nanoma e ials
A icle
A No el Biocompa ible Ti anium–Gadolinium
Quan um Do as a Bac e ial De ec ing Agen wi h
High An ibac e ial Ac i i y
Vishma P a ap Su 1,2,*,†, Aninda Mazumda 1,2,†, Ami mansoo Ash a i 1,2 ,
A ipan Mukhe jee 1,2, Ved an Milosa lje ic 1,2 , Hana Michalko a 1, Pa el Kopel 3,
Lukáš Rich e a 1,2 and Ami a a Moulick 1,2,*
1Depa men o Chemis y and Biochemis y, Facul y o Ag iSciences, Mendel Uni e si y in B no,
Zemedelska 1/1665, CZ-61300 B no, Czech Republic; [email p o ec ed] (A.M.);
ami mansoo [email p o ec ed] (A.A.); [email p o ec ed] (A.M.); [email p o ec ed] (V.M.);
[email p o ec ed] (H.M.); [email p o ec ed] (L.R.)
2Cen al Eu opean Ins i u e o Technology (CEITEC), B no Uni e si y o Technology, Pu kyno a 123,
CZ-62100 B no-K álo o Pole, Czech Republic
3Depa men o Ino ganic Chemis y, Facul y o Science, Palacky Uni e si y, 17. lis opadu 12,
CZ-771 46 Olomouc, Czech Republic; [email p o ec ed]
*Co espondence: [email p o ec ed] o [email p o ec ed]
(V.P.S.); [email p o ec ed] o [email p o ec ed] (A.M.);
Tel.: +420-545-133-350 (V.P.S.); +420-545-133-350 (A.M.)
†These au ho s con ibu ed equally o his wo k.
Recei ed: 18 Ma ch 2020; Accep ed: 16 Ap il 2020; Published: 17 Ap il 2020


Abs ac :
In his s udy, he i anium–gadolinium quan um do s (TGQDs) we e no el, i s o i s
ype o be syn hesized, and ully cha ac e ized o da e. Mul iple physical cha ac e iza ion includes
scanning elec on mic oscopy (SEM), scanning elec ochemical mic oscope (SCEM), x- ay luo escence,
spec opho ome y, and dynamic ligh sca e ing we e ca ied ou . The ob ained esul s con i med
app op ia e size and shape dis ibu ions in addi ion o p ocessing op ical ea u es wi h high quan um
yield. The syn hesized TGQD was used as a luo escen dye o bac e ial de ec ion and imaging
by luo escen mic oscopy and spec opho ome y, whe e TGQD s ained only bac e ial cells, bu
no human cells. The signi ican an ibac e ial ac i i ies o he TGQDs we e ound agains a highly
pa hogenic bac e ium (S aphylococcus au eus) and i s an ibio ic esis an s ains ( ancomycin and
me hicillin esis an S aphylococcus au eus) using g ow h cu e analysis and de e mina ion o minimum
inhibi o y concen a ion (MIC) analysis. Li e/dead cell imaging assay using phase-con as mic oscope
was pe o med o u he con i ma ion o he an ibac e ial ac i i y. Cell wall dis up ion and elease
o cell con en was obse ed o be he p ime mode o ac ion wi h he educ ion o cellula oxygen
demand (OD).
Keywo ds:
bac e ial esis ance; i anium–gadolinium quan um do s; bac e ial de ec ion; an ibac e ial
ac i i y; SECM
1. In oduc ion
An ibio ic esis ance in bac e ia is a majo challenge o medical sciences and he p io i y lis by
Wo ld Heal h O ganiza ion (WHO) ca ego izes i in o c i ical, high, and medium [
1
]. Resea ch o he
pas wo decades ha e p oduced only wo new ypes o an ibio ics: lipopep ides and oxazolidinones,
al hough majo ly o e coming an ibio ic esis ance has no been achie ed ye . The p esen si ua ion
o bac e ial in ec ions is conside ed o be a h ea o human socie y due o he e ol ing s ains o
Nanoma e ials 2020,10, 778; doi:10.3390/nano10040778 www.mdpi.com/jou nal/nanoma e ials
Nanoma e ials 2020,10, 778 2 o 18
S aphylococcus au eus wi h esis ance o b oad- ange an ibio ics such as me hicillin and ancomycin [
2
].
The in ec ions caused by he esis an bac e ia a e usually ea ed wi h exis ing an ibio ics, which
usually exe s side e ec s wi h hei incomple e dosage and imp ope use. The o e use and misuse
o an ibio ics a e he key ac o s con ibu ing o an ibio ic esis ance [
3
]. The o e use o an ibio ics,
especially aking an ibio ics e en when hey a e no he app op ia e ea men , p omo es an ibio ic
esis ance [
4
]. On he o he hand side, he e ec s can be a ied, o example, omi ing, nausea ( eeling
like one may omi ), dia hea, bloa ing and indiges ion, abdominal pain, loss o appe i e, a aised,
i chy skin ash (u ica ia, o hi es), coughing, wheezing, igh ness o he h oa , eeling ligh headed
o ain , b ea hing di icul ies such as as , shallow b ea hing, wheezing, a as hea bea , clammy
skin, con usion and anxie y, collapsing o losing consciousness [
5
]. Fu he mo e, some an ibac e ial
agen s can elici ad e se e ec s agains he hepa ic sys em, whe e he ypes o li e damage induced
by an ibac e ial agen s co e cy o oxic, inju y, choles a ic inju y, mixed cy o oxic and choles a ic
inju y, s ea osis, ch onic, ac i e hepa i is, and ci hosis [
6
]. This u he inc eases he complexi y in he
de elopmen o he esis ance agains hese molecules, assis ing i in being a global heal h p oblem [
7
,
8
].
