Preparation of functionalized magnetic nanoparticles conjugated with feroxamine and their evaluation for pathogen detection

P epa a ion o unc ionalized magne ic nanopa icles conjuga ed wi h e oxamine and hei e alua ion o pa hogen de ec ion† Diana Ma ´ ınez-Ma amo os, a Soco o Cas o-Ga c´ ıa, a Miguel Balado, b Ad iana Ma amo os-Veloza, c Mille Alonso Cama go-Vale o, d Osca Cespedes, e Jaime Rod ´ ıguez, * a Manuel L. Lemos b and Ca los Jim´ enez * a This wo k epo s he p epa a ion o a conjuga e be ween amino- unc ionalized silica magne i e and he side opho e e oxamine. The mo phology and p ope ies o he conjuga e and in e media e magne ic nanopa icles (MNPs) we e examined by powde X- ay diff ac ion (XRD), Fou ie T ans o m In a ed spec oscopy (FT-IR), Raman spec oscopy, X- ay pho oelec on spec oscopy (XPS), magne iza ion s udies, ze a po en ial measu emen s, T ansmission Elec on Mic oscopy (TEM) and Ene gy Dispe si e X- ay (EDX) mapping. Fu he mo e, his s udy in es iga ed he in e ac ion be ween he unc ionalized magne ic NPs and Ye sinia en e ocoli ica wild ype (WC-A) using Scanning Elec on Mic oscopy (SEM) and TEM images. In addi ion, he in e ac ion be ween MNPs and a Y. en e ocoli ica mu an s ain lacking e oxamine ecep o FoxA, was also used o s udy he binding specifici y. The esul s showed ha he cap u e and isola ion o Y. en e ocoli ica by he MNPs ook place in all cases. Mo eo e , he specific in e ac ion be ween he MNP conjuga e and bac e ia did no inc ease a e blocking he ee amine g oups wi h -bu oxyca bonyl (Boc) and ca boxylic acid (COOH) unc ional g oups. Elec os a ic su ace in e ac ions ins ead o molecula ecogni ion be ween MNP conjuga e and e oxamine ecep o seem o ule he a achmen o bac e ia o he conjuga e. In oduc ion A g owing in e es in magne ic nanopa icles (MNP) based on magne i e (Fe 3 O 4 ) has been obse ed o e he las 10 yea s in analy ical sensing and nanomedicine due o i s s ong magne ic p ope ies and biocompa ibili y. 1 The magne ic eld o MNP plays a key ole in he cap u e and bio-sepa a ion o analy es. The unc ionaliza ion o MNP's su ace allows he de elopmen o mul iple applica ions such as magne ic hype he mia, magne ic esonance imaging (MRI), a ge d ug deli e y and de ec ion o bac e ia, since MNP a e capable o cap u ing bac e ia using specic ecogni ion. In ac , MNP a e capable o in e ac ing wi h biological en i ies such as p o eins and bac e- ial memb anes, among o he s, can be manipula ed by an ex e nal magne ic eld, and a e easy o syn hesize. 2 The de elopmen o apid, sensi i e and eliable me hods o he de ec ion and iden ica ion o in ec ious mic oo ganisms is one o he main conce ns in ood and heal h indus ies. Nowadays, his in e es has become mo e impo an wi h he eme ge o i ulen s ains o common pa hogenic bac e ia and he need o limi ing he sp ead o ela ed con agious diseases. The adi ional me hods based on cell cul u ing a e usually e y slow and ime-consuming p ocesses. Nume ous apid and sensi i e me hods o mic obial de ec ion ha e been de eloped (e.g., immunoassays, enzyme-linked immunoso ben assays (ELISA) and polyme ase chain eac ion me hodologies (PCR)). 3 Howe e , hey a e no effec i e in complex sys ems when bac e ia a e p esen in low concen a ions. An eme ging esea ch a ea based on he magne ic, elec onic, pho onic, and op ical p ope ies and unc ionaliza ion o MNP has being adop ed o de elop al e na i e me hods based on he isola ion a Cen o de In es igaci´ ons Cien ´ ıcas A anzadas (CICA), Depa amen o de Qu´ ımica, Facul ade de Ciencias, Uni e sidade da Co u˜ na, 15071 A Co u˜ na, Spain. E-mail: [email p o ec ed]; [email p o ec ed] b Depa men o Mic obiology and Pa asi ology, Ins i u e o Aquacul u e, Uni e sidade de San iago de Compos ela, Campus Su , San iago de Compos ela 15782, Spain c Ins i u e o Func ional Su aces, School o Mechanical Enginee ing, Uni e si y o Leeds, Leeds LS2 2JT, UK d BioResou ce Sys ems Resea ch G oup, School o Ci il Enginee ing, Uni e si y o Leeds, Leeds LS2 9JT, UK e Facul y o Ma hema ics and Physical Sciences, School o Physics and As onomy, Uni e si y o Leeds, Leeds LS2 9JT, UK Depa amen o de Ingenie ´ ıaQu ´ ımica, Uni e sidad Nacional de Colombia, Campus La Nubia, Manizales, Colombia †Elec onic supplemen a y in o ma ion (ESI) a ailable: p-XRD, FT-IR spec a, magne iza ion hys e esis loops, he mog a ime ic analysis (TGA) o blocked NPs, SEM images, TEM images and EDX maps and Y. en e ocoli ica WC-A-MNP in e ac ion assay esul s o i on and i on deciency g ow h condi ions. See DOI: 10.1039/c8 a10440a Ci e his: RSC Ad .,2019,9, 13533 Recei ed 20 h Decembe 2018 Accep ed 22nd Ap il 2019 DOI: 10.1039/c8 a10440a sc.li/ sc-ad ances This jou nal is © The Royal Socie y o Chemis y 2019 RSC Ad .,2019,9, 13533–13542 | 13533 RSC Ad ances PAPER Open Access A icle. Published on 01 May 2019. Downloaded on 7/9/2020 6:24:03 PM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online View Jou nal | View Issue o pa hogenic bac e ia using nanoma e ials o biological iden ica ion. 4 The specici y o MNP is based on he chemical ecogni ion o pa hogenic bac e ia h ough he conjuga ion o MNP wi h an ibodies, ap ame s, biop o eins, ca bohyd a es and bac e- iophages. 