ion o 3-(4-ni ophenyl)-1,3-oxazolidin-2-one o 3-(4-aminophenyl)-1,3oxazolidin-
2-one ca alyzed by Au-NCs/SCNPs. In his sense, he aminophenyl– oxazolidinone
agmen is a common mo i con ained in se e al d ugs like Ri a oxaban, Su ezolid, and
Linezolid (see Figu e 65).
Figu e 65. Illus a ion o he educ ion o 3-(4-ni ophenyl)-1,3-oxazolidin-2-one o 3-
(4aminophenyl)- 1,3-oxazolidin-2-one by BH4 − in wa e a . . ca alyzed by Au-NCs/SCNPs. The
aminophenyl– oxazolidinone agmen is a common mo i con ained in se e al d ugs like
Ri a oxaban, Linezolid, and Su ezolid.
We ound he Au-NCs/SCNPs-ca alyzed educ ion o 3-(4-ni ophenyl)-1,3-
oxazolidin2one o p oceed in wo s eps ( as and slow, espec i ely), as summa ized in
Figu e 66.
4. Gold Nanoclus e Syn hesized wi hin Single-Chain Nanopa icles as Ca aly ic Nano eac o s in Wa e
133
Figu e 66. Reduc ion o 3-(4-ni ophenyl)-1,3-oxazolidin-2-one (in blue) o 3-(4-
aminophenyl)1,3oxazolidin-2-one (in g een) ca alyzed by Au-NCs/SCNPs. The as s ep
co esponds o he o ma ion o he diazene in e media e (in ed), and he slow s ep o he
gene a ion o he aminophenyloxazolidinone p oduc .
Figu e 67 shows he UV-Vis spec a eco ded a di e en eac ion imes co esponding
o he as s ep. We obse ed he comple e disappea ance o he UV-Vis abso p ion peak
o 3-(4-ni ophenyl)-1,3-oxazolidin-2-one (λmax ≈ 320 nm) in 15 min. and hen he
appea ance o a new UV-Vis abso p ion peak (λmax ≈ 360 nm), which can be a ibu ed o
he gene a ion o he diazene in e media e ((Z)-3,3ʹ-(diazene-1,2-
diylbis(4,1phenylene))bis(oxazolidin- 2-one)) in 3 h o eac ion ime. The in ensi y o his
new band was s able a e 3 h o eac ion ime, and i did no change un il 9 h o
addi ional eac ion ime. Then, du ing he slow s ep, a p og essi e disappea ance o he
UV-Vis peak o he diazene in e media e a λmax ≈ 360 nm was ound, as well as he
concomi an appea ance o a new UV-Vis abso p ion band a λmax ≈ 250 nm
co esponding o he 3(4-aminophenyl)-1,3oxazolidin-2-one p oduc (see Figu e 67).
Figu e 73 illus a es he e olu ion o he concen a ion o 3-(4-ni ophenyl)-1,3-
oxazolidin-2-one, (Z)-3,3ʹ(diazene-1,2-diylbis(4,1phenylene))bis(oxazolidin-2-one), and
3-(4-aminophenyl)-1,3oxazolidin-2-one o e ime, as es ima ed om da a in Figu e 67,
cis (Z) = 91 %
ans (E) = 9 %
FAST STEP
SLOW STEP
Yield: 89%

4. Gold Nanoclus e Syn hesized wi hin Single-Chain Nanopa icles as Ca aly ic Nano eac o s in Wa e
134
and he co esponding calib a ion cu es a e p o ided in he Suppo ing In o ma ion.
A e 20 h o eac ion ime, he eac ion yield was 89%.
a
b
Figu e 67. Kine ics o he educ ion o 3-(4-ni ophenyl)-1,3-oxazolidin-2-one o 3-
(4aminophenyl)- 1,3-oxazolidin-2-one ca alyzed by Au-NCs/SCNPs as de e mined by UV-Vis
spec oscopy co esponding o he as s ep (a) and he slow s ep (b) o he eac ion.
0
0.2
0.4
0.6
0.8
1
260 280 300 320 340 360 380 400
0 min
5min
15min
30min
1h
3h - 12h
Abso bance (a.u.)