Thus, he impo ance o de eloping new ea men s a egies o al e na i es o an ibio ics agains
hese mul id ug esis an bac e ial s ains a e he majo demands in 21s cen u y medical science [
9
–
11
].
To o e come he an ibio ic esis ance and absolu e necessi y o inding an al e na i e o exis ing
an ibio ics wi h negligible oxici y, p ope biocompa ibili y, and ul il he needs o clinical esea ch,
no el an ibac e ial agen s like nanopa icles, pep ides, me aboli es, oligonucleo ides, and o he biogenic
o chemical compounds ha e been in oduced [
8
,
12
–
17
]. Nanopa icles, nowadays, a e he ocus o
medical sciences wi h a ious applica ions like d ug deli e y, acking, and al e na i es o an ibio ics
such as nanomedicine [
18
–
20
]. Howe e , nanopa icles also ha e ad e se e ec s owa d human heal h.
Nanopa icles exe a ious kinds o oxici y, hey can be cance ous, a ec ou immune sys em, li e ,
spleen, can gene a e cellula ROS, damage ou DNA, o hey can also a ec ou me abolism h ough
blocking o dis up ing a ious enzyma ic pa hways [21,22].
The de ec ion and imaging o bac e ia by s aining has been a eliable me hod o mo e han a
cen u y. Comme cial una ailabili y o any bac e ia speci ic luo escen s ain has limi ed he p ocess o
luo escen labelling. Mos o he exis ing s ains ollow a gene al mode o ac ion, whe e i s ains almos
all ypes o cells including mammalian cells, o i is a i icially designed o speci ic a ge s aining. Fo
example, BacLigh g een is a bac e ial speci ic s ain ha con ains SYTO9, equen ly used in li e/dead
cell imaging o bac e ia, bu SYTO9 is an in e cala ing memb ane pe meable g een s ain ha s ains
all cells con aining nucleic acid [23]. Mo eo e he i al s ain, luo escein diace a e (FDA) is based on
in acellula hyd olysis o FDA and in he case o li ing cells, he non- luo escen FDA is con e ed
in o luo escein, a g een luo escen compound, indica ing he cell iabili y [
24
,
25
]. The de ec ion o
bac e ia in human issue du ing in ec ion de elopmen , o he hos pa hogen in e ac ion s udy by using
a luo escen dye is qui e challenging due o he gene alized mode o ac ion o comme cialized o ganic
dyes. Thus, he necessi y o de eloping a bac e ia speci ic s ain o imp o e he bac e ial de ec ion and
imaging is equi ed and long awai ed.
Nanopa icles (NPs) can be de i ed om a ious sou ces in a desi able nanome e (nm) size. Due
o hei enhanced e en ion and pe meabili y, NPs ha e a b oad ange o clinical applica ions along
wi h hei abili y o accumula e he si es o in ec ion by enhancing e en ion and he pe meabili y
e ec [
26
]. NPs wi h ew modi ica ions help in si e speci ic d ug deli e y by educing a ge oxici y [
27
].
Quan um do s (QDs) a e a ype o nanopa icles wi h he abili y o luo esce, which is helping in
he de elopmen o new de ec ion and imaging echniques [
27
–
29
]. A ew no el QDs ha e been
epo ed o ha e an ibac e ial [
30
–
32
] and an icance p ope ies [
33
,
34
], which ele a es hei alue e en
mo e in he ield o biomedicine wi h some d awbacks like oxici y, in lamma ion, and s abili y [
27
].
Ti anium dioxide (TiO
2
) based nanocomposi es ha e been epo ed o ha e an imic obial ac i i y
agains Esche ichia coli,S aphylococcus au eus,S ep ococcus sob inus,Pseudomonas pu ida,Pseudomonas
ae uginosa, and Lis e ia innoculim due o he p oduc ion o eac i e oxygen species (ROS), which can
inac i a e o ganic and ino ganic pollu an s and consequen ly inac i a e he mic oo ganisms, e en
Nanoma e ials 2020,10, 778 3 o 18
whe e en i onmen al and economic ac o s ha e aised hei olume [
35
–
38
]. The gadolinium based
nanopa icles also ha e an imic obial ac i i ies agains Bacillus sub ilis,E. coli, and S aphylococcus
au eus [
39
,
40
]. Though bo h gadolinium and TiO
2
nanopa icles a e biologically ac i e, no s udy has
es ed hei eal implica ions and e ec s agains ancomycin esis an S aphylococcus au eus (VRSA)
and me hicillin esis an S aphylococcus au eus (MRSA) ye .