1 Mo eo e , MNP can be also coupled o side opho es ha a e ecognized by specic memb ane ecep o s o mic o- o ganisms. 5 Side opho es a e small o ganic molecules ecog- nised o playing a ole in he mechanisms con olling Fe 3+ up ake by bac e ia. 6,7 So a , h ee diffe en app oaches ha e been documen ed o he de ec ion o mic obial pa hogens using side opho e scaffolds. The  s one u ilizes an immobi- lized side opho e o cap u e human pa hogens, in which a side opho e conjuga e is a ached o gold-pla ed glass chips h ough bo ine se um albumin (BSA). 8,9 O he example is a modied, a icial side opho e complex a ached o he su ace o an Au elec ode and placed on qua z c ys al mic o- balance (QCM) chips. 10,11 A ecen wo k using his app oach, documen ed he use o a side opho e-based ac i e bac e ial emo al in eg a ed in a localized su ace plasmon esonance (LSPR) sensing pla o m. 12 The second app oach employs a side opho e a ached o unc ionalized quan um do s (QDs) o he bac e ial in e ac ion wi h a specic ecep o . 13 The hi d app oach uses unc ionalized aga ose columns bound o a specic bac e ial side opho e o he cap u e o side opho e- binding p o eins. 14 Howe e , he conjuga ion o side opho es and magne ic nanopa icles o isola e and cap u e pa hogenic bac e ia has no been s udied ye . He ein, we epo he  s syn hesis o a conjuga e be ween amino- unc ionalized silica magne i e and he side opho e e oxamine, he blocking o ee amine g oups on he su ace o amino- unc ionalized silica magne i e and he conjuga e wi h - bu oxyca bonyl (Boc) and ca boxylic acid (–COOH) unc ional g oups and i s e alua ion o he cap u e o wild ype (WC-A) and a mu an lacking e oxamine ecep o FoxA (FoxA WC-A 12-8) Y. en e ocoli ica s ains. Expe imen al All s a ing ma e ials, eagen s and sol en s we e ob ained om comme cial supplie s and used wi hou u he pu ica ion. A gon gas was used o a oid he p esence o mois u e and oxygen in sensi i e eac ions. Size exclusion ch oma og aphy was pe o med on Sephadex™LH-20 esins. LREIMS and HRESIMS we e measu ed on Applied Biosys ems QSTAR Eli e. Syn hesis Syn hesis o Fe 3 O 4 magne ic nanopa icles (MNP). 15 A solu- ion o 0.5 g o i on(III) ace ylace ona e (Fe(acac) 3 )in10mLo benzyl alcohol was sonica ed o 2 min, ans e ed o a hea ing block and le o eac a 180 C o 72 h. Ae ha ime, he esul ing mix u e was allowed o cool down be o e he p ecipi- a es we e decan ed by cen i uga ion (5000 pm o 30 min), while he supe na an was disca ded. The solids we e insed h ee imes wi h 96% e hanol, sonica ed and eco e ed using a magne . Syn hesis o SiO 2 coa ing o MNP (MNP@SiO 2 ). 16 80 mL o isop opanol, 4 mL o ammonia (21%), 7.5 mL o dis illed wa e and 0.56 mL o e ae hyl o hosilica e (TEOS) we e ca e ully added in his o de o 2 g o MNP. The mix u e was hea ed a 40 C o 2 h wi h con inuous s i ing and hen sonica ed o 1h.Ae ha ime, he MNP we e emo ed om he solu ion using a magne and e-dispe sed in 30 mL o isop opanol. This coa ing p ocedu e was epea ed a second ime. Finally, he SiO 2 coa ed MNP we e insed wi h e hanol and sepa a ed om he dispe sion using a magne . Syn hesis o amino- unc ionalized silica magne i e (MNP@SiO 2 @NH 2 ). 17 A modied p ocedu e desc ibed by Chen e al. 17 was used o he unc ionaliza ion o MNP@SiO 2 . Fo ha , 500 mg o MNP@SiO 2 we e insed and sonica ed h ee imes wi h 3 mL o dime hyl o mamide (DMF). Then, he pa icles we e e-suspended in 9 mL o DMF and 9 mL o 3- aminop opyl ie hoxysilane (APTES). The esul ing mix u e was hen shaken a 60 C o 12 h. Finally, he unc ionalized pa icles (MNP@SiO 2 @NH 2 ) we e sepa a ed wi h a magne and sonica ed wi h 96% e hanol, h ee imes. Syn hesis o e oxamine (2). 18 100 mg (0.15 mmol) o de e - oxamine mesyla e (1) sal and 53.0 mg (0.15 mmol) o Fe(acac) 3 we e dissol ed in 5 mL o dis illed wa e and les i ing o e - nigh . The esul ing p oduc was washed h ee imes wi h 20 mL o E OAc and hen, he sol en was emo ed unde acuum using a o a apo . The aqueous phase was eeze-d ied o ob ain e oxamine as a ed solid (94.4 mg, 78% yield). (+)-HR-ESIMS m/ z614.2751 [M + H] + (calcula ed o C 25 H 45 FeN 6 O 8 : 614.2729). Syn hesis o N-succinyl e oxamine (3). 