Improved antifouling properties and selective biofunctionalization of stainless steel by employing heterobifunctional silane-polyethylene glycol overlayers and avidin-biotin technology

1 Scien i ic RepoR s | 6:29324 | DOI: 10.1038/s ep29324 www.na u e.com/scien i ic epo s Imp o ed an i ouling p ope ies and selec i e bio unc ionaliza ion o s ainless s eel by employing he e obi unc ional silane- polye hylene glycol o e laye s and a idin-bio in echnology Ville Hynninen1, Leena Vuo i2, Ma kku Hannula2, Kos i Tapio3, Kimmo Lah onen2, Tommi Isoniemi3, Elina Leh onen2, Mika Hi simäki2, J. Jussi Toppa i3, Mika Valden2 & Vesa P. Hy önen1,4 A s aigh o wa d solu ion-based me hod o modi y he bio unc ionali y o s ainless s eel (SS) using he e obi unc ional silane-polye hylene glycol (silane-PEG) o e laye s is epo ed. Reduced nonspeci ic bio ouling o bo h p o eins and bac e ia on o SS and u he selec i e bio unc ionaliza ion o he modi ied su ace we e achie ed. Acco ding o pho oelec on spec oscopy analyses, he silane-PEGs o med less han 10 Å hick o e laye s wi h close o 90% su ace co e age and ep oducible chemical composi ions. Consequen ly, he su aces also became mo e hyd ophilic, and he obse ed non- speci ic bio ouling o p o eins was educed by app oxima ely 70%. In addi ion, he a achmen o E. coli was educed by mo e han 65%. Mo eo e , he po en ial o he o e laye o be u he modi ied was demons a ed by success ully coupling bio inyla ed alkaline phospha ase (bAP) o a silane-PEG- bio in o e laye ia a idin-bio in b idges. The ac i i y o he immobilized enzyme was shown o be well p ese ed wi hou comp omising he achie ed an i ouling p ope ies. O e all, he simple solu ion- based app oach enables he ailo ing o SS o enhance i s ac i i y o biomedical and bio echnological applica ions. In bio echnology and ood and pha maceu ical indus ies he abili y o speci ically adjus and modi y a ma e ial’s in e ac ions wi h i s su oundings is ex emely desi able1–3. Un o una ely, ma e ials wi h bo h bene icial bulk p ope ies and app op ia e su ace unc ionali ies a e seldom eadily a ailable. The e o e, su ace modi ica ion o ma e ials ha demons a e o he wise po en ial p ope ies, such as s ainless s eel (SS), is o en p e e able2,4. Fo example, h ough co alen coupling eac ions and polyme g a ing, o he wise biologically challenging ma e ials can be adjus ed o comply wi h a ious ci cums ances5,6. SS is a commonly used me al in indus ial and medical se ings, whe e i becomes exposed o o ganic and biological agen s ha may esul , o example, in bio ouling and mic obial-induced co osion7. Fo ins ance, o hopedic implan s and ca dio ascula s en s a e cons an ly in con ac wi h bodily luids and in e ac wi h li ing cells1,8,9. Among o he s, SS has dis inguished he mal and mechanical p ope ies, g ea wo kabili y, a ailabili y, and inhe en co osion esis ance, which esul s om i s na i e, sel -healing oxide laye 3. The oxide laye also con ibu es o he biocompa ibili y o SS10. Howe e , o op imal biological pe o mance bio unc ionali y is also 1BioMediTech, Uni e si y o Tampe e, Bioka u 6, FI-33520 Tampe e, Finland. 2Su ace Science Labo a o y, Op oelec onics Resea ch Cen e, Tampe e Uni e si y o Technology, P.O. Box 692, FI-33101 Tampe e, Finland. 3Uni e si y o Jy askyla, Depa men o Physics, NanoScience Cen e , P.O. Box 35, FI-40014 Uni e si y o Jy äskylä, Finland. 4Fimlab Labo a o ies, Bioka u 4, FI-33520 Tampe e, Finland. Co espondence and eques s o ma e ials should be add essed o V.P.H. (email: [email p o ec ed]) Recei ed: 07 Ap il 2016 Accep ed: 15 June 2016 Published: 06 July 2016 OPEN www.na u e.com/scien i ic epo s/ 2 Scien i ic RepoR s | 6:29324 | DOI: 10.1038/s ep29324 equi ed. Fo ins ance, enzymes, such as lysozyme and alkaline phospha ase ha e been used o in oduce an ibac- e ial p ope ies o o he speci ic biological unc ions o su aces11,12. Silanes a e well known and widely used su ace- unc ionaliza ion agen s ha a e able o bind o he hyd ox- ide g oups o he SS oxide laye , and in sui able condi ions s ic ly o ganized monolaye s can be o med12,13. Fo example, Slaney e al.13 ha e used a omic laye deposi ion (ALD) o p ecoa SS su aces o acili a e he subsequen addi ion o bimolecula silane laye s. In addi ion, Si-O-Si siloxane bonds can be o med be ween adjacen silane molecules, which adds o he enaci y o he o e laye 12,14,15. Va ious ypes o unc ionalized silanes, such as he he e obi unc ional silane-polye hylene glycol (silane-PEG) used in his s udy, a e also eadily comme cially a ail- able and in gene al hey a e ela i ely inexpensi e and