Ti le: A linea -polyme -based lac o e in- selec i e ecogni ion elemen o
an ELISA mimic: A P oo o concep
Au ho s: M.A. Goicolea a*, A. Gómez-Caballe o a, M. Saumell-Esnaola b, d, G. Ga cía del
Caño c, d, N. Unce a a, J. Sallés b, d. e and R.J. Ba io a.
a Depa men o Analy ical Chemis y. Facul y o Pha macy, Uni e si y o he Basque
Coun y (UPV/EHU), 01006 Vi o ia-Gas eiz, Spain.
b Depa men o Pha macology. Facul y o Pha macy, Uni e si y o he Basque Coun y
(UPV/EHU), 01006 Vi o ia-Gas eiz, Spain.
c Depa men o Neu osciences. Facul y o Pha macy, Uni e si y o he Basque Coun y
(UPV/EHU), 01006 Vi o ia-Gas eiz, Spain.
d Ins i u o de In es igación Sani a ia Bioa aba, Neu o a macología Celula y Molecula ,
01008 Vi o ia-Gas eiz, Spain.
e Cen o de In es igación Biomédica en Red de Salud Men al (CIBERSAM), 28029. Mad id,
Spain.
*Co esponding au ho .
E-mail add esses: ma iaa [email protected]
Highligh s
A linea polyme o he selec i e ecogni ion o lac o e in is p esen ed.
The linea polyme wo ks as a plas ic an ibody in enzyme-linked immunoso ben assays.
Assay pe o mance o lac o e in was es edin he p esence o o he p o eins.
This is he accep ed manusc ip o he a icle ha appea ed in inal o m in Analy ica Chimica Ac a 1191 : (2022) //
A icle ID 339309, which has been published in inal o m a h ps://doi.o g/10.1016/j.aca.2021.339309. © 2021 Else ie
unde CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/)
Abs ac
The syn hesis o polyme s wi h ailo ed p ope ies o he ecogni ion o mac omolecules such
as p o eins is challenging. In his wo k, he syn hesis o a new polyme o ma , a linea polyme
(LP), as he selec i e ecogni ion elemen o he globula p o ein lac o e in (LF) is p oposed
as a p oo -o -concep s udy. Fo he syn hesis, a solid-phase s a egy using he e e sible
deac i a ion adical polyme isa ion (RDRP) mechanism is p oposed. This app oach, which is
usually used in molecula imp in ing, in ol es he immobilisa ion o LF on he su ace o a
solid suppo , bu , unlike classical imp in ing, a c oss-linke in he polyme isa ion mix u e is
no equi ed. Consequen ly, he copolyme is soluble and lexible, hus o e coming he
d awbacks associa ed wi h adi ional syn he ic polyme s o mac omolecule imp in ing. This
new polyme o ma has g ea po en ial o eplacing na u al an ibodies in bioassays such as
enzyme-linked immunoso ben assays (ELISA), do blo , wes e n blo , o pull-down. In ou
case, he linea polyme was used as a ecogni ion elemen o eplace na u al an ibodies in a
LF-selec i e ELISA. The esponses o he linea polyme be ween LF concen a ions o 0.1 nM
and 0.25 µM we e s udied, and a signi ican di e ence was obse ed be ween he non-speci ic
signals and he signals measu ed in he p esence o he polyme ic ma e ial. Fu he , he esponse
e sus log concen a ion cu es we e i ed o a logis ic equa ion, allowing es ima ion o he
EC50 alue: 11.8 ± 1.4 nM. We also con i med he selec i e de ec ion o LF using he
compe i i e inhibi ion o he selec i e LF-bio in conjuga e (LF-Bi) binding o he plas ic
ecep o (LP) o closely ela ed p o eins (e.g. hose ha ing simila molecula weigh s o
isoelec ic poin s) such as human lysozyme, ypsin, and albumin, which a e p esen in human
body luids. The sys em p esen s a c oss- eac i i y alue o selec i i y o 1.95% o lysozyme,
0.028% o ypsin, and 0.016% o albumin. The applicabili y o his me hod o he
de e mina ion o u ine LF le els in in lamma o y and in ec ious diseases o he human u ina y
ac is also demons a ed.
Keywo ds
Linea polyme , e e sible deac i a ion adical polyme isa ion, plas ic an ibody, lac o e in,
ELISA mimic, u ine sample
1. In oduc ion
Molecula ecogni ion is a undamen al e en in biochemical p ocesses in which a
ecep o ecognises and iden i ies one chemical species h ough s uc u ally well-de ined
molecula in e ac ions. The mos s udied molecula ecogni ion sys em is he immune sys em
o highe o ganisms in he o m o an igen–an ibody ecogni ion. This ecogni ion is he basis
o nume ous bioassays, including he enzyme-linked immunoso ben assay (ELISA), as well as
do -blo , wes e n blo ing, and immunop ecipi a ion assays.
These ecogni ion phenomena a e c ucial in biological sys ems, and much mode n
chemical esea ch is mo i a ed by he p ospec ha molecula ecogni ion by design could lead
o new echnologies o he eplacemen o na u al ecep o s by mo e du able and obus
syn he ic s uc u es[1,2].
To add ess his challenge, many e o s ha e been made o design, syn hesise, and es
a i icial ma e ials wi h biomime ic p ope ies o applica ions in binding assays.
Sup amolecula hos –gues in e ac ions ep esen one o he ea lies non-na u al molecula
ecogni ion sys ems. Howe e , his now- adi ional ield o chemis y is ela i ely limi ed in
e ms o he numbe and unc ion o po en ial ecep o s. Wi h he inc easing demand o
mul i unc ional ad anced ma e ials, he molecula imp in ing echnique has ecei ed much
a en ion as a s a egy o inco po a e speci ic molecula ecogni ion si es in o polyme ic
s uc u es, leading o a i icial polyme ic ecep o s ha mimic he mechanism o na u al
ecep o s [3]. Fu he , in p inciple, a ional molecula design and chemical syn hesis allow he
gene a ion o a o dable ma e ials ha a e easy o ab ica e and use and ha e high selec i i y
and obus ness[4]. Acco dingly, molecula ly imp in ed polyme s ha e been p oposed in ecen
yea s as subs i u es o na u al an ibodies in immunoassays[5-8].
In adi ional molecula imp in ing wi h small molecules, o p ese e he ca i ies c ea ed
du ing polyme isa ion, he imp in ed polyme s end o be e y dense, and he p esence o a
c oss-linking monome makes i possible o ob ain well-de ined binding ca i ies while
main aining ecogni ion.
To da e, mac omolecule imp in ing echnology has no ye eached i s ue po en ial
because o he inhe en need o pe o m imp in ing in aqueous media. Mo eo e ,
mac omolecula s uc u es, such as pep ides and p o eins, can exis in a mul i ude o
con o ma ions, leading o he de elopmen o he e ogeneous binding si es as opposed o well-
de ined ca i ies du ing he imp in ing o small o ganic molecules. In addi ion, he la ge size o
mac omolecules can educe he e ec i eness o ex ac ion om he polyme ma ix and
subsequen binding. Al hough some solu ions o hese p oblems in p o ein imp in ing
echniques ha e been published [9-11] se e al d awbacks emain.