19 350 mg (3.50 mmol) o succinic anhyd ide we e added o a solu ion o 100 mg (0.17 mmol) o e oxamine in 5 mL o py idine. The esul ing mix u e was s i ed a oom empe a u e o 16 h. Ae ha ime, he excess o py idine was elimina ed in a o a apo unde acuum. The ed solid p oduc was pu ied by size exclusion ch oma- og aphy using me hanol as eluen o sepa a e 93.1 mg o N- succinyl e oxamine (3) as a da k ed solid. (+)-HR-ESIMS m/z 736.2700 [M + Na] + ; (calcula ed o C 29 H 49 FeN 6 O 11 Na: 736.2706). Syn hesis o MNP@SiO 2 @NH@Fa (4). 30 mg o d y MNP@SiO 2 @NH 2 we e insed wice wi h DMF and sonica ed o 30 minu es. A solu ion o N-succinyl e oxamine (3; 200 mg, 0.30 mmol), benzo iazole-1-yl-oxy- is-(dime hylamino)- phosphonium hexauo ophospha e (BOP, 173 mg, 0.45 mmol), 1-hyd oxybenzo iazole (HOB , 46 mg, 0.39 mmol) and N,N-diisop opyle hylamine (DIPEA, 128.8 mg, 1.21 mmol) in 10 mL o DMF was added d opwise o a suspension o 30 mg o MNP@SiO 2 @NH 2 in 3 mL o DMF unde sonica ion in d y and oxygen ee condi ions using an a gon gas a mosphe e. 20 The mix u e was les i ing a oom empe a u e o e nigh . Finally, he esul ing conjuga e (MNP@SiO 2 @NH@Fa, 4) was sepa a ed om he suspension wi h a magne and he sepa a ed solid was insed and sonica ed 5 imes wi h 10 mL o e hanol. The solid was acuum d ied o 24 h. Syn hesis o MNP@SiO 2 @NHBoc and MNP@SiO 2 @- NHBoc@Fa (5). 28.5 mg o MNP@SiO 2 @NH 2 was insed wi h d y DMF and sonica ed wice o 5 minu es unde an a gon gas 13534 |RSC Ad .,2019,9, 13533–13542 This jou nal is © The Royal Socie y o Chemis y 2019 RSC Ad ances Pape Open Access A icle. Published on 01 May 2019. Downloaded on 7/9/2020 6:24:03 PM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online a mosphe e and hen suspended in 10 mL o d y DMF. Ae ha , 200 mg o Boc 2 O (di- e -bu yl dica bona e) we e dissol ed in d y DMF and mixed wi h he nanopa icles. The eac ion mix u e was sonica ed o 30 min and hen s i ed a oom empe a u e in an o bi al shake a 200 pm o 24 h. Finally, he solids we e sepa a ed using a magne and insed wi h 10 mL o e hanol and sonica ed  e imes. The solids we e hen acuum d ied o 24 h o yield 27.5 mg o MNP@SiO 2 @NHBoc. The same p ocedu e was epea ed o 13.6 mg o MNP@SiO 2 @- NH@Fa (4) o ob ain 13.8 mg o MNP@SiO 2 @NHBoc@Fa (5). Syn hesis o MNP@SiO 2 @NHCOOH and MNP@SiO 2 @- NHCOOH@Fa (6). 25 mg o MNP@SiO 2 @NH 2 we e  s insed wi h d y py idine and sonica ed wice o 5 minu es unde an a gon a mosphe e and hen suspended in 10 mL o d y py idine. Ae his, 200 mg o succinic anhyd ide we e added o he nanopa icles. The eac ion mix u e was sonica ed 30 min and hen s i ed a oom empe a u e in an o bi al shake a 200 pm o 24 h. Finally, he solids we e sepa a ed using a magne , insed and sonica ed  e imes using 10 mL o e hanol and acuum d ied o 24 h o yield 21.3 mg o MNP@SiO 2 @NHCOOH. The same p ocedu e was epea ed o 9.3 mg o MNP@SiO 2 @NH@Fa (4) o ob ain 8.7 mg o MNP@SiO 2 @NHCOOH@Fa (6). Cha ac e iza ion Powde X- ay diff ac ion (XRD). XRD analyses o samples con aining MNP we e pe o med using a B uke D8 diff ac- ome e (CuK a ) wi h a scan ange be ween 2 and 70 2qa 0.05 2qmin 1 . The MNP con aining samples we e e-dispe sed in e hanol and moun ed on o a poly(me hyl me hac yla e) spec- imen holde o analysis. Peak iden ica ion was pe o med by using X'Pe High Sco e Plus sowa e by compa ing he collec ed diff ac ion da a wi h he In e na ional Cen e o Diff ac ion Da a da abase. FT-IR and Raman spec oscopy. FT-IR analyses we e ca ied ou on powde ed nanopa icle samples using an A2-Technology Mic oLab Po able mid-IR spec ome e equipped wi h a dia- mond in e nal eec ion (DATR). Fo he analysis, he back- g ound was collec ed wi hou deposi ion o he sample and hen a sample was e-dispe sed in e hanol and placed on o he diamond window o he ins umen . Indi idual spec a (4096) we e acqui ed be ween 650 o 4000 cm 1 a a esolu ion o 1cm 1 and hen co-added and p ocessed using O igin 8 (O i- ginLab, No hamp on, MA, USA). Raman analyses we e ca ied ou in a LabRAM HR 800 Ho iba Scien ic spec ome e , wi h a 633 nm lase using a 10% powe (1 mW) and a diff ac ion g a ing o 600 ln pe mm. Each spec um included en measu emen s o 300 seconds ( o al measu ing ime 3000 seconds). X- ay pho oelec on spec oscopy (XPS). Analysis o he samples was pe o med using a The mo Scien ic K-Alpha ESCA ins umen equipped wi h aluminum K a mono- ch oma ized adia ion a 1486.6 eV X- ay sou ce. Due o he no conduc o na u e o samples, i was necessa y o use an elec on ood gun o minimize su ace cha ging. Neu aliza ion o he su ace cha ge was pe o med by using bo h a low ene gy ood gun (elec ons in he ange 0 o 14 eV) and a low ene gy A gon ions gun. The XPS measu