conside ed en i onmen ally sa e. He e, we epo a simple and s aigh o wa d solu ion based me hod o deposi an ex ensi e silane-PEG o e - laye on an SS subs a e o al e i s bio unc ionali y. The app oach is based on he p o ocol p e iously epo ed in Vuo i e al.12. Howe e , ins ead o implemen ing a s ep-by-s ep buildup o he silane o e laye , eady-made he - e obi unc ional silane-PEGs wi h ei he ca boxylic acid (silane-PEG-COOH, SPC) o bio in (silane-PEG-bio in, SPB) as he o he end g oup we e used. The concep o he s udy is depic ed in Fig.1. In addi ion o su ace bio unc ionali y, special ca e was placed on he ex ensi e cha ac e iza ion o he su ace wi h high esolu ion pho oelec on spec oscopy me hods complemen ed wi h imaging echniques. Ini ially, SS was elec ochemically (EC) hyd oxyla ed o ensu e he p esence o an ex ensi e oxide laye . This su ace p e ea men is essen ial o he p ope silaniza ion o SS and imposes addi ional challenges and eac- ion s eps when compa ed o, o example, he modi ica ion o mo e eac i e and esponsi e silica16,17 and glass su aces18,19. Insu icien su ace p e ea men migh lead o he undesi ed agg ega ion o he silane-PEGs and, consequen ly, esul in an une en silane o e laye . Since we ha e shown in Vuo i e al.12 ha EC ea men gen- e a es a high-quali y, ep oducible and la gely con aminan - ee passi a ed su ace and we ha e solid expe ience wi h he echnique, EC ea men was selec ed as he su ace p e ea men me hod o be used he e. Silaniza ion o he EC- ea ed SS was hen pe o med in one s ep by simple imme sion echnique, which makes he p oce- du e ex emely use iendly. Due o he silane g oups, he silane-PEGs bind SS i s ia hyd ogen bonds, which may hen be con e ed in o mo e igid co alen bonds20. SPC molecules we e used o imp o e SS an i ouling p ope ies, whe eas SPB o e laye s enabled u he speci ic immobiliza ion o a idin (neu al chime ic a idin, nChiA d21) on o he su ace. Cha ac e is ically, a idin binds e y igh ly o he bio in molecules a ailable on he su ace and, also, allows o u he addi ion o o he bio inyla ed molecules22. Consequen ly, a idin-bio in b idges we e used o unc ionalize he su ace wi h bio inyla ed alkaline phospha ase. All in all, he ex en o modi ica ions was assessed wi h a ious su ace sensi i e echniques, he an i ouling p ope ies wi h p o ein adso p ion and bac e ial adhesion es s, and he achie ed bio unc ionali y wi h spec oscopic enzyme ac i i y assay12. Me hods Ma e ials. Lase -cu elec ochemically polished 12.5 mm × 28 mm × 0.7 mm EN 1.4404 (AISI 316L) SS chips p oduced by Ou okumpu S ainless Oy (To nio, Finland) we e o de ed om Kaa inan T ime Oy (h p:// www.kaa inan ime . i/). Silane-PEG-COOH (SPC, MW 2000 Da) and silane-PEG-bio in (SPB, MW 2000 Da) we e acqui ed om Nanocs Inc. (New Yo k, NY, USA). Sul u ic acid, oluene and phospha ase subs a e (pNPP, P oduc No. S0942) we e bough om Sigma-Ald ich (S . Louis, MO, USA), and glacial ace ic acid and ac idine o ange zinc chlo ide double sal (P oduc No. 115931) om Me ck (Da ms ad , Ge many). Wild ype chicken a idin (Belo o, Belgium, MW = 16 kDa/monome ) and ib onec in (gela in-a ini y pu i ied om human se um, MW = 220 kDa/monome ) had been p e iously labeled wi h Alexa Fluo ® 488 NHS es e (Ca . No. A-20000, The mo Fishe Scien i ic, Inc. Wal ham, MA, USA) by D . J. Pä ssinen acco ding o manu ac u e ’s ins uc ions. Chemically compe en E. coli Top10 cells we e acqui ed om The mo Fishe scien i ic, Inc. Fo bio unc ion- aliza ion, neu al chime ic a idin (nChiA d21) and bio inyla ed alkaline phospha ase (Ca . No. B-2005, Vec o Labo a o ies (Bu lingame, CA, USA) we e used. Vec ashield Ha dse An i ade Moun ing Medium (Ca . No. H-1400, Vec o Labo a o ies) was used o mic oscope sample p epa a ion. Figu e 1. Schema ic illus a ion o silane-PEG-modi ied SS. (a) SPC o e laye esis s non-speci ic bio ouling o bo h bac e ia and p o eins. (b) SPB o e laye enables selec i e unc ionaliza ion o SS ia a idin-bio in b idges wi h bAP wi hou comp omising he an i ouling p ope ies. www.na u e.com/scien i ic epo s/ 3 Scien i ic RepoR s | 6:29324 | DOI: 10.1038/s ep29324 Hyd oxyla ion. An SS chip was washed by sonica ion in bo h e hanol and deionized (DI) wa e o 10 min each a oom empe a u e (RT). EC ea men was pe o med using an Au olab PGSTAT12 po en ios a /gal a- nos a (Me h om Au olab, The Ne he lands) wi h a h ee-elec ode elec ochemical cell, he SS sample as a wo k- ing elec ode, an Ag/AgCl e e ence elec ode and an SS (316L) coun e elec ode. N2 degassed 0.1 M sul u ic acid was used as elec oly e. Mild degassing was con inued h oughou he