The main p oblem acing he imp in ing o mac omolecules is he high deg ee o c oss-
linking equi ed o achie e ecogni ion. As c oss-linking inc eases, la ge empla es, such as
p o eins, can become apped in he ne wo k a e polyme isa ion. I he empla e molecule
canno be ex ac ed, he ne wo k is useless o ecogni ion applica ions. In addi ion, a high
deg ee o c oss-linking can dec ease he di usion o hese molecules in he ne wo k, esul ing
in slow ecogni ion kine ics. Consequen ly, mos esea ch on p o ein imp in ing has ocused on
he p oduc ion o ma e ials using su ace molecula imp in ing echniques [12]. In his s a egy,
he ca i ies ha e special shapes o s e ic e ec s ha a e complemen a y o pa s o agmen s
o he a ge p o ein. As a esul , mass ans e is easie and binding kine ics a e less es ic ed,
al hough possibly lowe , and selec i i y may also dec ease because only pa o he p o ein is
bound o ecognised. C ucially, he s abili y o he con o ma ion o mac omolecules du ing he
imp in ing p ocess mus be gua an eed, so unc ional monome s and c oss-linke s ha main ain
he ac i i y o he p o eins mus be selec ed [13].
Some au ho s ha e p oposed he use o p o ein–polyme hyb id s uc u es o achie e
con ol o he nanos uc u e and o ien a ion o he p o eins while main aining hei s abili y and
ac i i y. Fo example, he conjuga ion o syn he ic copolyme s o p o eins can con e bene icial
p ope ies such as ailo ed amphiphilici y, no el sel -assembly, and phase sepa a ion beha iou
o he esul ing hyb ids [14,15]. Copolyme s a e capable o complexing p o eins wi h
e iciencies depending on he copolyme composi ion and molecula a chi ec u e. These
complexes a e ypically assembled using linea copolyme s [16].
In he syn hesis o polyme s o he up ake o biomac omolecules, he deg ee o lexibili y
o he polyme mus be conside ed. Scha ade e al. [17] e ealed ha linea copolyme s ha ing
special unc ions and componen s could exhibi high binding p ope ies and selec i i y o
biomolecules. They demons a ed ha , o selec i e p o ein ecogni ion, a p e-es ablished
binding si e is no a equi emen as long as he e is a su icien numbe o in e ac ions be ween
he unc ional g oups o he polyme and he amino acid esidues on he p o ein su ace. In his
case, he ecogni ion esul s om induced i .
Speci ically, he copolyme adap s i s shape acco ding o he opology o he p o ein
su ace. In pa icula , linea copolyme s a e much simple and su e om less s e ic hind ance
han h ee-dimensional polyme ic s uc u es; he e o e, he amoun o immobilised p o ein can
be maximised. Typically, he polyme isa ion mix u e is composed o a se o excess monome s
wi h a la ge a ie y o unc ional g oups. Thus, he expense depends on he di e en amino acid
esidues ha p edomina e in he p o ein s uc u e. As a esul , lexible soluble polyme s lacking
a bulky h ee-dimensional s uc u e a e ob ained, and hese ha e p o en ad an ageous o he
de elopmen o new biochemical ools and bioassays [18,19].
C ucially, he linea copolyme s used o molecula ecogni ion a e no imp in ed, and
ecogni ion is en i ely he esul o induced i , which enhances he a ou able in e ac ions
be ween he polyme unc ionali y and amino acid esidues on he p o ein su ace. This
app oach is concep ually iden ical o he heo ies pos ula ed o accoun o he unlimi ed binding
di e si y o an ibodies. Tha is, he concep o induced i elaxes he equi emen o an exac
p e-exis ing i be ween he an ibody and an igen [20].
Simila ly, i has been shown ha andom he e opolyme s composed o monome s ha ing
chemical p ope ies designed o in e ac wi h he species p esen on he p o ein su ace adop
local con o ma ions ha maximise he a ou able in e ac ions wi h he p o ein, aking
ad an age o he con o ma ional lexibili y and designed complemen a i y in he polyme
backbone [21,22]. Molecula ecogni ion occu s when wo molecules a e bo h geome ically
and chemically complemen a y; ha is, when hey can bo h “ i oge he ” spa ially, as well as
bind o each o he using non-co alen o ces, including hyd ogen bonds, elec os a ic
in e ac ions, o hyd ophobic in e ac ions [14]. The ecogni ion abili y inc eased wi h he
numbe o di e en ypes o monome uni s and complemen a y adso p ion si es o he p o ein.
Thus, i could be said ha he he e opolyme is uned o amino acid esidues on he su ace o
he p o ein [23].
In his s udy, a lexible linea polyme o selec i e binding o a whole p o ein was
de eloped. As in he wo k o Scha ade [17], he s a is ical copolyme isa ion o monome s
complemen a y o he mos cha ac e is ic su ace esidues o he p o ein is p oposed, and his
esul s in a lexible linea polyme capable o adap ing o he opology o he a ge p o ein:
lac o e in (LF).
The aim o his wo k was o inc ease he size and lexibili y o he polyme o acili a e
p o ein binding, aking in o accoun he mul iple in e con e ing con o ma ions o he p o ein.
In his con ex , we desc ibe he de elopmen , cha ac e isa ion, and biological es ing o soluble
linea copolyme s (LPs) designed o LF. To alida e hese ma e ials as subs i u es o
an ibodies in diagnos ic es s, hei beha iou was s udied using an ELISA mimic.
Thus, as a p oo o concep , he syn hesis o a new LP is p oposed. In his s udy, he
monome polyme isa ion was ca ied ou in he p esence o a empla e molecule, bu , unlike
adi ional polyme isa ion o he gene a ion o a molecula ly imp in ed polyme s, no c oss-
linke was equi ed o main ain he speci ic ecogni ion. This esul s in soluble copolyme s
consis ing o ca bon backbones ha ing unc ional side chains ha a e andomly dis ibu ed as
a esul o he s a is ical polyme isa ion o he selec ed unc ional monome s. Tha is,
copolyme s a e o med in which he sequen ial dis ibu ion o he monome ic uni obeys
s a is ical laws, and he p ocesses leading o he o ma ion o a s a is ical sequence o
monome ic uni s do no occu wi h equal p obabili y.
C ucially, he linea and lexible polyme can adap i s shape o he la and ough su ace
opologies o he a ge p o ein. As discussed abo e, a key o his me hod is he op imisa ion o
he selec ion and p opo ion o he di e en unc ional monome s, which mus be selec ed o
hei a ou able in e ac ions wi h he a ge molecule [3]. In his s udy, a mix u e o di e en
ac ylamide monome de i a i es wi h a ious unc ionali ies was used o p epa e he LP and
a oid he o ma ion o homopolyme s, which would be incapable o selec i e binding. We
hypo hesised ha , o ob ain good a ini y and selec i i y, he sequence o monome s along he
polyme backbone should be di ec ed by p e‐polyme isa ion d i en by sel -assembly a ound
he empla e. To achie e his, polyme isa ion was conduc ed ollowing a solid-phase syn hesis
s a egy o he syn hesis o imp in ed ma e ials in which LF was immobilised on he su ace
o glass beads, which ac ed as he solid phase [24,25]. These we e hen placed in con ac wi h
he aqueous monome mix u e, and e e sible deac i a ion adical polyme isa ion (RDRP) was
ini ia ed in he p esence o an ini e e . This echnique o e s g ea con ol o e he
polyme isa ion p ocess, allowing he syn hesis o polyme s wi h a con olled molecula weigh
dis ibu ion and a mo e homogeneous dis ibu ion o binding si es compa ed o con en ional
adical polyme isa ion. In addi ion, RDRP enables he p oduc ion o ad anced ma e ials
con aining la en eac i a able species ha can be used o u he p ocessing in he pos -
syn he ic phase [19,21,26].