Wa eleng h (nm)
0
0.2
0.4
0.6
0.8
1
200 250 300 350 400 450
12h
13h
15h
16h
17h
18h
20h
Abso bance (a.u.)
Wa eleng h (nm)
4. Gold Nanoclus e Syn hesized wi hin Single-Chain Nanopa icles as Ca aly ic Nano eac o s in Wa e
135
In his case, a clea in e media e was obse ed again in he eac ion, so as in he
Reduc ion o ni obenzene o aniline, bo h he in e media e and he inal p oduc we e
cha ac e ized by 1H NMR as can be seen in Figu e 68 and Figu e 69.
Figu e 68. 1H MNR spec um a e isola ion ia p epa a i e TCL o he (Z)-3,3'-(diazene-1,2-
diylbis(4,1-phenylene))bis(oxazolidin-2-one) in e media e species.
Figu e 69. 1H MNR spec um a e isola ion ia p epa a i e TCL o he 3-(4-aminophenyl)-
1,3oxazolidin-2-one p oduc .
4. Gold Nanoclus e Syn hesized wi hin Single-Chain Nanopa icles as Ca aly ic Nano eac o s in Wa e
136
Acco dingly, in o de o ensu e ha ou moni o ing was eliable and accu a e, calib a ion
cu es we e cons uc ed o ob ain mola ex inc ion coe icien s o he subs ances
in ol ed in ou eac ion. This was necessa y in ou case because he mola ex inc ion
coe icien is highly speci ic o each chemical species and depends on a numbe o
pa ame e s ha a e di ec ly ela ed o he expe imen al condi ions. This moni o ing is
c ucial o being able o ob ain he ull pic u e o wha is happening du ing a chemical
eac ion. I can show us he eac ion kine ics, so we can op imize he condi ions in which
we pe o m he eac ion o he g ea es yield, and also o main ain he consis ency and
quali y o p oduc om ba ch o ba ch.
Figu e 70. Calib a ion cu e o de e mina ion o he UV-Vis mola ex inc ion coe icien o 3-(4-
ni ophenyl)-1,3-oxazolidin-2-one in wa e a . .: ε (λmax = 320 nm) ≈ 2074 M-1cm-1.
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1 10-4 2 10-4 3 10-4 4 10-4 5 10-4
y = -0.020172 + 2074.4x R2= 0.99287
Abso bance (a.u.)
[C] (mol/L)
4. Gold Nanoclus e Syn hesized wi hin Single-Chain Nanopa icles as Ca aly ic Nano eac o s in Wa e
137
Figu e 71. Calib a ion cu e o de e mina ion o he UV-Vis mola ex inc ion coe icien o
(Z)3,3'- (diazene-1,2-diylbis(4,1-phenylene))bis(oxazolidin-2-one) in wa e a . .: ε (λmax = 360
nm) ≈ 3060 M- 1cm-1.
Figu e 72. Calib a ion cu e o de e mina ion o he UV-Vis mola ex inc ion coe icien o 3-
(4- aminophenyl)-1,3-oxazolidin-2-one in wa e a . .: ε (λmax = 250 nm) ≈ 2801 M-1cm-1.
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
5 10-5 1 10-4 1.5 10-4 2 10-4 2.5 10-4 3 10-4
y = 0.001723 + 3060.4x R2= 0.99859
Abso bance (a.u.)
[C] (mol/L)
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1 10-4 2 10-4 3 10-4
y = 0.1775 + 2800.9x R2= 0.99369
Abso bance (a.u.)