emen s we e ca ied ou using monoch oma ic Al-K a adia ion (hn¼1486.6 eV). Pho oelec- ons we e collec ed om a ake-offangle o 90 ela i e o he sample su ace. The measu emen was done in a Cons an Analyse Ene gy mode (CAE) wi h a 100 eV pass ene gy o su ey spec a and 20 eV pass ene gy o high esolu ion spec a. Cha ge e e encing was done by se ing he lowe binding ene gy C1s pho o peak a 285.0 eV C1s hyd oca bon peak. Su ace elemen al composi ion was de e mined using he s anda d Scoeld pho oemission c oss sec ions. Da a analysis and quan ica ion we e pe o med using he A an age sowa e e sion 5 om he manu ac u e The mo Scien ic. Magne ic cha ac e iza ion. Magne iza ion was measu ed using an Ox o d Ins umen s VSM wi h a magne ic eld o 1 T and a sensi i i y o 10 mic o-emu. Ze a po en ial analysis. Ze a po en ial measu emen s we e pe o med wi h a NanoB ook 90 Plus om B ookha en Ins u- men s. Samples we e p epa ed wi h ul a-pu e wa e and analyzed immedia ely ae sonica ion. The mog a ime ic analysis (TGA). The mog a ime ic analyses we e ca ied ou using a diffe en ial scanning calo- ime e STA 449 F3 Jupi e (Ne zsch), equipped wi h a SiC o en. The samples we e analyzed in a ni ogen gas a mosphe e by an inc emen o he empe a u e o 5 C min 1 un il 900 C. Weigh loss o each sample was ob ained by measu emen s a diffe en empe a u es. T ansmission elec on mic oscopy (TEM) and ene gy dispe si e X- ay (EDX) mapping. B igh eld images and maps we e acqui ed a oom empe a u e using a Tecnai TF20 FEG- TEM wi h an ope a ing ol age o 200 keV  ed wi h a high angle annula da k eld (HAADF) de ec o and a Ga an O ius SC600 CCD came a. EDX maps we e collec ed a oom empe a u e using a FEI Ti an G2 S/TEM wi h an ope a ing ol age o 200 keV, a beam cu en o 0.1 nA, a con e gence angle o 18 m ad and a HAADF inne angle o 54 m ad. Bac e ial cap u e s udy wi h Y. en e ocoli ica s ains Ye sinia en e ocoli ica WC-A and FoxA WC-A 12-8 we e dona ed by P o esso Klaus Han ke (Uni e si y o T¨ ubingen, Ge many). T yp icase Soy B o h (TSB), T yp icase Soy Aga (TSA), Ringe s solu ion and PBS buffe we e p epa ed wi h dis illed wa e (DW) o biological assays. T yp icase soy b o h (TSB) cul u es o Y. en e ocoli ica (wild ype and mu an s ains) we e incuba ed up o an OD 600 be ween 0.5 and 0.8 in i on decien condi ions by adding 2,20- bipy idyl up o 100 mM. Then, 100 mLo a1mgmL 1 solu ion o ba e, MNP@SiO 2 , MNP@SiO 2 @NH 2 o MNP@SiO 2 @NH@Fa (4) was added o 1 mL o 1 : 100 dilu ion o each Y. en e ocoli ica s ain (equi alen o ca. 610 6 bac e ial cells pe mL) in Phospha e Buffe Saline (PBS) pH 7.4 and incuba ed o 1 h. The MNP/bac e ia agg ega es we e sepa a ed wi h a magne and he supe na an was ca e ully disca ded. The emaining agg ega es we e insed wice wi h PBS and e-suspended again in esh PBS. Se ial en- old dilu ions o his suspension we e pla ed on T yp icase Soy Aga (TSA) and incuba ed a 37 C o 24 h. Ae This jou nal is © The Royal Socie y o Chemis y 2019 RSC Ad .,2019,9, 13533–13542 | 13535 Pape RSC Ad ances Open Access A icle. Published on 01 May 2019. Downloaded on 7/9/2020 6:24:03 PM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online his ime, he colony o ming uni s (CFU) cap u ed wi h he MNP conjuga e we e coun ed. E alua ion o bac e ia–nanopa icle in e ac ion Scanning elec on mic oscopy (SEM). SEM images we e ob- ained using a FEI Quan a 650 FEGESEM en i onmen al SEM wi h an Ox o d Ins umen s INCA 350 EDX sys em/80 mm X-Max SDD de ec o , EBSD and KE Cen au us EBSD sys em. Image analysis was pe o med in ImageJ sowa e. 21 Y. en e ocoli ica WC- A was g own in 10 mL o TSB un il a OD 600 ¼0.5, hen he bac e ia in solu ion was dilu ed 1 : 10 and mixed wi h 1 mL o a suspension o MNP@SiO 2 @NH@Fa (4) in PBS. The bac e ia we e allowed o in e ac wi h he nanopa icles a oom empe - a u e o 1 h, hen he solids (bac e ia–nanopa icles) we e sepa a ed om he suspension wi h a magne , and insed wice wi h 1 mL o PBS. The cap u ed bac e ia we e mixed wi h 2.5% glu a aldehyde in 0.1 M phospha e buffe and allowed o eac o 2h.Ae ha ime, he solids we e insed wice wi h 0.1 M phospha e buffe o 30 min. Pos -xed samples we e mixed wi h 1% osmium e oxide in 0.1 M phospha e buffe o e nigh . Ae ha ime, he solids we e dehyd a ed using an ascending ace one se ies (20–40–60–80–100%) o 30 min, each un. Ae his, he samples we e d ied wi h a Pola on E3000 c i ical poin d ying appa a us using liquid ca bon dioxide as he ansi ion uid o affo d enough solid o be moun ed on 13 mm-diame e pin s ubs using double sided adhesi e ape. Finally, hese samples we e coa ed wi h pla inum o a hickness o 5 nm using a C essing on 208HR high esolu ion spu e coa ing uni . T ansmission elec on mic oscopy (TEM) and EDX maps. Bac e ia–nanopa icle in e ac ion was pe o med as desc ibed in he SEM analysis. The solids we e xed in 2.5% glu a alde- hyde and 0.1 M phospha e buffe o 2 h, and insed wice ( o 30 min each) wi h a 0.1 M phospha e buffe . 