2. Expe imen al
2.1. Ma e ials and eagen s
Human LF (90%), human lysozyme (≥ 100,000 uni s/mg), po cine panc ea ic ypsin,
a y acid- ee bo ine se um albumin (BSA, 99%), ac ylamide (AAm, 99%), N- e -bu yl
ac ylamide (TBAm, 97%), ac ylic acid (AAc, 99%), N-(3-aminop opyl) me hac ylamide
hyd ochlo ide (APMA, 98%), 3-aminop opyl ie hoxysilane (APTES, 99%), 1,2-
bis( ie hoxysilyl) e hane (BTESE, 95%), glu a aldehyde (GA, 50% w/ , e hanolamine
(99.5%), dansyl chlo ide (99%), bio in N-hyd oxysuccinimide es e (bio in-NHS) (98%), glass
beads (GB, 150 µm < d < 212 µm), and sil e colloidal dispe sion (60 nm, 0,02 mg mL-1) we e
pu chased om Sigma–Ald ich (Spain). Sodium hyd ogen phospha e, sodium dihyd ogen
phospha e, sodium hyd oxide, poly inylpy olidone, Amicon Ul a-15 cen i ugal il e uni s
(3 and 30 KDa), and BCA p o ein assay ki we e pu chased om Me ck (Spain). Sodium
chlo ide, sodium ni a e, d y oluene, dime hyl sulphoxide (DMSO, 99.5%), and hyd ochlo ic
acid (37%) we e acqui ed om Pan eac (Spain). Pie ce® Bio in Quan i a ion Ki , pe oxidase-
a idin conjuga e (HRP-a idin), 3,3',5,5'- e ame hylbenzidine (TMB), Tween 20 su ac an , 96-
well Cos a 3590 polys y ene mic opla es, and ni ocellulose memb anes (200-nm po e size)
o use as subs a e o su ace-enhanced Raman spec oscopy (SERS) we e pu chased om
The mo Fishe Scien i ic (Spain). Liquid ch oma og aphy–mass spec ome y g ade and high-
pe o mance liquid ch oma og aphy (HPLC) g ade e hanol we e ob ained om Schalab S.L.
(Spain). Deu e a ed wa e o NMR was ob ained om Eu iso op (F ance).
The calib a ion o he gel pe mea ion ch oma og aphy/ e ac i e index (GPC/RI) sys em
was ca ied ou using EasiVial polye hylene glycol (PEG)/polye hylene oxide (PEO) s anda ds
complian wi h he ISO9001:2008 ce i ica ion and supplied by Agilen Technologies (Spain).
All chemicals and sol en s we e o analy ical o HPLC g ade and we e used wi hou
u he pu i ica ion. Each bu e solu ion was p epa ed wi h ul apu e wa e ob ained om
Elix20 e e se osmosis and Milli-Q wa e pu i ica ion sys ems.
2.2. Equipmen
The silanisa ion o he solid suppo was e i ied by measu ing he luo escence a 365
nm using a Spec onics CM-26A cabine equipped wi h a model ENF-260C lamp (USA).
The immobilisa ion o he p o ein on he solid suppo was con i med by spec oscopic
measu emen s using a 1-m long, 10-mm op ical pa h leng h ib e op ic p obe coupled o an
Agilen Technologies Ca y 60 UV-Vis spec opho ome e . Ca y WinUV so wa e ( e sion 5.0)
was used o da a collec ion, s o age, analysis, and display.
Fo UV-ligh -induced polyme isa ion, a Summe Glow HB 175 lamp om Hap o (The
Ne he lands) was used.
The p econcen a ion o he polyme ic ma e ial was pe o med by e apo a ion and
cen i uga ion using a e ige a ed cen i uge (Alleg a X-15R, Beckman Coul e , USA) and a
Hei-VAP Ad an age o a y e apo a o (Ge many).
Emp y 60-mL p opylene ca idges pu chased om Supelco (Spain) we e used o solid-
phase ex ac ion (SPE) es s, and p ocess con ol was ca ied ou in a acuum mani old
connec ed o a acuum pump o p essu e con ol (Va ian, CA, USA).
Fo he polyme cha ac e isa ion, a 1260 In ini y gel il a ion ch oma og aphy sys em
wi h a 20 G1362A RI de ec o o concen a ion de e mina ion and mul i-angle (15° and 90°)
ligh sca e ing de ec o o molecula weigh de e mina ion om Agilen Technologies was
used. Fo he sepa a ion o he di e en ac ions o he polyme ic ma e ials, a PolySep-GFC-
P-4000 column om Phenomenex (To ance, Cali o nia, USA) ha ing a size ange o 3,000–
400,000 Da and 0.1 M NaNO3 as he mobile phase was used.
1H-NMR spec a we e acqui ed in a B uke A ance 400 spec ome e ope a ing a 400
MHz, and he spec a we e p ocessed using MNo a p og am (Mes elab Resea ch, Spain).
The SERS measu emen s we e pe o med using a Ho iba XploRa Raman mic oscope
(Jobin-Y on, Ho iba G oup, New Je sey, USA) equipped wi h lase s ha ing wa eleng hs o
532, 638, and 785 nm. Spec al da a we e ea ed using he OMNIC 7.2 Spec a So wa e
(The mo Scien i ic, Wal ham, MA, USA).
The ELISA es s we e conduc ed wi h empe a u e con ol and shaking using a Selec a
model o en (Selec a, Spain) and an IKA VIBRAX VXR o bi al shake (Scha lab, Spain). Signal
measu emen s we e ca ied ou on an abso bance mic opla e eade (Sun ise, Tecan Li e
Sciences, Spain). Da a acquisi ion was pe o med using Rd Ole4 e sion 4.5.1. Fi ings o he
binding kine ic, sa u a ion, and compe i i e assay esul s we e pe o med using PRISM®
(G aphPad e sion 5.01 So wa e Inc., San Diego, CA).
2.3. Syn hesis o he linea copolyme (LP)
The syn he ic s a egy o he gene a ion o he LP was based on he solid-phase app oach
using a pho oac i a ed e e sible deac i a ion adical polyme isa ion mechanism in he
p esence o an ini e e (pho o RDRP).
2.3.1 P epa a ion o LF-modi ied glass beads
Be o e copolyme syn hesis, immobilisa ion o LF on he solid suppo (glass beads) was
ca ied ou . The i s s age in ol ed he ac i a ion and unc ionalisa ion o he glass beads. Fo
his, 120 g o glass beads was suspended in 250 mL o 4 M NaOH solu ion. A e boiling he
mix u e o 30 min, he glass beads we e il e ed h ough a 50-µm sie e and washed wi h
ul apu e wa e un il he wash wa e eached pH o 7. The beads we e hen washed wi h
me hanol and allowed o d y o e nigh in an o en a 100 °C. Once d y, he glass beads we e
silanised by imme sing hem in 100 mL o 95:5 ( / ) e hanol:wa e solu ion acidi ied wi h 1
mL o ace ic acid and hea ed o 70 °C. Immedia ely a e , 3 mL o APTES and 0.5 mL o he
dipodal silanise BTESE (10:1 mola a io o APTES: BTESE) we e added, and he mix u e
was le o eac o e nigh a oom empe a u e. The ea e , he silanised glass beads we e
il e ed, washed wi h me hanol and ace one, and d ied in a desicca o unde acuum. Finally,
o ensu e comple e wa e emo al, he samples we e placed in an o en a 150 C o 1h. To
de e mine he deg ee o APTES g a ing, he ninhyd in es was pe o med using 1 mg o
silanised glass beads and a 2% ninhyd in solu ion om Me ck (Spain), e ealing a g a ing
deg ee o 438.4 ± 6.7 µg-APTES/g-glass beads.