[C] (mol/L)

5. Consecu i e One-Po Alkyne Semihyd ogena ion/Alkene Dioxygena ion Reac ions by P (II)/Cu(II)
Single-Chain Nanopa icles in G een Sol en
145
5.1. Mo i a ion
This wo k was spa ked by he p essing need o sus ainable p og esses in ca alysis,
pa icula ly o he design o nanoca alys s ha can ca y ou mul i-s ep chemical
eac ions all in one eac ion essel. Realizing hese en i onmen al and economic
d awbacks o using oxic sol en s and he di icul ies ha a e accompanied by pu i ying
p ocesses in he classical mul i-s ep syn hesis, we could ocus on o e coming hese
challenges. He e, we de eloped a acile me hodology ha a o ded he e obime allic
P (II)/Cu(II) single-chain polyme nanopa icles (SCNPs) in a sequen ial manne ,
capi alizing bo h on me al-ligand coo dina ion o in amolecula olding and hei
syne gis ic ca aly ic ac i i y. Thus, his s a egy is g eene in N-bu ylpy olidone use as a
non oxic sol en and displays he e iciency and selec i i y o such bime allic SCNPs in
consecu i e one-po alkyne semihyd ogena ion and alkene dioxygena ion eac ions. This
wo k, h ough his con ibu ion, ies o make a success ul a emp a making i s
con ibu ion o he la ge goal o sus ainable chemis y, as he abo e-said complex mul i-
me allic SCNPs may open new is as in he syn hesis and applica ion o hese highly
e sa ile and mul i- unc ional nanoca alys s.
5.2. In oduc ion
Me al-ligand coo dina ion allows in amolecula ly olding o ligand-deco a ed disc e e
syn he ic polyme chains o me allo olded single-chain polyme nanopa icles (SCNPs).1
SCNPs a e in a-chain c oss-linked single polyme chains wi h mani old p omising
applica ions, mainly as ca alys s, nanosenso s and d ug nanoca ie s.2 In gene al,
in amolecula olding o he isola ed syn he ic chains gene a es locally compac zones
wi hin he SCNPs o e icien immobiliza ion o ca aly ic ac i e species, luminopho es o
d ugs.3 To some ex en , he in achain olding o disc e e syn he ic polyme chains o
SCNPs esembles he olding o ce ain p o eins o hei p ecise unc ional con o ma ion
(i.e., na i e s a e).4 In pa icula , me allo- olded SCNPs le e age he dual ole played by
he me al: as a olding elemen ia in a-chain me al-ligand coo dina ion, and as an
immobilized unc ional cen e o subsequen ca alysis.5 The numbe and ca aly ic
5. Consecu i e One-Po Alkyne Semihyd ogena ion/Alkene Dioxygena ion Reac ions by P (II)/Cu(II)
Single-Chain Nanopa icles in G een Sol en
146
applica ions o me allo- olded SCNPs as enzyme-mime ic nanoen i ies ha e g own
signi ican ly in ecen yea s.6,7 In a seminal wo k, Te ashima e al. epo ed Rucon aining
amphiphilic SCNPs o ca alyse he educ ion o cyclohexanone o cyclohexanol in wa e .8
Pomposo e al. pionee ed he in oduc ion o me allo- olded Cu(II)-con aining SCNPs
showing ca aly ic selec i i y in alkyne homocoupling eac ions,5 and single-chain
globules mimicking he mo phology and polyme ase ac i i y o me alloenzymes.9 He e
al. p epa ed me allo- olded SCNPs con aining Ni- hiola e complexes showing excellen
he mal s abili y unde ae obic condi ions and excellen ac i i y and selec i i y du ing
he pho oca aly ic educ ion o CO2 o CO.10 Zimme man e al. de eloped “clickase”
SCNPs displaying enzyme-like “click” ca alysis in i o and enabling e icien cell su ace
glycan edi ing.11 Ta on e al. epo ed SCNPs con aining Ag(I)-N-he e ocyclic ca bene
(NHC) linkages as NHC p e-ca alys s o he benzoin condensa ion eac ion.12 Yang e al.
syn hesized me al-con aining SCNPs in concen a ed solu ions a oom empe a u e ( . .)