In his s udy, we used i anium and gadolinium oge he o syn hesize a i anium–gadolinium
quan um do (TGQG), he i s o i s kind, and i s cha ac e iza ion was unde aken using dynamic
ligh sca e ing, spec opho ome y, X- ay luo escence, scanning elec ochemical mic oscope (SECM),
and scanning elec on mic oscope (SEM). The ea e , TGQD was used as a s ain in he de ec ion o
VRSA using a luo escence mic oscope. Fu he mo e, he an ibac e ial ac i i y was es ed agains he
pa hogenic s ains o ancomycin esis an S. au eus (VRSA), me hicillin esis an S. au eus (MRSA),
and he mechanism o ac ion was also in es iga ed. Finally, he cy o oxici y es was pe o med and
conclusions we e d awn.
2. Ma e ials and Me hods
2.1. Chemicals
2-Py idineca boxaldehyde, die hylene iamine, gadolinium (III) ni a e, i anium (IV) bu oxide,
isop opanol, and me hanol we e ob ained om Sigma-Ald ich (S . Louis, MO, USA). Mulle Hin on
b o h (MH) was ob ained om HiMedia Labo a o ies P . L d., Thane, India.
2.2. Chemicals and Syn hesis o Ti anium–Gadolinium Quan um Do s (TGQDs)
The p epa a ion o he gadolinium Schi base was acco ding o ou p e ious s udies wi h necessa y
modi ica ion [
28
]. Ini ially, 1.08 mL o die hylene iamine and 1.9 mL o 2-py idineca boxaldehyde
we e mixed and hea ed unde e lux in 35 mL o me hanol (MeOH) o 6 h. To p epa e he desi ed
solu ion o Schi base [(2-[(E)-2-py idylme hyleneamino]-N-[2-[(E)-2-py idylme hylene-amino]e hyl]
-e hanamine)], he solu ion was cooled and he olume was made up o 50 mL using MeOH. In a
sepa a e beake , 5 mL o gadolinium ni a e aqueous solu ion (90 mg/mL) was mixed wi h 10 mL o
MeOH, which was subsequen ly mixed wi h 5 mL solu ion o he Schi base. Finally, he solu ions
we e mixed on ho pla e magne ic s i e o 2 h a 40
◦
C and he olume was made up o 100 mL
wi h deionized wa e . The Gadolinium (Gd)–Schi base solu ion was s o ed a 25
◦
C. To p epa e he
i anium dioxide (TiO
2
) solu ion, 50 mL o isop opanol and 50
µ
L o i anium bu oxide was pu in o a
glass beake and placed on a magne ic s i e o 48 h, and he clea solu ion u ned in o a milky whi e
solu ion wi hou p ecipi a ion.
Then, he aqueous phase syn hesis o quan um do s was ca ied ou by mixing he Gd–Schi base
solu ion wi h TiO
2
in a beake . The Gd–Schi base and TiO
2
solu ions we e mixed in he a io o 1:1.
La e , 2 mL o he solu ion was aken in a glass ube and hea ed o 10 min a 80
◦
C, 300 W, ( amping
ime, 10 min) unde mic owa e i adia ion (Mul iwa e 3000, An on Paa GmbH, G az, Aus ia) o
p epa e he TG quan um do s. The samples we e il e ed using 3 kDa (Amicon Ul a 0.5 mL cen i ugal
il e s) il e s and u he il e ed h ough a 0.22
µ
m memb ane. To emo e he un eac ed ini ia o s,
he solu ion was dialyzed agains deionized wa e se e al imes, hen he TGQD we e d ied unde a
acuum d y sys em.
2.3. Cha ac e iza ion o TGQD
The p epa ed TGQDs we e isualized unde a UV ansillumina o a exci a ion wa eleng hs
(
λex
) o 312 nm and 270 nm (T ansillumina o Mul iband TFX-35.MC, To cy, F ance). The luo escence
and abso bance spec a o he TG QDs we e ob ained using a mic opla e (UV pla e, 96 well; Co ning
Inco po a e, Co ning, NY, USA) in Tecan In ini e m200 PRO (Männedo , Swi ze land). A o al o
100
µ
L o he samples we e used o he measu emen s. The luo escence spec um was measu ed
using an exci a ion wa eleng h (
λex
) o 230 nm, he emission wa eleng h (
λem
) ange was 280–850
Nanoma e ials 2020,10, 778 4 o 18
nm, and he abso bance spec um o he TGQDs was measu ed om 230 o 1000 nm. The QDs we e
obse ed unde a luo escence mic oscope wi h a UV il e o isualiza ion. The pho oluminescence
quan um yield o he TGQDs was de e mined using he e e ence as hodamine 6G acco ding o a
epo ed p o ocol [41].