Fo LF immobilisa ion on he su ace o he ac i a ed and unc ionalised solid suppo ,
30 g o glass beads we e placed in a ound-bo omed lask wi h 100 mL o 0.1 M phospha e
bu e (pH = 7.4) con aining 0.1 M NaCl (phospha e bu e saline, PBS) and GA (5%, / ). To
ensu e an oxygen- ee a mosphe e, he mix u e was subjec ed o epea ed N2 pu ge– acuum
cycles using a Schlenk line. Subsequen ly, he mix u e was allowed o eac o 2 h in he da k.
Subsequen ly, he glass beads we e il e ed and washed wi h ul apu e wa e . They we e hen
ans e ed o a wo-necked lask wi h 25 mL o PBS, and LF (12.5 mg) was added. The mix u e
was again subjec ed o N2 pu ge– acuum cycling and kep in he da k o 2 h. Once he LF had
been immobilised, 12 µL o e hanolamine was added o he mix u e o 15 min o cap any
un eac ed aldehyde- e minal g oups ha had no eac ed wi h LF. Finally, he glass beads we e
il e ed and insed wi h Milli-Q wa e .
2.3.2. Syn hesis and cha ac e isa ion o he wa e -soluble ini e e 2-((die hylca bamoyl) hio)
ace ic acid.
Ini e e s beha e sequen ially as ini ia o s, ans e agen s, and e mina o s in he adical
polyme isa ion p ocess. Howe e , examples o he ini e e -induced polyme isa ion o
biomolecules a e sca ce because o hei poo wa e solubili y [27–29].
In ou case, he wa e -soluble ini e e was p epa ed ollowing a p ocedu e adap ed om
ha o Xu e al. [30]. B ie ly, in a wo-necked ound-bo omed lask, 5 mmol o sodium
die hyldi hioca bama e ihyd a e (1.265 mg) dissol ed in 50 mL o wa e was added and
deoxygena ed wi h high-pu i y ni ogen. Nex , 5 mmol o sodium chlo oace a e (98%, 582.5
mg) dissol ed in 50 mL o wa e was added d opwise, and he mix u e was kep unde a ni ogen
a mosphe e o 48 h. The esul ing p oduc was il e ed o emo e he insoluble sal s. Then, 1
mL o hyd ochlo ic acid (37%) was added o he solu ion o p ecipi a e he ini e e . The
esul ing p oduc was il e ed, edissol ed in ace one, and he sol en was e apo a ed wi h
ni ogen o c ys allisa ion. A e d ying a 45 °C, he c ys als we e s o ed a 4 °C in he da k.
The syn hesised ini e e was cha ac e ised by quad upole ime-o - ligh mass
spec ome y (QTOF-MS). The measu emen s we e acqui ed using an Agilen 6530 hyb id
in he cha ac e isa ion o he polyme ic ma e ial by GPC wi h RI and mul i-angle ligh
sca e ing de ec ion.
Ini ially, he monome concen a ion was se as desc ibed in Sec ion 2.3.3 (deno ed ×1),
and he in luence o he UV ac i a ion ime was s udied in he ange o 5–90 min. The syn hesis
ime was se o 45 min (15 min × 3 imes, he mix u e was main ained in an ice ba h) because
he polyme a e age molecula weigh did no inc ease wi h longe eac ion imes (Fig. 1a). In
addi ion, an i adia ion ime o 45 min was se , and he in luence o he monome con en in he
polyme isa ion mix u e was s udied. Fo all he composi ions es ed (×1, ×2, ×5, and ×10), an
inc ease in he a e age molecula weigh (Mw 7,749–37,741) was obse ed as he monome
con en in he polyme isa ion mix u e inc eased (Fig.1b).
The ×5 mix u e con aining AAc (11 mg), APMA (27.5 mg), TBAm (165 mg), and AAm
(122.5 mg) was selec ed because he highe concen a ion (×10) esul ed in no signi ican
inc ease in polyme molecula weigh and an inc ease in polyme he e ogenei y was obse ed.
The a e age molecula weigh o his monome mix u e was 34,277 ± 3,219. Rega ding
polyme homogenei y, he polydispe si y alue o 1.12 ± 0.08 is close o one, as expec ed o a
e e sible deac i a ion adical polyme isa ion because he molecula weigh and con e sion
inc ease wi h inc easing monome concen a ion a he expense o polydispe si y [35].
The concen a ion o polyme p esen in he syn hesis s ock solu ion was de e mined o
be 0.091 ± 0.004 mg·mL–1 based on a e ac i e index alue (dn/dc) 0.165 mL g-1 [36]. In
addi ion, om he esul s ob ained om he mul i-angle ligh sca e ing de ec o , he a e age
adius o gy a ion (Rg = 79 ± 2 nm) and b anching o he polyme we e de e mined by
calcula ing he adius o gy a ion con ac ion ac o gMw = Rg (b anched)/Rg (linea ) = 0.92 ±
0.03. The polyme adius o gy a ion desc ibes he dimensions o he polyme chain. When a
polyme 's adius o gy a ion is g ea e han 10 nm, i is conside ed o be a long polyme . On he
o he hand, om he alues o gMw close o one ( alue o linea polyme s) and conside ing he
b anched- e na y weigh a e age model, which indica es a single b anch poin o he backbone,
he a e age numbe o b anches (BnMw = 0.55 ± 0.05) was calcula ed, assuming a andom
dis ibu ion o b anches in he polyme . The lowe he gMw alue, he highe is he deg ee o
b anching. F om hese esul s, we can conclude ha i is a homogeneous linea polyme wi h
low b anching.
The LP was also cha ac e ised by SERS measu emen s, and di e en SERS spec a
co esponding o di e en LP bands we e ob ained using a con ocal Raman spec ome e . A
532-nm lase wi h a powe o 0.05 W (5 mW) was used o SERS exci a ion a he sample
posi ion. A mic oscope a achmen wi h a 100× objec i e was used o ocus he lase beam on o
a spo o 1 µm in diame e . The in eg a ion imes a ied be ween 2 and 10 s wi h 10
accumula ions in he spec al ange o 100–3,200 cm-1. The spec al esolu ion was 2 cm-1. In
addi ion a e es ing wi h di e en ma e ials, we ound ha a ni ocellulose (NC) memb ane
was a good subs a e o SERS because i o e s a low backg ound, minimal p epa a ion, and
op imal posi ion o Raman bands. The NC memb ane wi h a 200-nm po e size allows he
cap u e o subnanog am quan i ies o analy e and concen a es hem in a small a ea om applied
olumes o 0.5 µL. The subsequen s aining p ocess wi h colloidal sil e nanopa icles (60 nm,
0.08 mg mL-1) allowed he gene a ion o local “ho spo s” showing SERS enhancemen s. To
achie e he agg ega ion o he sil e colloid, he eagen suspension was p e-concen a ed by
cen i uga ion a 30,000g o 30 min. This s aining p ocess was pe o med by incuba ing he
sample-loaded ni ocellulose memb ane wi h he p econcen a ed sil e colloid o 2 h.
Fig. 2 shows a compa ison o he SERS spec a o he monome s used in he
polyme isa ion (Fig. 2a) and he LP (Fig. 2b). Fig. 2b shows wo spec a o he LP ob ained
om di e en a eas o he sample. The di e ence obse ed be ween he spec a may be due o
he polyme being adso bed on he su ace in di e en o ien a ions.