by in oducing elec os a ic epulsion and in a-chain c osslinking by coo dina ion wi h
Cu(II) o Fe(II) o Fe(III) ions.13 Tan e al. syn hesized enzyme-mime ic SCNPs wi h chi al
Fe(II)-oxazoline complexes o e icien asymme ic sul a-Michael addi ion o hiols o
α,β-unsa u a ed ke ones in wa e a . .14 Mo e ecen ly, Pomposo e al. de eloped a
me hod o upcycling poly( inyl chlo ide) (PVC) was e o e icien ca aly ic Cu(II)-
con aining SCNPs.15 Cu en ad ances in ca alysis u ilizing SCNPs ha e been ecen ly
e iewed by he Ba ne -Kowollik eam.16
Howe e , in oduc ion o a leas wo dis inc me al species in SCNPs al hough highly
desi able is s ill syn he ically challenging. In a pionee ing wo k, Lemco e al. syn hesized
Rh(I)/I (I) SCNPs al hough hei ca aly ic p ope ies we e no e alua ed.17 Subsequen ly,
Ba ne -Kowollik e al. syn hesized he e obime allic Eu(III)/P (II)-SCNPs18 and Au(I)/Y(III)-
SCNPs19 in which only one o he wo me al ions, P (II) o Au(I), was used o ca alysis,
while he o he was employed o sensing o in achain olding (Eu(III) o Y(III),
espec i ely). Mo e ecen ly, he same g oup deco a ed SCNPs olded h ough e ocene
uni s wi h Pd(II) a oms, which p o ed o be an ac i e ca alys o he in amolecula
hyd oamina ion o an aminoalkyne.20
5. Consecu i e One-Po Alkyne Semihyd ogena ion/Alkene Dioxygena ion Reac ions by P (II)/Cu(II)
Single-Chain Nanopa icles in G een Sol en
147
Ad anced he e obime allic nanoca alys s o ca ying ou mul is ep chemical p ocesses
in a single eac ion essel a e cu en ly o g ea in e es in academia and indus y.21
O en, howe e , majo cos s o many consume p oduc s syn hesized in mul is ep
p ocesses a e incu ed in he pu i ica ion and isola ion o in e media es.22 Addi ionally,
eplacemen o oxic o ganic sol en s by g een sol en s has a ac ed signi ican
in e es .23 To he bes o ou knowledge, bime allic SCNPs ha would allow consecu i e
one-po eac ions o be pe o med in a g een sol en ha e no been epo ed. To ill his
gap, we epo he ein he p oo o concep o he e obime allic SCNPs h ough he
syn hesis o P (II)/Cu(II)-SCNPs. We show ha hese can se e as ad anced so
nanoca alys s o pe o m consecu i e one-po alkyne semihyd ogena ion / alkene
dioxygena ion eac ions in N-bu ylpy olidone (NBP) as a g een sol en .
5.3. Ma e ials and me hods
5.3.1. Ma e ials
Me hyl me hac yla e (MMA) (99%), (2-ace oace oxy)e hyl me hac yla e (AEMA) (95%),
2.2ʹ-azo(2-me hylp opioni ile) (AIBN) (≥98%), p-ca boxybenzenesul onazide (p-CBSA)
(97%), ie hylamine (E 3N) (>99%), dichlo ome hane (CH2Cl2) (anhyd ous, ≥99.8%), e hyl
ace a e (E OAc) (anhyd ous, 99.8%), die hyl e he (E 2O) (ACS eagen , anhyd ous,
>99.0%), deu e a ed chlo o o m (CDCl3) (99.96 a om % D, con aining 0.03% ( / )
e ame hylsilane), N,Ndime hyl o mamide (DMF) (≥ 99.9%), n-hexane (Hex) (>95%),
Nbu ylpy olidinone (NBP) (≥99.5%), (1,5-cyclooc adiene)pla inum(II) dichlo ide
(P (COD)Cl2), coppe (II) ace a e (Cu(OAc)2) (98%), magnesium sulpha e (Mg2SO4)
(anhyd ous, ≥99.5%), phenylace ylene (1a) (98%) we e pu chased om Sigma-Ald ich
and used, unless speci ied, as ecei ed. 2-Cyanop op-2-yldi hiobenzoa e (CPDB) (≥97%)
was pu chased om S em Chemicals. Me hanol (MeOH) (syn hesis g ade) and THF
(HPLC g ade) we e pu chased om Scha lab. 1-E hynyl-4-me hoxybenzene (1b) (>98.0%
by GC), 3-b omophenylace ylene (1c) (>98.0% by GC), 4-e hynylbenzo i luo ide (1d)
(>98.0% by GC), 4-Py idylace ylene (1e) (>98.0% by GC) we e pu chased o m TCI
Chemicals. N-Hyd oxyph alimide (NHPI) (98%) was pu chased om BLDPha m. Pu i ied
5. Consecu i e One-Po Alkyne Semihyd ogena ion/Alkene Dioxygena ion Reac ions by P (II)/Cu(II)
Single-Chain Nanopa icles in G een Sol en
148
wa e was ob ained om a The mo Scien i ic appa a us (Ba ns ead TII Pu e Wa e
Sys em). AIBN was ec ys allized om me hanol. MMA was pu i ied by dis illa ion be o e
use. AEMA was pu i ied by passing h ough alumina.