1% osmium e oxide in 0.1 M phospha e buffe was added o he pos xed sample and leo e nigh . Ae ha ime, samples we e dehy- d a ed using an ascending ace one se ies (20–40–60–80–100%), o 30 min each un. Then, he sample was ea ed wice wi h p opylene oxide o 20 min each ime. A 50 : 50 p opylene oxide–a aldi e solu ion was added o he sample and leo e - nigh , hen 25 : 75 and le o se e al hou s, and nally 100% a aldi e was added and le o 8 h. The esul ing p epa a ion was ans e ed o embedding moulds wi h esh a aldi e and polyme ase o e nigh a 60 C. Ul a- hin sec ions (sil e –gold 80–100 nanome e s) we e picked up on 3.05 mm g ids and s ained wi h sa u a ed u anyl ace a e (120 min). Resul s and discussion Su ace modica ion and cha ac e iza ion The p epa a ion o he conjuga e be ween e oxamine and unc ionalized silica-coa ed magne i e nanopa icles h ough he o ma ion o an amide bond is shown in Fig. 1. Fi s , magne i e (Fe 3 O 4 , MNP) was syn he ized using i on(III) ace yla- ce ona e (Fe(acac) 3 ) and benzyl alcohol, 15,22 and hen, i s su ace unc ionaliza ion was accomplished ia a ligand addi ion mechanism. The silica-coa ed magne i e (MNP@SiO 2 ) was hen unc- ionalized wi h 3-aminop opyl ie hoxysilane (APTES) using a sol–gel me hod 17 ha p o ides abundan NH 2 e minal unc- ional g oups on he coa ed pa icle su ace. Silane chemis y was employed o he su ace modica ion o ba e Fe 3 O 4 (MNP). The coa ing wi h SiO 2 using e ae hoxysilane (TEOS) 16 p o ided an adequa e scaffold o c ea e ailo ed a ia ion in he su ace unc ional g oups such as amine g oups as well as o acili a e he dispe sion o he nanopa icles in wa e , and he pos e io unc ionaliza ion would esul o be mo e uni o m. In pa allel, comme cial de e oxamine mesyla e sal (1) was complexed wi h i on(III) using aqueous Fe(acac) 3 o p oduce e oxamine complex (2) ha was hen ea ed wi h succinic anhyd ide o o m he co esponding N-succinyl e oxamine (3). 18 The coupling be ween he amine unc ionalized silica coa ed MNP (MNP@SiO 2 @NH 2 ) and N-succinyl e oxamine (3) using BOP and HOB 20 nally o med he desi ed MNP@SiO 2 @NH@Fa conjuga e (4) h ough he o ma ion o a co alen amide bond. Fu he unc ionaliza ion o MNP@SiO 2 @NH 2 and MNP@SiO 2 @NH@Fa (4) ia nucleophilic eac ion o –NH 2 wi h Boc 2 O and succinic anhyd ide allowed us he in oduc ion o Boc and ca boxylic acid g oups, espec i ely, in hei ee amine g oups o ob ain he nanopa icles MNP@SiO 2 @NHBoc and MNP@SiO 2 @NHCOOH and he conjuga es MNP@SiO 2 @- NHBoc@Fa (5) and MNP@SiO 2 @NHCOOH@Fa (6). The nal MNP@SiO 2 @NH@Fa conjuga e (4) and he MNP in e media e solids we e cha ac e ized by diffe en me hods including powde XRD, Raman Spec oscopy, FTIR, XPS, magne iza ion s udies, Z po en ial measu emen s, TEM and EDX mapping. XRD analysis con med he c ys alline s uc u e o ou syn he ic magne i e (MNP) by compa ing wi h he diff ac ion peaks o a s anda d magne i e JCPDS le 00-003-0863 (Fig. S1†). Raman analyses o he MNP@SiO 2 @NH@Fa (4) conjuga e and in e media es allowed us o con m he silica coa ing and unc ionaliza ion o he ba e MNP (Fig. 2). The peaks p esen in all Raman spec a a 305.8, 537.2 and 665.6 cm 1 co espond o Fe–O ib a ions. 23 The appea ance o a shoulde on he peak a 713.5 cm 1 in all spec a o silica coa ed MNP ela es o Si–O–Si ib a ions. 24 The MNP@SiO 2 @NH 2 spec um shows wo in ense peaks a 1001.5 and 1027.4 cm 1 also associa ed o he p esence o SiO 2 . The p esence o wo in ense and well-dened peaks a 1578.6 and 1597.9 cm 1 in he MNP@SiO 2 @NH 2 spec um con med he o ma ion o Si–C bonds. Mo eo e , a shoulde obse ed a 703.0 cm 1 con med he p esence o APTES (Fig. 2C). 25,26 The wo in ense peaks in he MNP@SiO 2 @NH 2 spec um a 1570 and 1590 cm 1 ela ed o Si–C bonds become a single b oade peak cen ed a 1580 cm 1 in he Raman spec um o he MNP@SiO 2 @NH@Fa (4) conjuga e due o he now p esence o amide g oups (1630–1680 cm 1 ). 