In he LP spec um (Fig. 2b-A), an in ense signal a 1,597 cm-1 can be obse ed, and a
band is also p esen in he spec um o he ac ylamide monome (Figu e 2a-B), which o ms
he backbone o he LP. When ac ylamide polyme isa ion occu s, he C=C g oup is ans o med
in o a single bond, bu he p ima y amide g oup is main ained (band a 1,597 cm-1 Fig. 2b-A).
In he spec a o N- e -bu yl ac ylamide (Fig. 2a-C) and N-(3-aminop opyl)
me hac ylamide (Fig. 2a-D), a double signal was obse ed a 1,542 and 1,575 cm-1 and 1,525
and 1,560 cm-1 espec i ely, ela i es o he seconda y amide p esen in he s uc u e o bo h
monome s. A e polyme isa ion, his band emained in he LP spec um (Fig. 2b-A) bu
o e lapped wi h he signal o he p ima y amide a ising om he ac ylamide.
In he spec um o LP (Fig. 2b-A and 2b-B), a band a 1,431 cm-1 was obse ed,
a ibu able o he δC-H and δC-C de o ma ions o he e -bu yl g oups. The signals be ween
and 1,100 and 1,300 cm-1 p esen in he LP spec um (Figu es 2b-A and 2b-B) a e due o C-C
s e ching o n-alkanes, skele al ib a ion o he polyme (1,050–1,150), and 1,175–1,310 ωCH2
and δCH2 n-alkanes. The C-H s e ches also appea ed in he spec al egion be ween and 2,800–
3,000. A 2,900 cm-1, he e is a CH2 asymme ic s e ch, which could be b oadened because o
he o e ones o he δCH2 de o ma ion. The bands appea ing a 2,800–3000 a e due o C-H
s e ches and may be a ec ed by he eo ien a ion o he alkyl chains. Finally, he LP spec um
(Fig. 2b-B) shows a well-de ined band a 1,613 cm-1 a ising om he ca boxylic acid g oups o
he ac ylic acid monome (Fig. 2a-A). Thus, he cha ac e is ic SERS bands o he monome s
in ol ed in LP syn hesis a e also p esen in he SERS spec um o he copolyme .
Fig. 3 shows he 1H-NMR spec um o he LP. The s ong signal a 1.3 ppm can be
assigned o he p o ons o he e -bu yl g oup o igina ing om he N- e -bu yl ac ylamide
monome . The low in ensi y signals close o his co espond o he p o ons om he alkane
chain o med du ing polyme isa ion. The signal a 2.1 ppm is assignable o p o ons om alkanes
close o he ca bonyl g oups, whe eas he signal a 1.6 ppm a ises om p o ons in alkanes
u he away om he ca bonyl g oups o igina ing om he ac ylamide and ac ylic acid
monome s.
3.2. E alua ion o LP as a plas ic “an ibody” in he ELISA mimic
The appa en dissocia ion cons an o he in e ac ion be ween he labelled ligand (LF-Bi)
and he LP ecep o was de e mined using an ELISA mimic. Fo his pu pose, an empi ical
adjus men o he ELISA condi ions was pe o med o achie e he maximum selec i e binding
esponse be ween LF-Bi and he LP ecep o . To minimise expe imen al e o , all assays we e
pe o med in iplica e. The e o e, he signals ep esen ed a e he mean alues.
Fi s , a simple p ocedu e o he immobilisa ion o he linea polyme as an an ibody
subs i u e in a quan i a i e ELISA mimic was de eloped. B ie ly, a commonly used physical
adso p ion p ocedu e o he immobilisa ion o an ibodies on o polys y ene mic opla es by
hyd ophobic binding was used. A s able coa ing was achie ed by allowing he polyme solu ion
loaded in each well o e apo a e o d yness o e nigh a 37 °C in he da k. A e es ing he
in luence o he concen a ion o LP on he selec i e binding esponse signal, each well was
coa ed wi h 50 µL o a 0.0091 mg mL-1 solu ion o he LP (1:10 dilu ion o he s ock solu ion,
equi alen o 455 ng-LP/well).
The s abili y o he coa ing was con i med a e blocking wi h BSA (0.1% in 10 mM PB,
pH = 7.4) and se e al washing s eps (3 × 5 min) wi h Tween 20 (0.05% in 10 mM PB, pH =
7.4). Blocking e e s o he p e- ea men o he wells be o e he addi ion o he labelled p o ein
(LF-Bi) o educe non-speci ic binding and imp o e he signal- o-noise a io.
Poly inylpy olidone has also been es ed as a non-p o ein al e na i e o con en ional blocking
bu e . Howe e , he blocking was inadequa e p obably because poly inylpy olidone is
usually used o small p o eins ha could be masked by BSA; in ou case, he a ge p o ein
(LF) is a la ge p o ein han BSA.
The in luence o ionic s eng h on he signal was also es ed, and a 65% inc ease in non-
speci ic binding was obse ed when 0.1 M NaCl was added o he bu e ed medium. This
beha iou may be ela ed o he ac ha ionic s eng h induces a e e sal o he cha ge o LF
and esul s in agg ega e o ma ion [37]. The e o e, he assays we e pe o med in PB 10 mM
(pH 7.4).
Washing was applied a e he incuba ion pe iod o emo e non-bound conjuga es, so ha
he colo ime ic measu emen accu a ely e lec s he amoun o LF-Bi bound o he linea
polyme laye deposi ed in he well. A wash was also pe o med be ween he addi ion o he
di e en eagen s o emo e esidual ma e ial ha could cause c oss- eac i i y. The p oposed
concen a ion o Tween 20 (0.05%, 4.7 × 10-4 M) is highe han i s c i ical micella
concen a ion (4.88 × 10-5 M) which would p e en he adhesion o he p o eins o he
polys y ene [38]. Thus, o concen a ions highe han 0.05%, a dec ease in he selec i e signal
was obse ed.
A e he blocking s ep, he coa ed wells we e incuba ed wi h LF-Bi o 90 min a 37 °C
ollowed by washing and speci ic ecogni ion ia he high-a ini y binding o bio in o HRP-
a idin (100 µL o a 1:2,000 dilu ion o he eagen o 10 min). Subsequen ly, he sample was
washed, and he colou was de eloped by eac ion wi h TMB o 10 min (100 µL o eagen ).
The enzyma ic eac ion was s opped by he addi ion o 100 µL o H2SO4 0.5 M solu ion. The
uncoa ed wells we e ea ed in he same way as he con ols.
The measu emen o he cha ac e is ics o he in e ac ions be ween LF-Bi and LP was
achie ed using h ee ypes o selec i e esponse binding assays. The kine ic expe imen s
in ol ed he measu emen o he selec i e binding esponse o one o mo e concen a ions o
LF-Bi o he linea polyme a inc easing ime poin s. The analysis o a amily o associa ion
kine ics cu es allows he es ima ion o associa ion (kon) and dissocia ion (ko ) a e cons an s,
as desc ibed in Sec ion 2.4.2.1. We used ou concen a ions o LF-Bi (6, 10, 60, and 100 nM)
and measu ed he selec i e binding esponses o incuba ion imes up o 300 min. The i ing o
he indi idual selec i e esponse binding cu es using he app op ia e equa ion allowed he
es ima ion o he obse ed a e cons an (kobs) o each concen a ion o LF-Bi assayed (Fig.