1a
1b
1c
1d
1e
NBP
5.3.2. Techniques
1H nuclea magne ic esonance (NMR) spec a we e ob ained a oom empe a u e ( . .)
using a B uke spec ome e ope a ing a 400 MHz wi h CDCl3 as he sol en . Size
exclusion ch oma og aphy (SEC) da a we e acqui ed using a PL-GPC 50 ins umen om
Agilen Technologies, which in eg a es di e en ial e ac i e index (DRI) and mul i-angle
ligh sca e ing (MALS) de ec o s. DMF con aining 0.1% o LiB wi h a low o 1.0 mL/min
was used as an eluen . A PLgel MIXED, 7.5 x 50 mmm, 10 μm, gua d column, and a PLgel
500 Å, 7.5 x 300 mm, 5 μm, o a PL Pola Gel-M, 7.5 x 300 mm, 8 μm, HPLC columns we e
used. The molecula weigh s o he di e en samples we e de e mined using a dn/dc
alue o 0.0608. SEC da a we e analysed using Wya 's ASTRA So wa e ( e sion 8.1).
Dynamic ligh sca e ing (DLS) measu emen s we e ca ied ou a . . on a Mal e n
5. Consecu i e One-Po Alkyne Semihyd ogena ion/Alkene Dioxygena ion Reac ions by P (II)/Cu(II)
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149
Ze asize Nano ZS appa a us. Me al con en in he single-chain nanopa icles was
de e mined by induc i ely coupled plasma-mass spec ome y (ICP-MS). Fou ie
ans o m in a ed (IR) spec a we e eco ded a . . on a JASCO 3600 FTIR spec ome e .
Elemen al analysis (EA) we e pe o med in a Eu o EA3000 elemen al analyze (CHNS).
5.4. P ocedu es
5.4.1. Syn hesis o P0
In a ypical p ocedu e, MMA (3 mL, 28.2 mmol), AEMA (2.63 mL, 15.58 mmol), CPDB
(22.2 mg, 0.1 mmol) and AIBN (16.5 mg, 1 mmol) we e dissol ed in E OAc (10.45 mL).
The solu ion was degassed by bubbling N2 o 15 min. The copolyme iza ion eac ion was
ca ied a 65 °C o 24 h. A e isola ion o he esul ing copolyme P0 by p ecipi a ion in
MeOH, i was d ied unde dynamic acuum un il cons an weigh . Yield: 94.0%. β-
ke oes e con en (1H NMR): 30 mol%. Mw (SEC) = 73.6 kDa, Ɖ (SEC) = 1.17.
5.4.2. Syn hesis o P1
The syn hesis o P1 was conduc ed unde ligh -p o ec ed condi ions. The p ocess
in ol ed dissol ing 300 mg (0.47 mmol) o P0 in 10 mL o CH2Cl2 a . . Nex , p-CBSA
(12.25 mg, 0.35 eq.) and E 3N (0.4 mL, 2.87 mmol) we e added o he solu ion, which
was hen s i ed o 24 h p o ec ed om ligh . Upon comple ion o he eac ion, he
esul ing solu ion was concen a ed and p ecipi a ed in MeOH, and he unc ionalized
copolyme , P1, was d ied in a acuum o en in he absence o ligh . Yield: 76%. α-diazo-
β-ke oes e con en (EA): 19 mol%. Mw (SEC) = 75.3 kDa, Ɖ (SEC) = 1.15, Rh (DLS) = 12.5
nm.