EC ea men . The SS sample was educed o 10 min wi h a cons an ca hodic cu en o 5 mA/cm2 ollowed by passi a ion o 10 min wi h a cons an po en ial o Ep = 0.2 V agains he Ag/AgCl e e ence elec ode. The sample was washed by insing wi h DI wa e and d ied unde an N2 s eam. Silaniza ion. The silane e minus o he silane molecules is ie hoxy silane o bo h SPC and SPB. Silane-PEG (SPC o SPB) was dissol ed in oluene a he desi ed concen a ion (3 o 5 mg/ml). An SS chip was imme sed in he solu ion o app oxima ely 43 h ( wo nigh s) on a ocking shake a RT. The solu ion was emo ed and he chip annealed unde a mosphe ic condi ions a 100 °C o 10 min. The chip was insed wi h a s eam o oluene by using a pipe e, allowed o ai -d y, and hen washed by imme sion in an excess o wa e h ee imes o a leas 30 s wi h in ense shaking (230–250 pm). Finally, he sample was ai d ied unde lamina low. Con ac angle measu emen s. A cus om-made imaging sys em wi h Pisa a image-analyzing so wa e (Fo oComp Oy, Jy äskylä, Finland) was used o con ac angle measu emen s. D ops o 4 μ l o DI wa e , 7–12 d ops o each SS chip, we e used. The samples we e imaged immedia ely a e pipe ing he d ops o he chips. Con ac angles o SS-SPC, SS-SPB, and unmodi ied SS we e de e mined. Pho oelec on spec oscopy (PES). PES measu emen s we e conduc ed in wo di e en analysis sys- ems: one ea u ed a non-monoch oma ized Al Kα adia ion (pho on ene gy hν = 1486.6 eV) sou ce (XPS)23 and he o he u ilized synch o on (SR-PES) a he D1011-beamline D1011 o MAX II s o age ing in he MAX IV Labo a o y (Lund, Sweden). The de ec ion a ea o he XPS analyses was app oxima ely 0.28 mm2. The SR-PE spec a we e collec ed by a SCIENTA SES-200 elec on ene gy analyze in FAT mode wi h 200 eV pass ene gy a a no mal emission angle. The o al ene gy esolu ion was ~100 meV and he de ec ion a ea o he SR-PES analyses was app oxima ely 0.04 mm2. The su ace chemical s a es we e iden i ied by analyzing high- esolu ion PE spec a. Upon sub ac ing he linea backg ounds, he spec al componen s we e i wi h a combina ion o Gaussian and Lo en zian line shapes (GL30) using Casa XPS so wa e e sion 2.3.1624. The binding ene gies we e calib a ed o 285.0 eV o alipha ic ca bons. The su ace mo phologies o he samples we e de e mined by inelas ic elec on ene gy backg ound (IEEB) analysis using QUASES-Tougaa d so wa e package25–27, whe e he a enua ion o Fe 2p (hν = 1486.6 eV) in ensi y o he silanized samples was compa ed o ha o he SS-EC 12,14,15. The e o o he me hod was es ima ed o be 15%28. The IEEB analysis is desc ibed in u he de ail in Supplemen a y In o ma ion. A omic o ce mic oscopy (AFM). AFM measu emen s we e pe o med wi h a Dimension 3100 (B uke Co po a ion, Bille ica, MA, USA) oge he wi h Nanoscope Analysis so wa e (B uke ) and Aspi e CT300 Conical apping mode AFM p obes (Pa No. CT300R-25, Nanoscience Ins umen s, Phoenix, AZ, USA). Phase and heigh images we e aken in ai using apping mode. P o ein adso p ion. SS-SPC chips we e a ixed on o a mic oscope slide wi h double-sided adhesi e ape and lexiPERM mic o12 chambe s (Sa s ed , Nü nb ech , Ge many) we e secu ed he eupon o o m well pla es. Fluo escen ly labeled a idin and ib onec in we e added o he wells a wo di e en concen a ions (3 o 30 μ g/ml in PBS) o 1 o 3 h in he da k a RT. The labeling a ios o he p o eins we e 0.47 and 8.1 molecules o dye pe p o ein subuni o a idin and ib onec in, espec i ely. Unmodi ied SS was used as e e ence. The samples we e washed h ee imes wi h an excess o PBS, lexiPERM chambe s emo ed, and hen ai -d ied. Glass co e slips we e moun ed on o he samples, and hey we e imaged wi h an LSM-780 con ocal mic oscope equipped wi h Zen Black so wa e (Zeiss, Ge many) using 10× objec i e (Plan-Apoch oma 10× /0.45 M27). Fi e images pe lexiPERM well a ea we e aken om andom loca ions. The mean in ensi ies o he images we e eco ded as ob ained om he Zen Black so wa e. Fixed exposu e and gain se ings, ini ially adjus ed o p e en o e expo- su e, we e used o allow o compa ison o he in ensi y alues. The backg ound in ensi y measu ed o an SS-SPC sample imme sed in clean PBS was sub ac ed om he measu ed in ensi ies o all he samples. Adhesion o E. coli. E. coli Top10 cells we e p ecul u ed in LB medium o e nigh on a pla o m shake (150 pm) a 37 °C. The op ical densi y o he cul u e solu ion a 600 nm (OD600) was measu ed wi h a BioPho ome e Plus ins umen (Eppendo , Hambu g, Ge many), and i was adjus ed o OD600 = 2.0 by dilu ing he samples wi h LB medium. SS-SPC was imme sed in he bac e ial suspension and incuba ed o 1 o 6 h on a ocking shake a