26 Fig. 3 shows he FTIR spec a o MNP, MNP@SiO 2, MNP@SiO 2 @NH 2 and MNP@SiO 2 @NH@Fa (4). The beginning o a band wi hin he spec al ange o he analysis a 600 cm 1 in all he FTIR spec a ela es o he Fe–O ib a ions. The FTIR spec um o MNP@SiO 2 showed an in ense and b oad band a 1050 cm 1 co esponding o he Si–O–Si s e ching ib a ion con ming he silica coa ing, and i is was also p esen in he 13536 |RSC Ad .,2019,9, 13533–13542 This jou nal is © The Royal Socie y o Chemis y 2019 RSC Ad ances Pape Open Access A icle. Published on 01 May 2019. Downloaded on 7/9/2020 6:24:03 PM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online MNP@SiO 2 @NH 2 and MNP@SiO 2 @NH@Fa (4) spec a. The b oad band be ween 830 and 1275 cm 1 in he FTIR spec um o MNP@SiO 2 is a ibu ed o he Si–O bond, and he band becomes mo e in ense in he FTIR spec um o MNP@SiO 2 @- NH 2 as a esul o he unc ionaliza ion o MNP@SiO 2 wi h APTES and i is p obably due o he Si–C bond expec ed be ween 1175 and 1250 cm 1 . Finally, he FTIR spec um o MNP@SiO 2 @NH@Fa (4) shows bands a 2995 cm 1 (C–H s e ching bonds), 1640 cm 1 (O]C amide ib a ion) and 1577 cm 1 (O]C–N hyd oxamic acid ib a ion) ha con med he p esence o e oxamine conjuga ed wi h he nanopa icles. 27 FTIR spec a o MNP@SiO 2 @NHBoc@Fa (5) and MNP@SiO 2 @NHCOOH@Fa (6) a e shown in Fig. S2.† Magne iza ion s udies ae coa ing and unc ionaliza ion ea men s we e pe o med using hys e esis loop es s. The pa icles exhibi a supe pa amagne ic beha io , wi h only a li le emanence and coe ci i y, which sugges s he p esence o a long- ange magne ic dipole–dipole in e ac ion among he Fig. 1 Syn hesis o conjuga es MNP@SiO 2 @NH@Fa (4), MNP@SiO 2 @NHBoc@Fa (5) and MNP@SiO 2 @NHCOOH@Fa (6). Fig. 2 Raman spec a o ba e i on oxide (Fe 3 O 4 ) MNP (A), MNP@SiO 2 (B), MNP@SiO 2 @NH 2 (C) and MNP@SiO 2 @NH@Fa (D). (*) APTES, (**) o he i on oxide phases, likely o med om he ans o ma ion o magne i e by he lase powe . This jou nal is © The Royal Socie y o Chemis y 2019 RSC Ad .,2019,9, 13533–13542 | 13537 Pape RSC Ad ances Open Access A icle. Published on 01 May 2019. Downloaded on 7/9/2020 6:24:03 PM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online assemblies o supe pa amagne ic pa icles. The p og essi e dec ease in magne iza ion sa u a ion, 68.6 emu g 1 o MNP, 26.5 emu g 1 o MNP@SiO 2 , 30.5 emu g 1 and 2.53 emu g 1 o MNP@SiO 2 @NH@Fa (4), indica es he addi ion o diamagne ic ma e ial on he MNP su ace (Fig. S3†)and p obably, he elec on exchange be ween he su ace Fe a oms and he ligands. Despi e o magne iza ion dec ease, he MNP@SiO 2 @NH@Fa (4) e ained hei supe pa amagne ic beha io ae he ea men s, sugges ing ha hei magne ic p ope ies a e s ill ac i e o allow magne ic sepa a ion ae in e ac ion wi h bac e ia. Fig. 4 shows he XPS spec a o he ba e and diffe en unc- ionalized MNPs. The appea ance o a peak a 285 eV could be ela ed in pa o he ca bon in oduced du ing he unc ionali- za ion p ocess obse ed as C–CandC–H, bu also i could due o he p esence o ad en i ious ca bon on he samples. Ne e he- less, he inc easing in ensi y o he peaks obse ed a 286 eV as he unc ionaliza ion p og esses is a good indica o o he p es- ence o C–OH, C–O–C and C–N in MNP@SiO 2 @NH 2 , MNP@SiO 2 @NH@Fa (4), MNP@SiO 2 @NHBoc@Fa (5)and MNP@SiO 2 @NHCOOH@Fa (6). Likewise, he peak a 288 eV ela es o he p esence o C]O/O]C-bonds by he in oduc ion o -bu oxyca bonyl (Boc) and ca boxylic acid (–COOH). The peak a 399 eV in he N1s spec a con ms he o ma ion o amide bonds be ween MNP@SiO 2 @NH 2 and e oxamine as obse ed in he spec a o MNP@SiO 2 @NHBoc@Fa (5) and MNP@SiO 2 @- NHCOOH@Fa (6). Fu he mo e, he peak a 402 eV is a ibu ed o he N–O bond o hyd oxamic moie ies. In all unc ionalized MNP, a peak a 102 eV in Si2p spec a is obse ed, which is in good ag eemen wi h he he binding ene gy o he siloxane g oup. 28,29 The ze a po en ial o MNP and MNP@SiO 2 we e 25.21 and 29.35 mV, espec i ely. The unc ionaliza ion o MNP@SiO 2 wi h APTES o p oduce MNP@SiO 2 @NH 2 was con med by he change o su ace cha ge om nega i e o posi i e due o he p esence o amine g oups. 30 The ze a po en ial emains posi i e o he conjuga e MNP@SiO 2 @NH@Fa (4) and he blocked de i a i e MNP@SiO 2 @NHBoc@Fa (5). In he case o MNP@SiO 2 @NHCOOH@Fa (6), a dec ease o ze a po en ial alue (10.96 mV) was obse ed in compa ison wi h he alue ob ained o MNP@SiO 2 @NH@Fa (4) (22.14 mV) which was a ibu ed o he p esence o ca boxylic acid g oups (Table 1). The he mal loss o MNP ( ed line in Fig. 5) om 50 o 900 C was 1.5%, which migh be due o esidual loss o wa e and alcohol ( he empe a u e ange om 30 o 150 C, Fig. 5). The weigh loss in 5.6% o MNP@SiO 2 @NH 2 was a ibu ed o APTES deg ada ion and also o he loss o small amoun o wa e abso bed. The la ges weigh loss (11.1%) was ound o be o he conjuga e MNP@SiO 2 @NH@Fa (4), clea ly indica ing he p esence o o ganic ma e ial on he su ace. Fu he mo e, he TGA allowed us o es ima e ha MNP@SiO 2 @NH@Fa (4) we e ob ained wi h app oxima ely 7.62 10 5 mmol o e ox- amine pe 1 mg MNP@SiO 2 @NH 2 . The TGA da a o MNP@SiO 2 @NHBoc@Fa (5) and MNP@SiO 2 @NHCOOH@Fa (6) conjuga es can be seen in Fig. S4.