Table 3. Elemen al analysis (EA) esul s o P0 and P1
Sample
C (%)
H (%)
N (%)
P0
57.41
7.51
-
P1
55.62
6.65
4.16

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150
5.4.3. Syn hesis o P1-SCNPs (as a con ol)
The syn hesis o P1-SCNPs om P1 was achie ed ia i adia ion wi h ul a iole ligh . 15
mg (0.07 mmol) o P1 we e dissol ed in 15 mL o DMF and he solu ion was exposed o
UV ligh i adia ion (365 nm) o 1 h. Subsequen ly, he eac ion was quenched by he
addi ion o liquid ni ogen. The esul ing P1-SCNPs we e p ecipi a ed wi h die hyl e he
and d ied unde dynamic acuum. Yield: 92%. Mw (SEC) = 75.6 kDa, Ɖ (SEC) = 1.11, Rh
(DLS) = 10.0 nm.
5.4.4. Syn hesis o P (II)-SCNPs
The syn hesis o P (II)-SCNPs om P1 was achie ed ia i adia ion wi h ul a iole ligh .
Speci ically, 15 mg (0.07 mmol) o P1 and 1.96 mg (0.00525 mmol) P (COD)Cl2 we e
dissol ed in 15 mL o DMF and he solu ion was exposed o UV ligh i adia ion (365 nm)
o 1 h. Subsequen ly, he eac ion was quenched by he addi ion o liquid ni ogen. The
esul ing P (II)-SCNPs we e p ecipi a ed wi h die hyl e he and d ied unde dynamic
acuum. Yield: 86%. P (II) con en (ICP-MS): 0.13 mol%. Mw (SEC) = 78.4 kDa, Ɖ (SEC) =
1.15, Rh (DLS) = 9.9 nm.
5.4.5. Syn hesis o P (II)/Cu(II)-SCNPs
P1 (15 mg, 0.07 mmol) and P (COD)Cl2 (1.96 mg, 0.00525 mmol) we e dissol ed in 15 mL
o DMF and he solu ion was exposed o UV ligh i adia ion (365 nm) o 1 h. The
eac ion was p omp ly quenched by he addi ion o liquid ni ogen. A concen a ed
solu ion o Cu(OAc)2 in DMF (0.954 mg, 0.00525 mmol o Cu) was hen added d opwise
and he eac ion mix u e was s i ed o 24 h. The esul ing P (II)/Cu(II)-SCNPs we e
p ecipi a ed wi h die hyl e he and d ied unde dynamic acuum. Yield: 79%. P (II)
con en (ICP-MS): 0.14 mol%. Cu(II) con en (ICP-MS): 0.43 mol%. Mw (SEC) = 81.6 kDa,
Ɖ (SEC) = 1.18, Rh (DLS) = 8.6 nm.
5. Consecu i e One-Po Alkyne Semihyd ogena ion/Alkene Dioxygena ion Reac ions by P (II)/Cu(II)
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151
Table 4. Induc i ely coupled plasma-mass spec ome y (ICP-MS) esul s o P (II)-SCNPs,
Cu(II)SCNPs and P (II)/Cu(II)-SCNPs
Sample
P (II) con en (g/mg)
Cu(II) con en (g/mg)
P (II)-SCNPs
9.29
-
Cu(II)-SCNPs
-
9.11
P (II)/Cu(II)-SCNPs
9.73
9.54
5.4.6. Syn hesis o Cu(II)-SCNPs (as a con ol).
P1-SCNPs (15 mg, 0.07 mmol) was dissol ed in DMF (15 ml) a . . A solu ion o Cu(OAc)2
in DMF (0.954 mg, 0.00525 mmol) was hen added d opwise, and he eac ion mix u e
was s i ed o 24 h. The esul ing Cu(II)-SCNPs we e p ecipi a ed wi h die hyl e he and
d ied unde dynamic acuum. Yield: 95%. Cu(II) con en (ICP-MS): 0.41 mol%. Mw (SEC)
= 77.1 kDa, Ɖ (SEC) = 1.21, Rh (DLS) = 10.0 nm.