The scanning elec on mic oscopy (SEM) ins umen (TESCAN Company, B no, Czech Republic,
EU) was used o obse e he TGQDs unde he ollowing condi ions: high acuum mode (10–3 Pa),
ol age o 15 kV, and wo k dis ance o 3 mm [
28
].The scanning elec ochemical mic oscopy (SECM)
measu emen s we e pe o med using a CHI 900 se up (CH Ins umen Inc., Aus in, TX, USA). A P
ul amic oelec ode (d=10 mm) was he SECM ip (RG ac o =10), a ba e glassy ca bon (GC) co e ed
wi h 10
µ
L Na ion 1%, and a GC immobilized wi h TGQDs and co e ed wi h 10
µ
L Na ion 1% se ed as
he SECM subs a es. The ip was posi ioned nea he subs a e using he p obe-app oach cu e (PAC)
echnique and a po en ial o 500 mV was applied o he ip wi h a scan a e o 0.5
µ
m/s. A pla inum
wi e was he coun e elec ode and he e e ence elec ode was Ag/AgCl (3 M KCl). A solu ion o
1 mM o e oceneme hanol (FcOH) in 0.1 M KCl was used as he edox media o . All po en ials we e
e e ed o he e e ence elec ode.
TGQDs we e also cha ac e ized using an elemen al analyze SPECTRO XEPOS ene gy dispe si e
x- ay luo escence (ED-XRF) spec ome e (SPECTRO Analy ical Ins umen s GmbH, Kle e, Ge many)
equipped wi h a 10 mm
2
Si-D i De ec o wi h Pel ie cooling and a 75
µ
m Be side window was
employed. The ins umen uses a Pd- a ge end window ube a a maximum powe o 50 W and
a maximum ol age o 50 kV. Spec al esolu ion o he ins umen (FWHM) was <170 eV o Mo
K
α
(measu ed unde inpu coun a e 10,000 pulses). SPECTRO XEPOS was ope a ed and da a
we e e alua ed by means o he so wa e Spec o X-Lab P o, Ve sion 2.5, Kle e, Ge many. Fo he
exci a ion o ligh elemen s (Mg–V, 25 kV), a HOPG (highly o ien ed py oli hic g aphi e) c ys al
a ge was used. Fo he de e mina ion o hea ie elemen s, a Mo seconda y a ge (C –Y, H –U,
45 kV) and Al
2
O
3
pola iza ion a ge (Z –Ce, 49.5 kV) we e used. The sample (1.0 mL) was d ied
di ec ly a 70
◦
C in a sample cup (32 mm in diame e ) on polyp opylene hin- ilm (Specac L d., Ken ,
UK) and measu ed in acuum using he so-called Tu boquan me hod ( undamen al pa ame e s
me hod) [
42
]. Fou ie ans o m in a ed (FTIR) spec a we e measu ed using a The mo Scien i ic
Nicole iS5 spec ome e (The mo Fishe Scien i ic, Wal ham, MA, USA) equipped wi h an iD5 Diamond
ATR accesso y o e a wa e numbe ange o 4000–550 cm
–1
. The a e age size o he TG QDs, he size
dis ibu ion, and ze a po en ial we e de e mined by quasielas ic lase dynamic ligh sca e ing (DLS)
wi h a Mal e n Ze asize (NANO-ZS, Mal e n Ins umen s L d., Wo ces e shi e, UK). Ini ially, 1.5 mL
o an aqueous solu ion o TGQDs (1 mg/mL) was pou ed in o a polys y ene la ex cell and measu ed a
a empe a u e o 25
◦
C wi h a de ec o angle o 173
◦
, a wa eleng h o 633 nm, a e ac i e index o 0.30,
and a eal e ac i e index o 1.59 [28].
2.4. Applica ion o TGQD on Bac e ia o De ec ion and Killing
The bac e ial s ains S aphylococcus au eus (S. au eus) (NCTC 8511) [
43
,
44
], ancomycin esis an
S aphylococcus au eus (CCM 1767) [
19
,
45
], and me hicillin esis an S aphylococcus au eus (MRSA)
ST239:SCCmec IIIA) [
46
] we e ob ained om he Czech Collec ion o Mic oo ganisms, Facul y o
Science, Masa yk Uni e si y, B no, Czech Republic and om England, wi h he coope a ion o he
Uni e si y o Camb idge [
19
,
43
–
46
]. The bac e ial cells we e cul i a ed in Mulle Hin on (MH) b o h
medium, pH 7.4. The bac e ial cul u es we e cul i a ed o e nigh a 37
◦
C in a shaking incuba o .
Bac e ial op ical densi y was adjus ed o 0.1 abso bance (0.5 MacFa land s anda ds) a 600 nm o he
subsequen expe imen s [45–50].
The abso bance spec al scan was eco ded o he bac e ial sample o hei de ec ion by using
Tecan In ini e m200 PRO immedia ely a e p epa ing he samples. Bac e ial cul u e was adjus ed up
o 0.012 abso bance (OD600) and incuba ed wi h TGQD o 5 min, hen he bac e ial sample incuba ed
wi h TGQD was placed on a spec opho ome ic pla e and he pla e was scanned unde he Tecan
In ini e m200 PRO.