† Fig. 6 shows b igh eld TEM images a medium and high esolu ion o ba e MNP and MNP@SiO 2 @NH 2 @Fa (4). Fig. 6A e ealed ha MNP we e 10 nm in diame e , al hough bigge pa icles (20 nm) we e also p esen . High esolu ion images con med he c ys allini y o hese nanopa icles as p e iously seen in he XRD analyses (Fig. 6B). The inges obse ed in he TEM image co espond o d-spacings o 2.9 and 2.4 ˚ A o he c ys al planes (220) and (311) o magne i e. 31 The elec on diff ac ion pa e n showed b igh spo s ha ma ch wi h he (111), (220), (311), (400), (422), (511) and (440) diff ac ion planes o magne i e co esponding o d-spacings o 4.9, 2.9, 2.4, 2.0, 1.7, 1.6 and 1.4 ˚ A, espec i ely (Fig. 6C). In addi ion, he p esence o ings along wi h small spo s demon- s a ed he o ma ion o a polynanoc ys alline magne i e. TEM images o MNP@SiO 2 @NH 2 @Fa (4) showed dispe sed MNP pa icles (10 nm) embedded in he amo phous ino ganic– o ganic ma e ial (Fig. 6D and E). The c ys alline inges o magne i e a e s ill isible in high esolu ion and in he elec on diff ac ion images (Fig. 6F). Chemical composi ion o he nanopa icles ae he Si coa ing was de e mined by ene gy dispe si e X- ay (EDX) maps. Fig. 7A shows he high angle annula da k eld (HAADF) image o MNP@SiO 2 and he maps o Fe, Si, O and C. They show ha Fe is homogeneously dis ibu ed all o e he MNP@SiO 2 while Si appea s widely dis ibu ed no only h oughou he nano- pa icle, bu also ex ended o he sides (i.e., coa ing laye is obse ed in he da ke a ea o he HAADF image). On he o he hand, C was de ec ed in low concen a ions likely due o con amina ion. Al hough simila esul s we e obse ed o Fe, Si and O in he MNP@SiO 2 @NH@Fa (4) esul s, he concen- a ion o C inc eased homogenously due o he addi ion o ca bon laye s on he nanopa icle su ace (Fig. 7B). Bac e ia cap u e s udies Once he MNPs we e cha ac e ized, we ca ied ou expe imen s o e alua e he capabili ies o ba e and unc ionalized magne ic Fig. 3 FT-IR spec a o ba e i on oxide (Fe 3 O 4 ) MNP (black), MNP@SiO 2 ( ed), MNP@SiO 2 @NH 2 (blue) and MNP@SiO 2 @NH@Fa (4) (g een). 13538 |RSC Ad .,2019,9, 13533–13542 This jou nal is © The Royal Socie y o Chemis y 2019 RSC Ad ances Pape Open Access A icle. Published on 01 May 2019. Downloaded on 7/9/2020 6:24:03 PM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online nanopa icles o cap u e wild ype (WC-A) and a mu an lacking e oxamine ecep o FoxA (FoxA WC-A 12-8) Y. en e ocoli ica s ains. Ba e MNP and unc ionalized MNPs we e incuba ed in a PBS solu ion con aining each Y. en e ocoli ica s ain. The agg ega es we e hen sepa a ed om he bac e ia suspension by using a magne . Ae insing he sepa a ed agg ega es wo imes wi h PBS, hey we e e-suspended in PBS, o p epa e se ial dilu ions ha we e pla ed o colony coun ing. The esul s ob ained om colony coun ing a e shown in Fig. 8. Bo h Y. en e ocoli ica s ains e alua ed did no show a signican binding specici y o he unc ionalized MNP in ela ion o ba e MNP. The lack o binding specici y is likely Fig. 4 XPS na ow spec a o MNP, MNP@SiO 2 @NH 2 , MNP@SiO 2 @NH@Fa (4) MNP@SiO 2 @NHBoc@Fa (5) and MNP@SiO 2 @NHCOOH@Fa (6). Table 1 Ze a po en ial measu emen s Sample Zpo en ial MNP 25.21 MNP@SiO 2 29.35 MNP@SiO 2 @NH 2 17.03 MNP@SiO 2 @NH@Fa (4) 22.14 MNP@SiO 2 @NHBoc@Fa (5) 19.16 MNP@SiO 2 @NHCOOH@Fa (6) 10.96 Fig. 5 The mog a ime ic analysis o MNP ( ed), MNP@SiO 2 @NH 2 (g een), and MNP@SiO 2 @NH@Fa (pink). Fig. 6 B igh field TEM images and elec on diff ac ion o ba e MNP (A, B and C), and o MNP@SiO 2 @NH@Fa (4) (D, E and F). Images a medium and high esolu ion. This jou nal is © The Royal Socie y o Chemis y 2019 RSC Ad .,2019,9, 13533–13542 | 13539 Pape RSC Ad ances Open Access A icle. Published on 01 May 2019. Downloaded on 7/9/2020 6:24:03 PM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online caused by su ace in e ac ions be ween nanopa icles and bac e ia. Mos bac e ia ha e a ne nega i e su ace cha ge, pa icula ly du ing he ea ly s a iona y phase o cell g ow h, 32,33 ha makes hem o p e e en ially in e ac wi h posi i ely cha ged su aces such as MNP@SiO 2 @NH 2 , due o he p esence o ee amine g oups h ough he p o ona ion in physiologic solu ion. The e o e, ou esul s a e in good ag eemen wi h p e ious wo ks epo ing bac e ia adso p ion h ough ee amine g oups o unc ionalized MNP. 30,34,35 The bac e ia adso p ion achie ed wi h MNP@SiO 2 pa icles can be a ibu ed o mu ually hyd ophobic in e ac ion. 36 In o de o educe he non-specic binding beha io due o he elec os a ic in e ac ions be ween he ee amine unc ion- alized nanopa icles and bac e ia, we made a emp s o block he su ace o he pa icles wi h wo diffe en g oups, one o hem o neu al na u e (Boc) and a second g oup wi h pola cha ac e (COOH). Boc g oups we e in