5.4.7. Gene al p ocedu e o he consecu i e one-po alkyne
semihyd ogena ion/alkene dioxygena ion eac ions ca alysed by
P (II)/Cu(II)-SCNPs in NBP a . .
In a 10 mL ound bo omed, o en-d ied lask, which was p e iously equipped o
magne ic s i ba , 2 mL o P (II)/Cu(II)-SCNPs solu ion in NBP ([polyme ] = 1 mg mL-1)
we e added, ollowed by 0.07 mmol o he alkyne compound (1a-1e) and he sys em was
sealed wi h ubbe sep um. The solu ion was hen degassed by pu ging a gon low o 5
min. A e displacemen o a mosphe ic oxygen, he lask was equipped o a secu i y
elas ic balloon o ensu e bo h no ex a-p essu e in he sys em and, simul aneously, o
educe ola ili y o sol en s and subs a es. A low-p essu e molecula hyd ogen low was
hen bubbled in he eac ion mix u e a . . a de ini e in e al o imes (10 min bubbling
each 20 min un il eac ion comple ion). The p og ess o he eac ion was moni o ed by
hin laye ch oma og aphy (TLC) using E OAc and Hex as eluen s. A e he
semihyd ogena ion eac ion was comple e, 13.68 mg (0.084 mmol) o N-
hyd oxyph halimide was added o he solu ion a . . and unde ai (open lask). The
5. Consecu i e One-Po Alkyne Semihyd ogena ion/Alkene Dioxygena ion Reac ions by P (II)/Cu(II)
Single-Chain Nanopa icles in G een Sol en
152
p og ess o he eac ion was moni o ed by TLC using E OAc and n-hexane as eluen s.
A e eac ion comple ion, 10 mL o wa e was added o he solu ion. The esul an
mix u e was ex ac ed wi h E OAc (3 x 5 mL) and successi ely washed wi h wa e (3 x 5
mL). The o ganic solu ion was d ied o e anhyd ous Mg2SO4, il e ed and e apo a ed o
gi e a esidue ha was pu i ied on silica gel column ch oma og aphy using Hex and
E OAc as eluen s o a o d he co esponding β-Ke o-N-alkoxyph halimide p oduc
(3a3e).
5.5. Resul s and discussion
The syn he ic p ocedu e ollowed in his wo k o p oduce P (II)/Cu(II)-SCNPs is depic ed
schema ically in Figu e 74. Ini ially, he monome s (2-ace oace oxy)e hyl me hac yla e
(AEMA) and me hyl me hac yla e (MMA), which unc ions as a space , we e
copolyme ized ia e e sible addi ion agmen a ion chain- ans e (RAFT)
polyme iza ion5 yielding he andom copolyme P0 (Figu e 74A). Subsequen ly, P0 wi h
a con en o β-ke oes e unc ional g oups o 30 mol%, a weigh -a e age molecula
weigh o Mw =73.6 kDa and a na ow dispe si y o Ɖ = 1.17 was deco a ed wi h 19 mol%
o α-diazo-β-ke oes e unc ional g oups -using p-ca boxybenzenesul onazide (p-CBSA)
as he diazo ans e eagen -23 lea ing 11 mol% β-ke oes e unc ional g oups un eac ed
(see Figu e 74B).
5. Consecu i e One-Po Alkyne Semihyd ogena ion/Alkene Dioxygena ion Reac ions by P (II)/Cu(II)
Single-Chain Nanopa icles in G een Sol en
153
Figu e 74A. P epa a ion o a andom copolyme P0 con aining naked β-ke oes e unc ional
g oups (30 mol%) ia e e sible addi ion agmen a ion chain- ans e (RAFT) polyme iza ion
(MMA = me hyl me hac yla e; AEMA = (2ace oace oxy)e hyl me hac yla e; AIBN =
azobisisobu y oni ile; CPDB = 2-cyanop op-2-yldi hiobenzoa e; E OAc = e hyl ace a e).
Figu e 74B. Deco a ion o P0 wi h α-diazo-β-ke oes e unc ional g oups (19 mol%) o gi e
polyme ic p ecu so P1 (p-CBSA = p-ca boxybenzenesul onazide; E 3N = ime hylamine; CH2Cl2 =
dichlo ome hane).