Nanoma e ials 2020,10, 778 5 o 18
Fu he mo e, he bac e ial de ec ion ia TGQD was ca ied ou h ough mic oscopic analysis.
The bac e ial sample (VRSA) was s ained wi h TGQD (15
µ
g/mL) and Images we e ob ained using an
op ical Olympus BX51 luo escence mic oscope equipped wi h 40×phase con as lens.
The ea e , u he con i ma ion was ob ained by ea ing bac e ia, bac e ia–human cell co-cul u e
wi h TGQD, and mic oscopic images we e cap u ed. Fo he co-cul u e and he bac e ial sample,
VRSA was added in a 24-well cell cul u e pla e in he p esence o PNT1A (human cells) cells wi h 60%
con luency, incuba ed o 1 h a 37
◦
C. The co-cul u e sys em con aining bac e ia and human cells
oge he was s ained by TGQD (15
µ
g/mL), hen he pla e was used o mic oscopy. The mic oscopy was
pe o med by using an mic oscopic in e ed Olympus IX 71S8F3 luo escence mic oscope (Olympus
Co po a ion, Tokyo Japan), which was equipped wi h an Olympus UIS2 se ies objec i e LUCplanFLN
40X (N.A. 0.6, WD 2.7–4 mm, F.N.22), and a me cu y a c lamp X-ci e 12 (120W; Lumen Dynamics,
Mississauga, ON, Canada) was used. As he con ol, bac e ial cells and he human cell line PNT1A
we e also s ained wi h TGQD and obse ed unde a mic oscope.
The bac e icidal e ec o TGQD was analyzed by scanning elec ochemical mic oscopy (SECM).
whe e a Pe i dish con aining he VRSA ea ed wi h TGQD was subjec ed o he SECM s udy. The Pe i
dish was coa ed wi h Poly-L-Lysine and hen he VRSA cells we e added o he Pe i dish o hei
a achmen wi h he Poly-L-Lysine coa ed Pe i dish su ace. The Pe i dish was illed wi h MH
B o h, which was composed o bee in usion o m 300.00 g/L, casein acid hyd olysa e 17.50 g/L, s a ch
1.50 g/L, and KCl 7.45 g/L. The p obe elec ode (P 10
µ
m) was inse ed in he Pe i dish and he cyclic
ol amme y (CV) was eco ded.
S anda d isola es o S. au eus, VRSA, and MRSA we e cul i a ed and used o his expe imen .
All bac e ial isola es we e g own in Muelle Hin on b o h in a shaking incuba o a 37
◦
C. The suscep ibili y
o bac e ial cul u es agains TGQD de ec ion was pe o med by he unaided eye [
45
,
46
,
51
,
52
].
The minimum inhibi o y concen a ion (MIC) is he lowes concen a ion ha inhibi s bac e ial g ow h.
The MIC o TGQD was ob ained by adding TGQD om lowe o highe concen a ions in mic opla e
wells, mixed wi h bac e ial cul u es (0.1 O.D. equi alen o 0.5 MacFa land), and incuba ed a 37
◦
C
o 24 h. The inal wo king concen a ion g adien o TGQD in he mic opla e wells we e 45
µ
g/mL,
55
µ
g/mL, 62.5
µ
g/mL, 85
µ
g/mL, and 125
µ
g/mL. The lowes concen a ion o TGQD added in he
mic opla e well and he mic opla e well showed almos no bac e ial g ow h ( anspa en medium wi h
no u bidi y), which was coun ed as he MIC alue o TGQD agains ha speci ic bac e ium. The con ol
was he bac e ia wi hou TGQD ea men .
The g ow h cu e analysis o S. au eus, VRSA, and MRSA we e pe o med o u he con i ma ion
o an ibac e ial ac i i y o TGQD. S anda d isola es o S. au eus, VRSA, and MRSA we e cul i a ed
and used o his expe imen . All bac e ial isola es we e g own in MH b o h in a shaking incuba o
a 37
◦
C. The con ol was he bac e ial cul u e wi h no TGQD ea men . The an ibac e ial ac i i y
o he TGQDs we e measu ed by Biosc een C MBR (Oy G ow h Cu es Ab L d., Helsinki, Finland)
o 24 h a 37
◦
C using a mul ichannel pipe e sys em in he 10
×
10 Honeycomb op ical mic opla e
well sys ems (Oy G ow h Cu es Ab L d., Helsinki, Finland) [
51
,
53
–
56
]. Di e en concen a ions o
TGQDs (45
µ
g/mL, 55
µ
g/mL, 62.5
µ
g/mL, 85
µ
g/mL, 125
µ
g/mL) we e added o bac e ial cul u es
(0.1 abso bance equi alen o 0.5 McFa land) in he mic opla e wells. The mic opla e wi h he samples
was hen incuba ed in he g ow h cu e analyze (Biosc een C MBR) o 24 h and he esul s we e
e alua ed acco dingly [46].