4a). Then, om he linea eg ession i o he kobs alues e sus he LF-Bi concen a ion, he
equa ion kobs = 0.0002 × [LF-Bi] + 0.0040 (R2 = 0.9825) was ob ained (Fig. 4b). This allowed
he es ima ion o kon (slope) = 0.0002 min-1 nM-1, and ko (y-in e cep ) = 0.0040 min-1, and he
es ima ion o he appa en a ini y cons an Ka o 20 nM as he a io o ko /kon.
The selec i e LF-Bi binding esponse was measu ed o 13 inc easing concen a ions o
LF-Bi (0.1–250 nM), and he esul s a e plo ed as a unc ion o he loga i hm o he LF-Bi
concen a ion, and a cha ac e is ic sigmoidal cu e is shown in Fig. 5a. Fi ing his
concen a ion– esponse cu e wi h a logis ic equa ion allowed es ima ion o he loca ion
pa ame e o he cu e as EC50 alues and he co esponding Hill coe icien s (nH, slope
ac o s). The de i ed EC50 alues we e 11.8 ± 1.4 nM, and he nH alues we e 1.2 ± 0.3
(n = 10). The da a we e also analysed using an equa ion adap ed o he pa icula si ua ion o
he p esen wo k om he model o he Langmui iso he m o one-si e binding equa ion:
B = Bmax [L] / Ka + [L] (eq. 2)
whe e B and L ep esen bound and ee concen a ions o a ligand, Bmax is he maximum
binding, and Ka is he appa en equilib ium dissocia ion cons an . The de i ed Ka alues we e
8.4 ± 0.8 nM (n = 10). These esul s a e compa ible wi h he no ion ha LF-Bi ecognises a
homogeneous popula ion o independen si es on he syn he ic ecep o (i.e. LP). Fu he mo e,
hese esul s a e consis en wi h he Ka alue (20 nM) ob ained o LF-Bi in he kine ic
expe imen s.
Finally, in homologous compe i i e binding expe imen s, he selec i e binding o a ixed
concen a ion o LF-Bi can be measu ed a equilib ium in he p esence o inc easing
concen a ions o non-labelled LF. F om hese compe i i e inhibi ion cu es, i is possible o
de e mine he po ency o LF indi ec ly om he ob ained IC50 alues, ha is, he unlabelled
ligand concen a ion ha inhibi s 50% o he esponse o he labelled molecule, as well as he
simple o complex na u e o he inhibi ion mechanism h ough he ob ained nH alues (slope
ac o o Hill slope). Ou esul s show ha unlabelled LF ully inhibi ed he selec i e LF-Bi
esponse binding (a 10 nM, a concen a ion close o i s EC50 alue), and we ob ained a alue
o 12.1 ± 1.5 nM o he IC50 alue and nH alue o 1.00 (Fig. 5b). Again, he esul s a e
indica i e o he absence o a he e ogeneous popula ion o si es in he syn he ic LP ecep o
labelled by LF-Bi and a e consis en wi h he beha iou o a noncoope a i e syn he ic ecep o .
Fu he mo e, he IC50 alue o LF indica es ha LF bio inyla ion does no ad e sely a ec he
binding o LF o LP because he IC50 is e y close o he EC50 alues ob ained in he
concen a ion dependence assays. Because o i s small molecula weigh (244 Da), bio in
labelling does no signi ican ly inc ease he size o he labelled p o ein and does no cause s e ic
hind ance. Speci ically, by using he bio inyla ion p ocedu e desc ibed in Sec ion 2.4.1,
app oxima ely h ee bio in molecules we e inco po a ed pe LF molecule.
Thus, he h ee ypes o LF-Bi binding esponse assays pe o med wi h ou ELISA-mimic
p o ocol allowed he calcula ion o consis en sensi i i y alues o he syn he ic ecep o (LP)
o he binding o he bio inyla ed a ge p o ein (LF-Bi). The esul s ob ained o he Ka, EC50,
and IC50 alues o LF-Bi, i espec i e o he ype o binding assay, a e compa able, indica ing
ha he me hod has a high deg ee o epea abili y.
Using ou ELISA-mimic p o ocol, compe i i e inhibi ion binding assays we e pe o med
o se e al p o eins (Fig. 5b). In he LP speci ici y assays, he selec i e inhibi ion o he LF-Bi
(10 nM) binding esponse by o he compe ing p o eins was examined. In e es ingly, lysozyme
and ypsin ully inhibi ed he selec i e LF-Bi binding esponse, yielding slope ac o s close o
uni y. Fo bo h p o ein ligands, he IC50 alues showed ela i ely low binding a ini ies, whe eas
he nH alues indica e a compe i i e mechanism o ligand inhibi ion o LF-Bi selec i e binding
o LP. Howe e , albumin was able o inhibi only 70% o he selec i e LF-Bi binding a a
concen a ion o 75 µM, he maximal albumin concen a ion assayed (Fig. 5b). The IC50 alues
de i ed om he ull compe i ion cu es we e 0.62 ± 0.03 and 43.3 ± 1.7 µM o lysozyme and
ypsin. In summa y, he o de o po ency was LF, lysozyme, ypsin, and albumin. On he basis
o hese esul s, i was possible o calcula e he c oss- eac i i y alues o hese p o eins in ou
LF-Bi/LP assay: CRLZ = 1.95%, CRT = 0.028%, and CRBSA = 0.016%. Fu he mo e, he da a
is use ul o demons a e he an i-in e e ence abili y o LF-Bi o he de e mina ion o LF in he
p esence o o he p o eins ha ing simila molecula weigh o isoelec ic poin . As obse ed in
Fig. 5b, i 10 nM o LF-Bi is used, concen a ions highe han 100 nM o Lysozyme, 1,000 nM
o T ypsin, o 10,000 nM o BSA a e equi ed o dec ease he abso bance in ensi y p o ided by
he p obe, which e idences ha i p esen s excellen an i-in e e ence abili y o de ec LF in he
p esence o o he coexis ing p o eins as BSA and ypsin (app oxima ely 3–4 loga i hmic uni s).
In addi ion, he in e e ing lysozyme concen a ion (1 log uni ) could be eally impo an in
cases whe e lysozyme concen a ions a e ele a ed, o ins ance, in he case o acu e
myeloblas ic leukemia [39] whe e u ina y lysozyme le els exceed a leas h ee imes he
expec ed no mal alues (60–136 nM).
3.3. Valida ion o he LF ELISA mimic o u ine analysis
U ine is an abundan and easily accessible body luid, p o iding an ideal ou e o he
non-in asi e diagnosis o a numbe o human in lamma o y and in ec ious diseases,
pa icula ly hose o he u ina y ac . The use ulness o he measu emen o u ina y LF o he
diagnosis o u ina y ac in ec ions [40-42] and as a no el u ine bioma ke o he diagnosis
and p ognosis o u o helial ca cinoma o he bladde has been epo ed p e iously [43]. Fo
example, he mean concen a ion o LF has been ound o be 30.4 ng mL−1 in heal hy u ine and
3,300 ng mL−1 in he u ine o indi iduals su e ing om in ec ions [40,44,45].
