A linear-polymer-based lactoferrin- selective recognition element for an ELISA mimic: A Proof of concept

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). 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