Fu he mo e, he cell leakage assay was ca ied ou . The bac e ial sample was ea ed wi h TGQD
and incuba ed a 37
◦
C o 4 h, which showed he cells ea ed wi h he TGQD we e p ecipi a ed
a e 4 h. The ea ed samples as well as he un ea ed samples we e cen i uged o cell deb is
emo al and he supe na an was sepa a ely kep in a mic o-cen i uge ube o quan i ica ion o DNA
and RNA by a spec opho ome e (Tecan In ini e m200 PRO). The wa eleng h o 260 nm was used
o he DNA and RNA quan i ica ion. Fo he DNA quan i ica ion, he samples we e ea ed wi h
RNase, and o he RNA quan i ica ion samples, we ea ed hem wi h DNase, and pu i ied hem in
a column pu i ica ion sys em. A e column pu i ica ion, he abso bance spec a we e eco ded a

Nanoma e ials 2020,10, 778 6 o 18
260 nm. The p esence o DNA and RNA we e calcula ed acco ding o a 260/280 a io. Acco ding o
he 260/280 a io, ~1.8 is gene ally accep ed as pu e o DNA and ~2.0 is gene ally accep ed as
pu e o RNA [
57
–
61
]. The DNA in he supe na an was used as a empla e wi h 16S RNA p ime s
(16S Fo wa d-ACTGGGATAACTTCGGGAAAC, and 16S e e se-CAGCGCGGATCCATCTATAA) o
pe o m PCR ampli ica ion, and he con i ma ion was done using aga ose gel elec opho esis wi h 1.3%
aga ose gel [
45
]. The posi i e con ol was genomic DNA and he nega i e con ol was he supe na an
om he un ea ed bac e ial sample.
To con i m he p esence o all ypes o li e cells (bac e ial and human cells), SYTO9 was used.
The ea e , an op ical Olympus BX51 luo escence mic oscope equipped wi h a 40
×
phase con as
lens was used o s udy he an ibac e ial ac i i y o he TGQD agains VRSA. Ini ially, he samples
we e incuba ed wi h bac e ia and TGQDs ( espec i e MIC) o 4 h a 37
◦
C in a shaking incuba o .
Two di e en kinds o luo escen dyes we e used: SYTO9 and p opidium iodide (PI) o li e and
dead cell s aining in equal p opo ion. Fluo escence dyes we e added o he sample and obse ed
unde mic oscope.
2.5. In Vi o Cy o oxici y Tes ing Assay
The MDA-MB-231 (mamma y gland adenoca cinoma cells) and HBL-100 (mamma y gland
epi helial cells) human cell line we e used o s udy he cy o oxici y o TGQDs. The MTT
3-(4,5-dime hyl hiazol-2-yl)-2,5-diphenyl e azolium b omide) assay was used o s udy he cell iabili y.
The cells we e main ained in RPMI-1640 medium wi h 10% e al bo ine se um, supplemen ed wi h
penicillin (100 u/mL) and s ep omycin (0.1 mg/mL). In a mic o i e pla e, each well was illed wi h
5000 cells in 50
µ
L medium, ollowed by 24 h incuba ion a 37
◦
C wi h 5% CO
2
. A e 24 h incuba ion
wi h TGQDs, 10
µ
L o MTT (5 mg/mL in PBS) was added, and incuba ed o 4 h a 37
◦
C wi h 5% CO
2
.
When his incuba ion pe iod was o e , MTT con aining medium was eplaced and 100
µ
L o 99.9%
dime hyl sul oxide ( / ) was added o 5 min. The abso bance was aken a 570 nm by an In ini e m200
PRO eade [46,62]
3. Resul s and Discussion
3.1. Syn hesis and Cha ac e iza ion o TGQDs
The cha ac e iza ion was ini ia ed by measu ing he luo escence and abso bance spec a o he
TGQDs, as shown in Figu e 1a. The Gd–Schi base was mixed wi h TiO
2
in a 1:1 a io and cooked
a 80
◦
C o o m TGQDs, which when obse ed unde mic oscopy showed a blue ield due o i s
high luo escence as obse ed unde a UV il e (Figu e 1b). The TGQD unde UV ansillumina o
(
λ=312 nm
) showed high luo escence in ensi y wi h a bluish whi e colo (Figu e 1c). The luo escence
emission maximum and he abso bance maximum o he TGQDs we e obse ed a
λ
=375 nm and
λ=262 nm
, espec i ely. Fu he mo e, i was cha ac e ized by x- ay luo escence spec opho ome y
using he seconda y a ge as Mo. The esul s indica e speci ic peaks ha showed he p esence o Gd
and Ti in he sample, as shown in Figu e 1d. Mo eo e , he pa icle size dis ibu ion, a e age pa icle
size, and he ze a po en ial o he p epa ed TGQDs we e analyzed using SEM and DLS (Figu e 2).
The esul s e ealed ha he size o he TGQDs we e ound o be in he ange o 45
±
2 nm o
95 ±2 nm
(Figu e 2a) and he alue o he ze a po en ial showed ha he alue was highes a 58.7
±
0.13 mV
(Figu e 2c). The size de e mined by DLS was in good ag eemen wi h he SEM image and da a, as
shown in Figu e 2b.