oduced on o MNP@SiO 2 @NH 2 and MNP@SiO 2 @NH@Fa (4) by using (Boc) 2 O o gi e MNP@SiO 2 @NHBoc and conjuga e MNP@SiO 2 @NHBoc@Fa (5), espec i ely. Ca boxylic acid g oups we e also in oduced on o he same conjuga e by using succinic anhyd ide o gi e he co esponding MNP@SiO 2 @- NHCOOH and MNP@SiO 2 @NHCOOH@Fa (6) simila o hose epo ed by Gunawan and cowo ke s. 34 When es ing he bac e ia cap u e wi h hese new conju- ga es, he colony coun ing did no show any signican changes o he adso p ion o Y. en e ocoli ica WC-A (wild ype s ain) indica ing ha he molecula ecogni ion o he side opho e again was no obse ed (Fig. 9A and B). Thus, hese modica- ions we e no enough o a enua e he elec os a ic in e ac ions be ween bac e ia and he modied nanopa icles as con med wi h he low dec ease alue o ze a po en ial (Table 1). Simila esul s we e ob ained when he expe imen s we e epea ed wi h and wi hou i on deciency g ow h condi ions (Fig. S8†). Fig. 10A shows he a achmen o he nano-sized conjuga e MNP@SiO 2 @NH@Fa (4) o he su ace o Y. en e ocoli ica WC-A. Fig. 7 EDX maps o MNP@SiO 2 : HAADF image and he co esponding Fe, Si, O and C maps o (A) MNP@SiO 2 and (B) MNP@SiO 2 @NH@Fa (4). Fig. 8 CFU o Y. en e ocoli ica cap u ed pe 100 mg o magne ic nanopa icles: ba e, MNP@SiO 2 , MNP@SiO 2 @NH 2 and MNP@SiO 2 @- NH@Fa (4). (A) WC-A (wild ype) (B) FoxA WC-A 12-8 (mu an lacking e oxamine ecep o FoxA). Fig. 9 CFU o Y. en e ocoli ica WC-A (wild ype) cap u ed pe 100 mg o magne ic nanopa icles (A) MNP@SiO 2 @NH 2 , MNP@SiO 2 @NHBoc, MNP@SiO 2 @NH@Fa (4), MNP@SiO 2 @NHBoc@Fa (5), (B) MNP@SiO 2 @NH 2 , MNP@SiO 2 @NHCOOH, MNP@SiO 2 @NH@Fa (4), MNP@SiO 2 @NHCOOH@Fa (6). Fig. 10 (A) SEM images o Y. en e ocoli ica WC-A in e ac ing wi h MNP@SiO 2 @NH@Fa (4). (B) TEM images o Y. en e ocoli ica WC-A in e ac ing wi h MNP@SiO 2 @NH@Fa (4), (B1) a achmen o nano- pa icles o he su ace o a single bac e ia, (B2) de ail o he a ach- men on he bac e ial memb ane. 13540 |RSC Ad .,2019,9, 13533–13542 This jou nal is © The Royal Socie y o Chemis y 2019 RSC Ad ances Pape Open Access A icle. Published on 01 May 2019. Downloaded on 7/9/2020 6:24:03 PM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online The co esponding hin-sec ioned samples measu ed by TEM (Fig. 10B1 and B2) con med he capabili y o he modied nanopa icles o a ach o he bac e ial memb ane. Addi ional images and EDX maps a e shown in Fig. S7 o he ESI.† Conclusions In his s udy, we desc ibe he p epa a ion o he conjuga e MNP@SiO 2 @NH@Fa (4) using su ace modied magne ic nanopa icles and de e oxamine i on(III) complex ( e oxamine) and i s s uc u al cha ac e iza ion using se e al echniques. The in e ac ion o MNP@SiO 2 @NH@Fa (4) wi h Y. en e - ocoli ica WC-A and FoxA WC-A 12-8 showed no signican diffe ence in he numbe o colonies cap u ed in ela ion o ba e, MNP@SiO 2 , and MNP@SiO 2 @NH 2 . The lack o binding specici y was a ibu ed o he p esence o elec os a ic o ces such as he posi i e cha ged ee amine g oups p esen in MNP@SiO 2 @NH 2 and he low concen a ion o side opho e memb ane ecep o in bac e ia. These esul s sugges ha he elec os a ic and o he su ace in e ac ions a e dominan o e hose due o he molecula ecogni ion be ween MNP conjuga e and e oxamine ecep o . The effec o ee amine g oups and he change o cha ge on he su ace we e e alua ed wi h Boc and COOH g oups in MNP@SiO 2 @NHBoc@Fa (5) and MNP@SiO 2 @NHCOOH@Fa (6), espec i ely. Un o una ely, hese new conjuga es did no imp o e bac e ia cap u e. Fu he effo s a e needed o explo e o he blocking ma e ials in o de o emo e o dec ease non-specic binding o magne ic nano- pa icles su ace o bac e ia. While he epo ed side opho e- based me hods o de ec ion o mic obial pa hogens allow he de ec ion o he a ge bac e ia, he de elopmen o he p esen s a egy would also allow bac e ia isola ion om a complex mix u e o mic oo ganisms o hei pos e io iden ica ion. Conflic s o in e es The e a e no conic s o decla e. Acknowledgemen s The au ho s g a e ully acknowledge P o esso Klaus Han ke (Uni e si y o T¨ ubingen, Ge many) o kindly supply he Ye sinia en e ocoli ica s ains used in his wo k. This wo k was suppo ed by g an s AGL2015-63740-C2-2-R and AGL2015-63740-C2-1-R (AEI/FEDER, EU) om he S a e Agency o Resea ch (AEI) o Spain, bo h co- unded by he FEDER P og amme om he Eu opean Union. No es and e e ences 1 Y. Pan, X. Du, F. Zhao and B. Xu, Chem. Soc. Re ., 2012, 41, 2912–2942. 2 R. A. Boha a, N. D. Tho a and S. H. Pawa , RSC Ad ., 2016, 6, 43989–44012. 3 O. Lazcka, F. J. Del Campo and F. X. Mu˜ noz, Biosens. Bioelec on., 2007, 22, 1205–1217. 4 T. Mocan, C. T. Ma ea, T. Pop, O. Mos eanu, A. D. 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