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. Buzoianu,
C. Puia, C. Iancu and L. Mocan, J. Nanobio echnol., 2017, 15,
25.
5 T. Zheng and E. M. Nolan, Me allomics, 2012, 4, 866–880.
6 R. C. Hide and X. Kong, Na . P od. Rep., 2010, 27, 637–657.
7 M. Sandy and A. Bu le , Chem. Re ., 2010, 109, 4580–4595.
8 D. D. Doo newee d, W. A. Henne, R. G. Rei enbe ge and
P. S. Low, Langmui , 2010, 26, 15424–15429.
9 Y. Kim, D. P. Ly e s, A. Wei, R. G. Rei enbe ge and P. S. Low,
Lab Chip, 2012, 12, 971–976.
10 T. Inoma a, H. Eguchi, Y. Funahashi, T. Ozawa and
H. Masuda, Langmui , 2012, 28, 1611–1617.
11 T. Inoma a, H. Tanabashi, Y. Funahashi, T. Ozawa and
H. Masuda, Dal on T ans., 2013, 42, 16043–16048.
12 J. Hu, M. Ghosh, M. J. Mille and P. W. Bohn, Anal. Me hods,
2019, 11, 296–302.
13 S. Wu, Z. Zhang, X. Wang, M. Zhang, J. Peng, Z. Xie and
D. Pang, J. Phys. Chem. C, 2009, 113, 9169–9174.
14 N. Bugdahn, F. Peucke , A. G. Alb ech , M. Mie hke,
M. A. Ma ahiel and M. Obe h¨
u , Angew. Chem., In . Ed.,
2010, 49, 10210–10213.
15 N. Pinna, S. G ancha o , P. Bea o, P. Bon ille, M. An onie i
and M. Niede be ge , Chem. Ma e ., 2005, 17, 3044–3049.
16 Y. S. Li, J. S. Chu ch, A. L. Woodhead and F. Moussa,
Spec ochim. Ac a, Pa A, 2010, 76, 484–489.
17 J. P. Chen, P. C. Yang, Y. H. Ma, S. J. Tu and Y. J. Lu, In . J.
Nanomed., 2012, 7, 5137–5149.
18 D. Goswami, M. T. Machini, D. M. Sil es e, C. S. Nomu a
and B. P. Esposi o, Bioconjuga e Chem., 2014, 25, 2067–2080.
19 J. D. He scheid, a Hoeks a and C. M. Vos, Eu . J. Nucl. Med.,
1984, 9, 508–510.
20 K. El-Boubbou, C. G uden and X. Huang, J. Am. Chem. Soc.,
2007, 129, 13392–13393.
21 M. D. Ab `
amoff, P. J. Magalh˜
aes and S. J. Ram, Biopho onics
In ., 2004, 11,36–42.
22 J. Ya˜
nez-Vila , M. S´
anchez-Anduja , S. Cas o-Ga c´
ıa, J. Mi a,
J. Ri as and M. A. Se˜
na ´
ıs-Rod ´
ıguez, Bol. Soc. Esp. Ce am.
Vid io, 2010, 49,81–88.
23 O. N. Shebano a and P. Lazo , J. Solid S a e Chem., 2003, 174,
424–430.
24 P. Gonz´
alez, J. Se a, S. Lis e, S. Chiussi, B. Le´
on and
M. P´
e ez-Amo , J. Non. C ys . Solids, 2003, 320,92–99.
25 M. Ve es, M. Ko´
os, S. T´
o h, M. F¨
ule, I. P´
ocsik, A. T´
o h,
M. Mohai and I. Be ´
o i, Diam. Rela . Ma e ., 2005, 14,
1051–1056.
26 Y. You, T. Yu, J. Kasim, H. Song, X. Fan, Z. Ni, L. Cao,
H. Jiang, D. Shen, J. Kuo and Z. Shen, Appl. Phys. Le .,
2008, 93, 103111–103113.
27 O. Coza , N. Leopold, C. Jelic, V. Chis, L. Da id, A. Mocanu
and M. Tomoaia-Co isel, J. Mol. S uc ., 2006, 788,1–6.
28 N. G a , E. Yegen, T. G oss, A. Lippi z, W. Weigel, S. K ake ,
A. Te o and W. E. S. Unge , Su . Sci., 2009, 603, 2849–
2860.
29 W. Michaeli, C. J. Blomeld, R. D. Sho , F. R. Jones and
M. R. Alexande , Appl. Su . Sci., 2002, 137, 179–183.
30 W. Fang, C. Han, H. Zhang, W. Wei, R. Liu and Y. Shen, RSC
Ad ., 2016, 6, 67875–67882.
This jou nal is © The Royal Socie y o Chemis y 2019 RSC Ad .,2019,9, 13533–13542 | 13541
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