Finally, he FTIR spec um o TGQD (Figu e S3) indica es he p esence o he di e en ib a ions
o he unc ional g oup, showing he p esence o all he bond and unc ional g oups p esen in he
gadolinium ni a e and i anium bu oxide, ini ial componen o TGQD (Figu es S1 and S2). In i anium,
he unc ional g oup NO
3
has asymme ic and symme ic s e ching om 1369 o 1381 cm
−1
and 1340
o 1267 cm
−1
[
63
]. In he case o i anium bu oxide abso p ions a 1125 cm
−1
(Ti–O–C ib a ion), he
in ensi y o he bands was om 1490 o 1340 cm
−1
[
64
]. The SECM images ob ained a e shown in
Nanoma e ials 2020,10, 778 7 o 18
Figu e 3. The scale o he cu en measu ed on he ba e glassy ca bon (GC) was om 0.09 o 0.2 nA.
The GC immobilized wi h TGQD showed a cu en ange mainly om 0.6 o 0.7 nA. Thus, i can be
concluded ha he cu en in e al in bo h cases was he same (almos 1 nA). Hence, he inhomogenei y
o bo h elec odes is compa able. I can be concluded ha he homogenei y o he bo h elec odes is
mainly a ec ed by he Na ion laye . Howe e , he cu en inc eased signi ican ly in he p esence o
he TGQD. This can be a ibu ed o he good elec ochemical conduc i i y o he syn hesized TGQD,
which is due o low elec ochemical esis ance o hei me allic componen s. Mo eo e , he cha ge
o he QDs may in luence hei elec ochemical conduc i i y. Howe e , we ound ha he TGQDs
we e wa e soluble and showed high s abili y ( luo escence measu emen ) in solu ion, e en a e ou
mon hs om i s p epa ed ime.
Nanoma e ials 2020, 10, x FOR PEER REVIEW 7 o 19
ound ha he TGQDs we e wa e soluble and showed high s abili y ( luo escence measu emen ) in
solu ion, e en a e ou mon hs om i s p epa ed ime.
Figu e 1. (a) The luo escence spec um is gi en in he blue line and he abso bance spec um is gi en
in he g een line. (b) Ti anium-Gadolinium Quan um do (TGQD) obse ed unde he UV il e o he
luo escence mic oscope. (c) TGQD shows a bluish whi e colo unde UV ansillumina o ; (d) X– ay
luo escence (XRF) spec um con i ms ha he syn hesized TGQDs con ained bo h gadolinium and
i anium me al.
Figu e 1.
(
a
) The luo escence spec um is gi en in he blue line and he abso bance spec um is gi en
in he g een line. (
b
) Ti anium-Gadolinium Quan um do (TGQD) obse ed unde he UV il e o he
luo escence mic oscope. (
c
) TGQD shows a bluish whi e colo unde UV ansillumina o ; (
d
) X– ay
luo escence (XRF) spec um con i ms ha he syn hesized TGQDs con ained bo h gadolinium and
i anium me al.
Nanoma e ials 2020,10, 778 8 o 18
Nanoma e ials 2020, 10, x FOR PEER REVIEW 8 o 19
Figu e 2. (a) Ze a size o he TGQD. (b) SEM analysis o TGQD wi h 20 µm scale ba ;.(c) Ze a po en ial
o TGQDs. Da a ep esen he mean ± SD, n = 5.
Figu e 2.
(
a
) Ze a size o he TGQD. (
b
) SEM analysis o TGQD wi h 20
µ
m scale ba ;.(
c
) Ze a po en ial
o TGQDs. Da a ep esen he mean ±SD, n=5.
Nanoma e ials 2020, 10, x FOR PEER REVIEW 9 o 19
Figu e 3. (a) Scanning elec ochemical mic oscopy (SECM) image o glassy ca bon elec ode co e ed
wi h Na ion. (b) SECM image o glassy ca bon immobilized wi h QDs and co e ed wi h Na ion. The
applied pa ame e s: he ip elec ode scan a e: 20 µm/s, he applied po en ial on ip: 0.5 V, he ip—
subs a e dis ance: 10 µm, quie ime: 30 s, he scanned su ace a ea 100 µm × 100 µm. The
measu emen s we e ca ied ou in a solu ion o 1 mm FcOH and 0.1 M KCl.
3.2. De ec ion o Bac e ia
Fu he mo e, acco ding o he spec opho ome y i is clea ha he bac e ial cells we e de ec ed
by applying TGQDs, whe e bac e ial cells p esen ed a 0.012 O.D. alue, whe eas abso bance om
only bac e ia and only QDs we e lowe han he QD mixed sample. Lowe concen a ions (0.012 O.D.
concen a ion) o he bac e ial sample we e de ec ed h ough he spec opho ome ic me hod (Figu e
S4). The bac e ial sample was s ained wi h TGQD, which ga e a high bluish whi e luo escence unde
he UV il e o he luo escence mic oscope (Figu e S5).
Figu e 3.