RT. Unmodi ied SS was used as e e ence. The sample placed in 6-well pla e was washed h ee imes o 30 s by imme sion in 1.5 ml o PBS unde s i ing (230–250 pm) o emo e loosely a ached bac e ia, and was hen ai -d ied. The bac e ia we e ixed and s ained by imme sing he sample in 3 mg/ml ac idine o ange in 2% glacial ace ic acid o 2 min in he da k a RT. The sample was a ached o a mic oscope slide wi h double-sided adhesi e ape and a co e slip was moun ed on o i . Imaging was pe o med wi h an LSM-780 con ocal mic oscope (Zeiss, Ge many) wi h Zen Black so wa e using a 63× oil imme sion objec i e (Plan-Apoch oma 63× /1.40 Oil DIC M27). Mosaic images (10 × 10) we e aken om andom loca ions on he samples, and he amoun o bac e ial cells pe image was calcula ed using ImageJ29. The adhesion o E. coli on SS was also e alua ed wi h scanning elec on mic oscopy (SEM). He e, 3 mg/ml SS-SPC samples and an unmodi ied SS e e ence we e used. E. coli had been ixed on he su aces wi h 4% pa a- o maldehyde. The samples we e coa ed wi h a 4 nm laye o a gold/palladium mix u e (3 + 1) using an ul a-high www.na u e.com/scien i ic epo s/ 4 Scien i ic RepoR s | 6:29324 | DOI: 10.1038/s ep29324 acuum elec on beam e apo a o (Ins umen i Ma ila, Mynämäki, Finland). Fo he imaging, a Rai h eLiNE 50 sys em (Rai h Inc., Do mund, Ge many) was used a an accele a ion ol age o 10 kV. Fo compa ison, bac e ial adhesion es s we e also conduc ed on glass and BSA-coa ed glass. Co e slips (18 × 18 mm; Zeiss) we e used as subs a es. Be o e he expe imen s, he co e slips we e washed by successi ely imme sing hem in 0.1 M NaOH, H2O, 70% e hanol and H2O; hen, he co e slips we e ai -d ied. Fo he BSA coa ing, a 100 μ g/ml BSA in H2O was p epa ed. The solu ion was added on o he co e slips o 1 h a RT. The co e slips we e hen washed h ee imes wi h an excess o PBS and ai -d ied. The bac e ial adhesion es s we e conduc ed as desc ibed abo e. Bio unc ionaliza ion wi h a idin. 3 μ g/ml o neu al chime ic a idin21 in PBS was added on o SS-SPB o 1 h a RT. The samples we e washed six imes wi h an excess o TBS- ween20 (T is-bu e ed saline wi h 0.05% Tween20) unde igo ous shaking. The a achmen o a idin was assessed wi h AFM. To bio unc ionalize he SS-SPC-nChiA d, bio inyla ed alkaline phospha ase was dilu ed 1:5000 in 100 mM T is-HCl + 150 mM NaCl bu e (pH 7.5). This solu ion was added on o he SS-SPC-nChiA d o 1 h a RT. The samples we e washed six imes wi h an excess o TBS- ween20 wi h igo ous shaking o emo e unbound enzymes. Subsequen ly, 50 μ l d ops o 1 mg/ml o pNPP phospha ase subs a e in 1 M die hanolamine (DEA) bu e con aining 0.5 mM MgCl2 (pH 9.8) we e applied on o he su aces and he samples we e p o ec ed om ligh . 2 μ l samples we e hen ob ained om he d ops a ime poin s o 10, 20, 30, 40, 50 and 60 min a e he addi ion o pNPP o measu e he espec i e abso bances a 405 nm using a NanoD op 2000 Spec ome e (The mo Scien i ic, Wilming on, DE, USA). Neu al chime ic a idin blocked wi h 2 μ M ee bio in was used o p epa e he e e ence samples. Resul s and Discussion Topog aphy and chemical p ope ies o silane-peg-modi ied SS. Pho oelec on spec oscopy esul s a e shown in Fig.2. Figu e2(a) depic s he su ey spec a o he SS-EC (SS a e EC ea men ) and SS-SPC samples wi h 3 and 5 mg/ml o SPC examined wi h con en ional XPS (hν = 1486.6 eV). The sampling dep h o each elemen al PES ansi ion depended on he kine ic ene gy o he pho oelec ons and he ma e ial pa ame e s. The sampling dep h (i.e., 95% o he PES in ensi y o igina es om his dep h) in Fig.2(a) a ied be ween ~6 and 10 nm. Hence, he su ey spec um consis ed o pho oelec ons om he SPC o e laye , he oxide laye o he SS su ace and, o a small deg ee, he SS bulk phase. In he SS spec um, elemen s o 316L s ainless s eel we e de ec ed (i.e., Fe, C , Ni, Mn, Mo, S, N and C). On he SS-SPC, an inc eased amoun o ca bon was obse ed. F om he su ey spec a, i was e iden ha he SPC o e laye was a he hin on he SS subs a e because he signals om he me allic elemen s o he SS we e no a enua ed o a g ea deg ee on he silanized samples. High- esolu ion con en ional XP spec a (hν = 1486.6 eV) showed ha Si was p esen on he SS-SPC samples (See Supplemen a y Tables 1 and 2). Wi h con en ional XPS, he expe imen s we e limi ed o one o wo pho on ene gies, and he e o e only a ew di e en sampling dep hs could be de e mined o each elemen i hey we e ope a ed a a cons an emis- sion angle. SR-PES p o ides an excellen me hod o p obing in o he su ace chemis y o hin o ganic coa ings on solid subs a es because he ene gy o he pho on lux ha is used o he