F om he esul s ob ained in he concen a ion-dependen assays, i was possible o
es ablish he limi o de ec ion and quan i ica ion o he p oposed ELISA mimic in PB as 18
and 60 ng mL−1, espec i ely. Fi s , he eliabili y o he p oposed assay o he de e mina ion
o LF was es ed using di ec o p e-concen a ed (10- o 20- imes) u ine es samples. Fo he
di ec and 10- old concen a ed es u ine samples, LF le els lowe han he de ec ion limi o
he me hod we e ob ained. Howe e , in he 20- old concen a ed samples, i was possible o
de e mine LF le els a 7.3 ± 0.3 ng mL−1 in he selec ed u ine es samples. These esul s
indica e ha he es u ine can be used as a con ol ma ix o e alua e he ma ix e ec s o his
me hod. Fo his, con ol u ine samples we e spiked wi h di e en concen a ions o LF (0.1–
250 nM), and he de eloped p ocedu e was applied. The quan i ica ion limi in u ine was
es ima ed a 1.5 nM (120 ng/mL). Me hod accu acy was also de e mined by he addi ion o a
known amoun o analy e o blank u ine, and besides, o compa ison, he same addi ion was
made o PB ins ead o u ine. Reco e y was pe o med in iplica e and o h ee concen a ion
le els (a concen a ion close o he limi o quan i ica ion, a concen a ion close o he EC50 y
and ano he one en imes highe han his). The eplica es we e p epa ed independen ly om
he beginning o he p ocess, a oiding se ial dilu ions. The accu acy is exp essed as a
pe cen age calcula ed om he measu ed analy e concen a ion e sus he added amoun o
analy e. Ob ained eco e ies we e ound o be 92.1 ± 2.8% o a concen a ion o 2 nM,
96.8 ± 1.8% o 10 nM and 94.2 ± 1.2% o 100 nM.
The in a-day p ecision o he me hod was es ima ed om successi e measu emen s
unde he same ope a ing condi ions o he h ee concen a ion le els men ioned abo e. The
esul s exp essed as RSD we e 5.7% o 2 nM, 2.9% o 10 nM and 3.6% o 100 nM. The
p ecision ound o di e en assays pe o med ho ough se e al weeks (in e -day p ecision)
was 11.4%, 8.6% and 4.2% espec i ely o 2 nM, 10 nM and 100 nM concen a ion le els.
Nex , compe i i e binding expe imen s in he spiked es u ine we e pe o med, and, om
he ob ained compe i i e inhibi ion cu e, he IC50, he concen a ion ha inhibi s 50% o he
esponse o he labelled molecule in he u ine ma ix, was 54.0 ± 1.3 nM. This alue is
app oxima ely ou imes highe han ha obse ed when he assay we e pe o med in pa allel
in PB medium (Fig. 6). Based on hese esul s, which e eal ma ix e ec s, he LF le els in he
selec ed es u ine sample (20- ime concen a ed) we e de e mined o be 22.4 ± 1.8 ng mL−1.
Ne e heless, despi e he obse ed ma ix e ec s, he p oposed me hod is alid o he
de e mina ion o u ine LF le els in human pa ien s su e ing om in lamma o y and in ec ious
diseases o he u ina y ac . In addi ion, we de e mined ha he de ec ion limi o he me hod
can be imp o ed by p e-concen a ing he u ine sample wi h Amicon Ul a-15 cen i ugal il e s
wi h a 50-kDa cu -o .
4. Conclusions
As a p oo o concep , he syn hesis o a new linea polyme by e e sible deac i a ion
adical polyme isa ion is p oposed. The linea and lexible polyme can adap i s shape o he
la and ough su ace opology o lac o e in, ou a ge p o ein. Thus, we p oposed a me hod
o p oduce and alida e a obus , inexpensi e, non-biological polyme ic an ibody o LF. This
polyme was, hus, used as plas ic an ibody in an ELISA mimic o he de e mina ion o he LF
concen a ion in eal u ine samples. The selec i i y o he ELISA mimic o LF in he p esence
o o he p o eins o simila molecula weigh o simila isoelec ic poin alues was also es ed
and ound he be accep able. The ad an ages o using he p oposed plas ic an ibody a e ha i s
syn hesis is as (hou s) compa ed o na u al an ibodies (mon hs), i is s able unde s o age a
oom empe a u e o se e al yea s and i s linea ange is in e media e compa ed o he
comme cial p oposals (8 ng/mL-20 g/mL) wi h an analysis ime o 3 h 20min and a p ecision
o 3–11% (RSD). The syn hesis could be easily pe o med in any labo a o y and he polyme
may be unc ionalized wi h luo escen , ca aly ic o magne ic labels o be used in di e en
biochemical assays.
Decla a ion o compe ing in e es
The au ho s decla e ha hey ha e no known compe ing inancial in e es s o pe sonal
ela ionships ha could ha e appea ed o in luence he wo k epo ed in his pape .
Acknowledgemen s
Funding o his esea ch was p o ided by he Spanish Minis y o Science, Inno a ion
and Uni e si ies (p ojec CTQ2017-85686-R) and by he Basque Go e nmen (Resea ch
G oups o he Basque Uni e si y Sys em, P ojec No IT 1186-19). The au ho s would like o
hank he Ala a Cen al Se ice Analysis belonging o SGIke (UPV/EHU) o hei echnical
assis ance.
Re e ences
[1] V.J.B. Ruig ok, M. Le isson, M.H.M. Eppink, H. Smid , J. Van De Oos , Al e na i e
a ini y ools: Mo e a ac i e han an ibodies?, Biochem. J. 436 (2011) 1–13.
h ps://doi.o g/10.1042/BJ20101860.
[2] A. Ya man, K.J. Je zschmann, B. Neumann, X. Zhang, U. Wollenbe ge , A. Co din, K.
Haup , F.W. Schelle , Enzymes as ools in MIP-senso s, Chemosenso s. 5 (2017).
h ps://doi.o g/10.3390/chemosenso s5020011.
[3] W. Chen, X. Tian, W. He, J. Li, Y. Feng, G. Pan, Eme ging unc ional ma e ials based
on chemically designed molecula ecogni ion, BMC Ma e . 2 (2020) 1–22.
h ps://doi.o g/10.1186/s42833-019-0007-1.
[4] L. Chen, X. Wang, W. Lu, X. Wu, J. Li, Molecula imp in ing: Pe spec i es and
applica ions, Chem. Soc. Re . 45 (2016) 2137–2211.
h ps://doi.o g/10.1039/c6cs00061d.
[5] I. Chianella, A. Gue ei o, E. Moczko, J.S. Caygill, E. V. Pile ska, I.M.P. De Va gas
Sansal ado , M.J. Whi combe, S.A. Pile sky, Di ec eplacemen o an ibodies wi h
molecula ly imp in ed polyme nanopa icles in ELISA - De elopmen o a no el assay
o ancomycin, Anal. Chem. 85 (2013) 8462–8468. h ps://doi.o g/10.1021/ac402102j.
[6] C. Cáce es, F. Can a o a, I. Chianella, E. Pe ei a, E. Moczko, C. Esen, A. Gue ei o, E.
Pile ska, M.J. Whi combe, S.A. Pile sky, Does size ma e ? S udy o pe o mance o
pseudo-ELISAs based on molecula ly imp in ed polyme nanopa icles p epa ed o
analy es o di e en sizes, Analys . 141 (2016) 1405–1412.
h ps://doi.o g/10.1039/c5an02018b.
[7] J. Xu, F. Me lie , B. A alle, V. Vieilla d, P. Deb é, K. Haup , B. Tse Sum Bui,
Molecula ly Imp in ed Polyme Nanopa icles as Po en ial Syn he ic An ibodies o
Immunop o ec ion agains HIV, ACS Appl. Ma e . In e aces. 11 (2019) 9824–9831.
h ps://doi.o g/10.1021/acsami.8b22732.
[8] K. Smolinska-Kempis y, A. Gue ei o, F. Can a o a, C. Cace es, M.J. Whi combe, S.