emission o pho oelec ons can be selec ed om a con inuous ange o pho on ene gies. The e o e, wi h SR-PES, i was possible o selec he sampling dep h o emi ed elec ons o each elemen . Also, he SR-PES enables he be e ecogni ion o he chemical s a es due o highe ene gy esolu ion. In Fig.2(b,c), he SR-PES ansi ions o C 1s and O 1s a e p e- sen ed, espec i ely. The pho on ene gies we e chosen o yield high su ace sensi i i ies. Fo bo h C 1s and O 1s, he sampling dep hs we e app oxima ely 2 nm. Figu e2(b) shows he C 1s SR-PE spec a (hν = 430 eV) o he SS and SS-SPC (5 mg/ml) samples. The C 1s spec um o SS (bo om) was ypical o s ainless s eel ha has been exposed o a mosphe ic condi ions15. The peaks a 285.0, 286.5 and 288.3 eV could be assigned o he C-O and C=O bonds, espec i ely. The p esence o he SPC molecules on he SS-EC su ace ( op) was e iden om he signi ican inc ease in he C-O in ensi y a e silaniza ion. Figu e2(c) demons a es ha he O 1s SR-PE spec- um (hν = 715 eV) o SS-EC (bo om) consis ed o me al oxides a 530.1 eV, hyd oxides a 531.6 eV, sul a es a 532.6 eV and o ganic impu i ies o H2O a 534.1 eV15. The la ge amoun o hyd oxides and he sul a e esidues o igina ed om he EC ea men 14,15. The O 1s spec um o SS-SPC exhibi ed h ee peaks a 530.0 eV (me al oxides), 531.4 eV (hyd oxides) and 532.8 eV. The peak a 532.8 eV could be assigned o he C-O bonds in PEG30. Me al oxides and hyd oxides a he SS su ace we e a enua ed upon he adso p ion o SPC. Sul a e esidues and possible Si-O-Si bonds we e also obse ed in he same binding ene gy ange as he C-O bonds in PEG, and hence could no be esol ed. Thus, he ex ensi e co alen siloxane bonding o he o e laye could no be con i med by SR-PES. Howe e , no conside able de e io a ion o he su ace coa ings we e de ec ed du ing he expe imen s, which sugges igh a achmen o he silane-PEGs. Mo eo e , he e we e no signi ican di e ences in he ela i e elemen al su ace concen a ions and chemical s a es be ween samples wi h 3 mg/ml and 5 mg/ml o be ween hea ed and unhea ed samples (see Supplemen a y Table S2). None heless, IEEB analyses we e used o de e mine su ace mo phologies. The bes i was ob ained using a su ace mo phology wi h a hin silane o e laye in addi ion o low co e age wi h highly clus e ed islands wi h hicknesses > 200 Å (see Supplemen a y Figu e S1). This was in acco dance o ou ea lie obse a ions o hinne silane monolaye s (mo e speci ically aminop opyl ime hoxysilane and me cap op opyl ime hoxysilane) on s ainless s eel12,15. A possible eason o he o ma ion o clus e ed islands is he s uc u al i egula i ies o he SS subs a e15. The hickness o he 3 mg/ml SS-SPC sample o e laye was 4.6 Å wi h 86.9% co e age and ha o he 5 mg/ml SS-SPC sample o e laye was 7.7 Å wi h 83.2% co e age (see Supplemen a y Table S3). These IEEB analyses indica ed ha he en i e su ace o he SPC-SS su ace was co e ed wi h SPC molecules. The hicknesses o he deposi ed silane-PEG laye s we e close o he hickness o APS coa ings epo ed in Vuo i e al.12. Addi ionally, Zhang e al.31 ha e epo ed app oxima ely 0.5 nm- hick PEG-SiCl coa ings on sili- con su aces wi h smalle (600 Da) molecules. Because he leng h o an ex ended PEG 2000 would be close o www.na u e.com/scien i ic epo s/ 5 Scien i ic RepoR s | 6:29324 | DOI: 10.1038/s ep29324 10 nm, i is likely ha he molecules we e no s anding in up igh posi ions bu ins ead sp ead ou la e ally on he su ace32,33. Thus, hey we e able o e ec i ely co e he en i e su ace e en hough he silane g oups appea ed o be loosely packed. The IEEB analysis is based on XPS da a measu ed in ul a high acuum condi ions. The molecula b ush ype silane molecules may adop a di e en su ace o ien a ion and appa en hickness in gas- eous o liquid en i onmen . Thus, he de e mined SPC o e laye hicknesses should be e alua ed wi h cau ion. S ill, densely packed o e laye s would yield signi ican ly highe hicknesses in he IEEB analysis. Wi h dense packing, he silane-PEGs would ha e been o ced o ex end upwa ds which would ha e inc eased he hickness o he o e laye . This could possibly be achie ed by using, o example, addi ional sho e space molecules o mo e e ec i ely o ganize he silane-PEG molecules o by inc easing he silane-PEG concen a ion32. Indeed, also he e he highe SPC concen a ion o 5 mg/ml esul ed in hicke SPC o e laye . Howe e , in e ms o chemical com- posi ion and su ace co e age, no signi ican di e ences we e obse ed be ween he wo applied concen a ions. Also, inc easing he silane-PEG concen a ion u he was ound di icul due o solubili y limi s. Thus, he lowe 3 mg/ml concen a ion was