Pile sky, A compa ison o he pe o mance o molecula ly imp in ed polyme
nanopa icles o small molecule a ge s and an ibodies in he ELISA o ma ., Sci. Rep. 6
(2016) 37638. h ps://doi.o g/10.1038/s ep37638.
[9] E. Ve heyen, J.P. Schillemans, M. Van Wijk, M.A. Demeniex, W.E. Hennink, C.F. Van
Nos um, Challenges o he e ec i e molecula imp in ing o p o eins, Bioma e ials. 32
(2011) 3008–3020. h ps://doi.o g/10.1016/j.bioma e ials.2011.01.007.
[10] S. Amb osini, S. Beyazi , K. Haup , B. Tse Sum Bui, Solid-phase syn hesis o molecula ly
imp in ed nanopa icles o p o ein ecogni ion, Chem. Commun. 49 (2013) 6746.
h ps://doi.o g/10.1039/c3cc41701h.
[11] P. Çaki , A. Cu i e , M. Resmini, B.T.S. Bui, K. Haup , P o ein-size molecula ly
imp in ed polyme nanogels as syn he ic an ibodies, by localized polyme iza ion wi h
mul i-ini ia o s, Ad . Ma e . 25 (2013) 1048–1051.
h ps://doi.o g/10.1002/adma.201203400.
[12] Y. Ge, A.P.F. Tu ne , Too la ge o i ? Recen de elopmen s in mac omolecula
imp in ing, T ends Bio echnol. 26 (2008) 218–224.
h ps://doi.o g/10.1016/j. ib ech.2008.01.001.
[13] M. Pan, L. Hong, X. Xie, K. Liu, J. Yang, S. Wang, Nanoma e ials-Based Su ace P o ein
Imp in ed Polyme s: Syn hesis and Medical Applica ions, Mac omol. Chem. Phys. 222
(2021) 1–18. h ps://doi.o g/10.1002/macp.202000222.
[14] A. Huang, J.M. Paloni, A. Wang, A.C. Obe meye , H. V. Su eka, H. Yao, B.D. Olsen,
P edic ing P o ein-Polyme Block Copolyme Sel -Assembly om P o ein P ope ies,
Biomac omolecules. 20 (2019) 3713–3723. h ps://doi.o g/10.1021/acs.biomac.9b00768.
[15] A. Rod iguez-Abe xuko, D. Sánchez-deAlcáza , P. Muñume , A. Beloqui, Tunable
Polyme ic Sca olds o Enzyme Immobiliza ion, F on . Bioeng. Bio echnol. 8 (2020) 1–
27. h ps://doi.o g/10.3389/ bioe.2020.00830.
[16] A. Nie o-O ellana, M. Di An onio, C. Con e, F.H. Falcone, C. Bosquillon, N.
Childe house, G. Man o ani, S. S olnik, E ec o polyme opology on non-co alen
polyme -p o ein complexa ion: mik oa m e sus linea mPEG-poly(glu amic acid)
copolyme s, Polym. Chem. 8 (2017) 2210–2220. h ps://doi.o g/10.1039/c7py00169j.
[17] S.J. Koch, C. Renne , X. Xie, T. Sch ade , Tuning Linea Copolyme s in o P o ein-
Speci ic Hos s, Angew. Chemie. 118 (2006) 6500–6503.
h ps://doi.o g/10.1002/ange.200601161.
[18] A. Mo ib, A. Gue ei o, F. Al-Baya i, E. Pile ska, I. Manzoo , S. Sha eeq, A. Kadam, O.
Kuipe s, L. Hille , T. Cowen, S. Pile sky, P.W. And ew, H. Yesilkaya, Modula ion o
Quo um Sensing in a G am-Posi i e Pa hogen by Linea Molecula ly Imp in ed Polyme s
wi h An i-in ec i e P ope ies, Angew. Chemie. 129 (2017) 16782–16785.
h ps://doi.o g/10.1002/ange.201709313.
[19] Y. Wada, H. Lee, Y. Hoshino, S. Ko ani, K.J. Shea, Y. Miu a, Design o mul i- unc ional
linea polyme s ha cap u e and neu alize a oxic pep ide: A compa ison wi h c oss-
linked nanopa icles, J. Ma e . Chem. B. 3 (2015) 1706–1711.
h ps://doi.o g/10.1039/c4 b01967a.
[20] J.M. Rini, U. Schulze-Gahmen, I.A. Wilson, S uc u al e idence o induced i as a
mechanism o an ibody-an igen ecogni ion, Science (80-. ). 255 (1992) 959–965.
h ps://doi.o g/10.1126/science.1546293.
[21] B. Panganiban, B. Qiao, T. Jiang, C. Del e, M.M. Obadia, T.D. Nguyen, A.A.A. Smi h,
A. Hall, I. Si , M.G. C osby, P.B. Dennis, E. D ockenmulle , M. Ol e a, D. C uz, T. Xu,
Random he e opolyme s p ese e p o ein unc ion in Fo eign En i onmen s., Science
(80-. ). 1243 (2018) 1239–1243.
[22] A. Alexande -Ka z, R.C. Van Lehn, Random copolyme s ha p o ec p o eins, Science
(80-. ). 359 (2018) 1216–1217. h ps://doi.o g/10.1126/science.aa 0155.
[23] Y.A. K iksin, P.G. Khala u , A.R. Khokhlo , Recogni ion o complex pa e ned
subs a es by he e opolyme chains consis ing o mul iple monome ypes, J. Chem. Phys.
124 (2006). h ps://doi.o g/10.1063/1.2191849.
[24] A. Poma, A. Gue ei o, S. Caygill, E. Moczko, S. Pile sky, Au oma ic eac o o solid-
phase syn hesis o molecula ly imp in ed polyme ic nanopa icles (MIP NPs) in wa e ,
RSC Ad . 4 (2014) 4203–4206. h ps://doi.o g/10.1039/c3 a46838k.
[25] A. Poma, A. Gue ei o, M.J. Whi combe, E. V. Pile ska, A.P.F. Tu ne , S.A. Pile sky,
Solid-Phase Syn hesis o Molecula ly Imp in ed Polyme Nanopa icles wi h a Reusable
Templa e-"Plas ic An ibodies", Ad . Func . Ma e . 23 (2013) 2821–2827.
h ps://doi.o g/10.1002/ad m.201202397.
[26] A. Baghe i, C.M. Fellows, C. Boye , Re e sible Deac i a ion Radical Polyme iza ion:
F om Polyme Ne wo k Syn hesis o 3D P in ing, Ad . Sci. 8 (2021) 1–16.
h ps://doi.o g/10.1002/ad s.202003701.
[27] H. Zhang, Recen Ad ances in Mac omolecula ly Imp in ed Polyme s by Con olled
Radical Polyme iza ion Techniques, Mol. Imp in ing. 3 (2016) 35–46.
h ps://doi.o g/10.1515/molim-2015-0005.
[28] P. Bonomi, M.D. A ieh, C. Gonza o, K. Haup , A New Ve sa ile Wa e -Soluble Ini e e
Pla o m o he P epa a ion o Molecula ly Imp in ed Nanopa icles by
Pho opolyme isa ion in Aqueous Media, Chem. - A Eu . J. 22 (2016) 10150–10154.
h ps://doi.o g/10.1002/chem.201600750.
[29] M.J. Ga cia-So o, K. Haup , C. Gonza o, Syn hesis o molecula ly imp in ed polyme s
Figu e 4
Figu e 5
Figu e 6