selec ed and used as he p ima y wo king concen a ion. Acco ding o he con ac angle measu emen s, silaniza ion makes he SS su aces mo e hyd ophilic. Fo all he silane-modi ied samples, app oxima ely 10-deg ee educ ions in he con ac angle alues we e obse ed com- pa ed o clean unmodi ied SS (see Supplemen a y Figu e S2). This ela i ely small di e ence was ound o be Figu e 2. Pho oelec on analyses o he silane-PEG-modi ied SS. (a) XPS su ey spec a o he SS-EC and SPC-SS samples. (b) C 1s SR-PE spec a (hν = 430 eV) (c) and O 1s SR-PE spec a (hν = 715 eV) o he SS- SPC and SS-EC samples. The spec a shown in (b,c) a e no malized. The sampling dep hs a e schema ically illus a ed in he inse . www.na u e.com/scien i ic epo s/ 6 Scien i ic RepoR s | 6:29324 | DOI: 10.1038/s ep29324 consis en be ween nume ous samples and s a is ically signi ican (one-way ANOVA). In con as , s a is ically signi ican di e ences be ween he wo di e en silane-PEG-modi ied su aces (SPC and SPB) we e no ound. O e all, he a e age con ac angle o he unmodi ied SS e e ence was de e mined o be 48° ± 6°, whe eas o SPC and SPB modi ied SS, he measu ed alues we e 39° ± 7° and 41° ± 3°, espec i ely. As mo e hyd ophilic su - aces a e o en also conside ed o be mo e an i ouling, he sligh ly highe hyd ophilici y o he modi ied SS, hus, likely posi i ely con ibu ed o he imp o ed bio ouling esis ance o he coa ed su aces as was obse ed in he an i ouling es s34,35. Howe e , as he con ac angle alues o unmodi ied SS we e also a he hyd ophilic o begin wi h, i could no be unambiguously de e mined how signi ican he con ibu ion o inc eased hyd ophilici y was o he bio ouling. In AFM, he silane-PEG o e laye could no be di ec ly obse ed, apa om some occasional agg ega es, bu he ypical opog aphy and su ace cha ac e is ics o SS we e clea ly isible in all he samples. This migh be caused by he ac ha , since he AFM imaging was pe o med in d y s a e, he con o ma ion o he PEGs migh ha e collapsed simila ly o UHV condi ions o he XPS measu emen s, which could make he coa ing ex emely hin. None hless, he images concu wi h he PES analyses and imply ha he o e laye is indeed e y hin and mos ly homogenous. Howe e , in heigh images he SPC-modi ied su aces appea ed smoo he han he unmod- i ied SS, and phase images showed di e ences in he su ace g oo e dips, which indica ed ha he dips we e illed wi h ma e ial di e ing om SS. The ep esen a i e images a e shown in Fig.3. Possibly, silane-PEG had Figu e 3. AFM images o SS-SPC. Typical AFM heigh (a) and phase (b) images o a silane-PEG-COOH- coa ed SS su ace. Scale ba s 200 nm. (c) La ge scale image o he same sample as in (a,b). Scale ba 1 μ m. (d,e) Heigh and phase images o an unmodi ied SS, espec i ely. Scale ba s 200 nm. ( ) A la ge-scale image o he same sample as in (d,e). Scale ba 1 μ m. www.na u e.com/scien i ic epo s/ 7 Scien i ic RepoR s | 6:29324 | DOI: 10.1038/s ep29324 accumula ed in he g oo es and pa ially illed hem, which also ag ees wi h he hypo hesis o ex ensi e la e al sp eading o he su ace-bound silane-PEGs as sugges ed by he IEEB analyses. An i ouling p ope ies. Su ace-g a ed PEG coa ings a e able o p e en bio ouling o p o eins and bac e- ia on dis inc su aces31,36–38. The e o e, he abili y o he SPC-coa ed SS su aces o esis bio ouling was es ed using wo di e en ypes o p o ein, a idin and ib onec in, and E. coli bac e ia. Two di e en incuba ion imes and concen a ions we e used o bo h o he p o eins, and in each case, he SPC o e laye signi ican ly educed bio ouling on he SS su aces (one-way ANOVA wi h Bon e oni pos -hoc es , p < 0.05). Fo example, SS-SPC showed a 70% educ ion in a idin (30 μ g/ml) adso p ion compa ed o he unmodi ied SS a e an incuba ion ime o 1 h. The espec i e educ ion in he case o ib onec in (30 μ g/ml) a e 1 h o exposu e was 72%. The co esponding luo escence mic oscope images o he samples and he measu ed mean luo escence in ensi ies a e shown in Fig.4. Howe e , he adso p ion o he p o eins could no be comple ely p e en ed, and he amoun o adso bed p o ein seemed o posi i ely co ela e wi h inc easing incuba ion ime (1 o 3 h) and p o ein con- cen a ion (3 o 30 μ g/ml) o some ex en . None heless, e en he highes de ec ed a e age in ensi y alues o he SS-SPC o bo h p o eins s ayed below he alues obse ed om he unmodi ied SS unde he mildes condi ions. Al oge he , he obse ed in ensi y alues o bo h p o eins we e e y simila , sugges ing ha e en hough a idin and ib onec in a e e y di e en in e ms o s uc u e and size, he coa ing was able o easonably e ec i ely educe binding o bo h o he p o eins. Al hough he eco ded alues o ib onec in appea ed o be sligh ly lowe , hese could possibly be accoun ed by he lowe luo escence labeling densi ies pe mass o p o ein ha esul ed om a ia ions du ing sample p epa a ion. Di e ences we e de ec ed in he a angemen o he p o eins on he su ace; a idin was ypically e enly dis ibu ed, whe eas ib onec in demons a ed agg ega ion and o ma ion o ib il-like s uc u es. Fib onec in clus e s and ib ils we e also mo e p ominen in he samples wi h longe expo- su e imes and highe p o ein concen a ions. Spon aneous ib illogenesis is cha ac e is ic o ib onec in, and i is known ha ib onec in ib ils can be gene a ed e en in he absence o cells, o ins ance, u ilizing a wa e -ai in e ace39. Simila educ ions in p o ein adso p ion ha e been epo ed, o example, by Yang e al.38 and Ha de e al.40. Yang and colleagues coa ed SS wi h a poly(e hylene oxide)-poly(p opylene oxide)-poly(e hylene oxide) (PEO-PPO-PEO) iblock copolyme , and p esen ed ha he coa ed SS su aces we e capable o signi ican ly educing he adso p ion o bo ine se um albumin (BSA). Mo eo e , hey no iced ha wi hou ini ial condi ion- ing o he SS wi h a su ace hyd ophobiza ion s ep, compa able in e ms o signi icance o he EC su ace passi a- ion me hod used he e, he PEO-PPO-PEO simply adso bed on o su aces in ine ec ual con o ma ion and was unable o p e en non-speci ic p o ein adso p ion. Ha de and colleagues, in u n, c ea ed ib inogen- esis an laye s on gold su aces u ilizing oligo(e hylene glycol)- e mina ed sel -assembled monolaye s. Addi ionally, hey obse ed ha simila sel -assembled monolaye s on sil e ailed o esis ib inogen adso p ion as a consequence o di e ences in he con o ma ion and a angemen o he monolaye assemblies. The e o e, he an i ouling p op- e ies o PEG and PEG-like polyme s appea o be con o ma ion dependen . Hence, ou posi i e esul s he e p o ide indi ec e idence o he p ope a achmen and a angemen o he SS-bound SPC. E. coli a achmen was also signi ican ly educed on SS-SPC, and a s a is ically signi ican di e ence was ound wi h bo h o he used ime poin s (1 and 6 h) when compa ed o he unmodi ied SS (K uskal-Wallis es wi h Dunn’s pos -hoc es , p < 0.05). Un o una ely, comple e p e en ion o bac e ial a achmen was no achie ed. The bac e ia we e coun ed using luo escence mic oscope images, and ep esen a i e images along wi h SEM images a e shown in Fig.5. The median bac e ial coun s pe image unde di e en condi ions we e as ollows: unmodi- ied SS wi h 1 h exposu e, 43 cells (In e qua ile ange (IQR) = 25.3–92.0 cells); SS-SPC wi h 1 h exposu e, 14 cells (IQR = 3.6–32.4); unmodi ied SS wi h 6 h exposu e, 892 cells (IQR = 512.0–1401.0); SS-SPC wi h 6 h exposu e, 39 cells (IQR = 30.5–76.5). Thus, o 1 h exposu e ime, he SPC o e laye educed bac e ial adhesion by app oxi- ma ely 65%. Co espondingly, a e exposu e imes o 6 h, he educ ion in bac e ial adhesion was obse ed o be close o 95%, which sugges s he e ec becomes mo e p onounced in longe ime scales. Mo eo e , SS-SPB we e also exposed o E. coli in he 1 h exposu e es o examine i di e en unc ional end g oups o silane-PEGs had an e ec on he bac e ial a achmen . As a esul , a compa able no iceable educ ion in E. coli adhesion was obse ed on SS-SPB han on SS-SPC. The change was de e mined o be o e 85%, which was in good acco dance wi h he SS-SPC esul s. Thus, bo h he SPC and SPB o e laye s demons a ed signi ican po en ial in p e en ing bac e ial a achmen and no no able e ec due o he di e en end g oups was de ec ed. SEM imaging also showed simila end as unmodi ied SS had much highe amoun s o a ached E. coli han espec i e SS-SPC samples. On SS-SPC, he bac e ia we e ypically ound in a ew clumps a ached o each o he , whe eas on unmodi ied SS he bac e ia ended o sp ead ou mo e e enly, as is shown in Fig.5(d– ). The opo- g aphical ea u es o SS o de ec s in he SPC coa ing we e expec ed o a ec and p omo e bac e ial a achmen on he modi ied su aces. Howe e , apa om occasional alignmen along he SS g ain bounda ies, no o he p e e en ial opog aphically-d i en a achmen was obse ed. Thus, di ec conclusions canno be d awn based on his da a and u he s udy is needed o elucida e he de ails o bac e ial binding on modi ied SS. Bac e ial adhesion es s we e also conduc ed on glass and BSA-coa ed glass o allow o he ela i e compa - ison o he silane-PEG-modi ied SS o a mo e well-known and gene ally u ilized ma e ial. BSA-coa ed glass was used as a model o adso p ion- esis i e su ace because BSA is ou inely used as a blocking agen o p e en non-speci ic su ace binding41. The esul s a e shown in Fig.5(a). Clean glass was obse ed o ha e app oxima ely 80% less bac e ia a ached a e 1 h o bac e ial exposu e han unmodi ied SS, and wi h he longe 6 h exposu e he di e ence was e en mo e p onounced. Thus, i seems ha SS is ma kedly mo e suscep ible o bac e ial adhesion, which emphasizes he need o e ec i e SS ea men . F om he p ac ical poin o iew, howe e , SS and glass a e a ely eadily in e changeable and a e o o ally di e en pu poses, e en hough hey a e o en used oge he o complemen each o he . As SS and glass a e widely used ma e ials in bio echnological labo a o ies, he es gi es a good poin o e e ence o he usabili y and po en ial o he modi ied SS in hose se ings. www.na u e.com/scien i ic epo s/ 8 Scien i ic RepoR s | 6:29324 | DOI: 10.1038/s ep29324 Bio unc ionaliza ion. To examine he bio unc ionaliza ion po en ial o he silane-PEG-modi ied SS, SS-SPB subs a es we e manu ac u ed, and neu al chime ic a idin was a ached o he bio in g oups o he coa - ing. Bio in-a idin links a e highly speci ic and hei ex emely high a ini y (dissocia ion cons an (Kd) ~ M) p o ides a ou e o a b oad ange o applica ions ia a achmen o bio inyla ed molecules42. Bio inyla ed alkaline Figu e 4. P o ein adso p ion on SS-SPC. (a) Fluo escence mic oscopy images o Alexa Fluo ® 488 labeled a idin and ib onec in adso bed on unmodi ied SS (SS-c l) and SS-SPC in di e en condi ions. The used p o ein concen a ion was ei he 3 μ g/ml (C1) o 30 μ g/ml (C2), and he exposu e ime 1 h (T1) o 3 h (T2). Scale ba s 100 μ m. (b,c) The boxplo s show he mean luo escence in ensi ies o he adso bed a idin and ib onec in, espec i ely, on SS-SPC (SPC) and unmodi ied SS (C l) in he condi ions depic ed in (a). www.na u e.com/scien i ic epo s/ 9 Scien i ic RepoR s | 6:29324 | DOI: 10.1038/s ep29324 phospha ase enzyme was linked o su ace-bound a idin, as depic ed in Fig.6(a), and he unc ionaliza ion e ec- i i y was assessed by spec opho ome ically measu ing he enzyma ic ac i i y o he immobilized bAP. In he nega i e con ol samples, a idin a achmen was blocked wi h ee bio in. The analysis e ealed clea di e ences be ween he ea men s, indica ing success ul selec i e a achmen o bAP ia a idin-bio in bonds, and simul- aneous no able esis ance agains non-speci ic adso p ion. The bAP enzyma ic ac i i y o he unc ionalized samples p oceeded smoo hly un il app oxima ely he 50 min ime poin , as shown in Fig.6(b), whe e a pla eau was eached. The abso bance a ha ime poin was measu ed o be 1.59. On he o he hand, he abso bance o he bio in-blocked con ols emained close o ze o (maximum a e age a he 60 min was 0.05). Thus, he SPB-coa ing was able o esis bio ouling, while simul aneously allowing selec i e su ace unc ionaliza ion. The a achmen o a idin on SPB su aces was also s udied using AFM. Pa icles wi h a diame e o 5–10 nm we e de ec ed on he a idin ea ed SS-SPB, while hey we e absen om he nega i e con ol su aces, as is shown in Fig.6(c– ). As he size o an a idin molecule is close o 5 nm, he pa icles we e mos likely indi idual a idin, sugges ing success ul unc ionaliza ion. The SS-SPB su aces ha had no been ea ed wi h a idin appea ed sim- ila o he SS-SPC su aces shown in Fig.3. The da a a e in good acco dance wi h he esul s epo ed p e iously in Vuo i e al.12, whe e a idin-bio in echnology was used o unc ionalize bimolecula o ganosilane-modi ied SS. Simila ly, hey used an AP enzy- ma ic eac ion o inspec he ex en o unc ionaliza ion and eco ded abso bance alues o 0.2 a 405 nm, which sugges ed success ul unc ionaliza ion. This alue is signi ican ly lowe han he maximum alues ob ained in his s udy. Howe e , alues a e no di ec ly compa able wi h each o he due o di e ences in he unc ional- iza ion app oaches used. While in Vuo i e al. spa se and unable unc ionaliza ion o SS was aimed, a mo e Figu e 5. A achmen o E. coli on SS-SPC. (a) Amoun (median wi h in e qua ile ange) o igh ly a ached bac e ia on unmodi ied SS (SS-C l), SS-SPC and SS-SPB a e 1 o 6 h exposu e. Also alues o unmodi ied and BSA-coa ed glass in simila condi ions a e shown o e e ence. (b) Rep esen a i e luo escence mic oscope image o E. coli on unmodi ied SS a e 1 h o incuba ion. Scale ba 25 μ m. (c) Rep esen a i e luo escence mic oscope image o E. coli on SS-SPC a e 1 h o incuba ion. Scale ba 25 μ m. (d) SEM image o E. coli on SS-SPC. The sample was no coa ed wi h conduc i e coa ing o imaging. Scale ba 10 μ m. (e) SEM o bac e ia on unmodi ied SS. The sample had been coa ed wi h AuPd p io o imaging. Scale ba 10 μ m. ( ) Highe magni ica ion o he same sample as in (e). Scale ba 1 μ m.