(
a
) Scanning elec ochemical mic oscopy (SECM) image o glassy ca bon elec ode co e ed
wi h Na ion. (
b
) SECM image o glassy ca bon immobilized wi h QDs and co e ed wi h Na ion.
The applied pa ame e s: he ip elec ode scan a e: 20
µ
m/s, he applied po en ial on ip: 0.5 V,
he ip—subs a e dis ance: 10
µ
m, quie ime: 30 s, he scanned su ace a ea 100
µ
m
×
100
µ
m.
The measu emen s we e ca ied ou in a solu ion o 1 mm FcOH and 0.1 M KCl.
Nanoma e ials 2020,10, 778 9 o 18
3.2. De ec ion o Bac e ia
Fu he mo e, acco ding o he spec opho ome y i is clea ha he bac e ial cells we e de ec ed
by applying TGQDs, whe e bac e ial cells p esen ed a 0.012 O.D. alue, whe eas abso bance om
only bac e ia and only QDs we e lowe han he QD mixed sample. Lowe concen a ions (0.012 O.D.
concen a ion) o he bac e ial sample we e de ec ed h ough he spec opho ome ic me hod (Figu e S4).
The bac e ial sample was s ained wi h TGQD, which ga e a high bluish whi e luo escence unde he
UV il e o he luo escence mic oscope (Figu e S5).
De ec ion ( h ough imaging) o he VRSA in he p esence o PNT1A (cocul u e imaging) was
ca ied ou unde luo escence mic oscopy. The samples we e s ained wi h a e y small amoun (10 o
15
µ
g/mL) o TGQD. The VRSA cells in he p esence o PNT-1A we e s ained wi h TGQD and SYTO9
o unde s and he dye speci ici y (whe he TGQD is speci ic owa d bac e ia o no ) owa d bac e ia
and human cells. The b igh ield image (Figu e 4a) showed he p esence o bac e ia and human cells
(indica ed by a ow) in he ield o obse a ion. Figu e 4b shows ha TGQD posi i ely s ained he
bac e ial cells, bu PNT1A cells we e uns ained and no isible in he ield. In Figu e 4c,e (me ged
image o Figu e 4a,c), i can be seen ha he SYTO9 s ained bo h he VRSA and he PNT1A cells, which
con i med ha SYTO9 does no ha e a cell speci ic s aining na u e. In con as , TGQD speci ically
s ained he bac e ial cells, which was clea ly isible in Figu e 4b,d (me ged image o Figu e 4a,b)
and (me ged image o Figu e 4b,c). Finally, om Figu e 4 ( he me ged image o Figu e 4b,c), i can
be clea ly concluded ha STYO9 is nei he speci ic owa d bac e ia no human cells (because bo h
bac e ial and human cells we e s ained), whe eas he TGQD is a bac e ia speci ic s ain (only bac e ial
cells we e s ained). Acco ding o he bes o ou knowledge, he e is no such TGQD ha can s ain
only bac e ia and no he human cells. This expe imen al da a was u he alida ed by con ol cell
imaging (Figu e S6) whe e TGQD s ained VRSA cells showed high luo escence unde a luo escence
mic oscope, bu he PNT1A cells we e uns ained and no luo escence was obse ed.
Nanoma e ials 2020, 10, 778 9 o 18
only bac e ia and only QDs we e lowe han he QD mixed sample. Lowe concen a ions (0.012 O.D.
concen a ion) o he bac e ial sample we e de ec ed h ough he spec opho ome ic me hod (Figu e
S4). The bac e ial sample was s ained wi h TGQD, which ga e a high bluish whi e luo escence unde
he UV il e o he luo escence mic oscope (Figu e S5).
De ec ion ( h ough imaging) o he VRSA in he p esence o PNT1A (cocul u e imaging) was
ca ied ou unde luo escence mic oscopy. The samples we e s ained wi h a e y small amoun (10
o 15 µg/mL) o TGQD. The VRSA cells in he p esence o PNT-1A we e s ained wi h TGQD and
SYTO9 o unde s and he dye speci ici y (whe he TGQD is speci ic owa d bac e ia o no ) owa d
bac e ia and human cells. The b igh ield image (Figu e 4a) showed he p esence o bac e ia and
human cells (indica ed by a ow) in he ield o obse a ion. Figu e 4b shows ha TGQD posi i ely
s ained he bac e ial cells, bu PNT1A cells we e uns ained and no isible in he ield. In Figu es 4c,e
(me ged image o Figu es 4a,c), i can be seen ha he SYTO9 s ained bo h he VRSA and he PNT1A
cells, which con i med ha SYTO9 does no ha e a cell speci ic s aining na u e. In con as , TGQD
speci ically s ained he bac e ial cells, which was clea ly isible in Figu es 4b, d (me ged image o
Figu es 4a,b) and (me ged image o Figu es 4b,c). Finally, om Figu e 4 ( he me ged image o
Figu es 4b,c), i can be clea ly concluded ha STYO9 is nei he speci ic owa d bac e ia no human
cells (because bo h bac e ial and human cells we e s ained), whe eas he TGQD is a bac e ia speci ic
s ain (only bac e ial cells we e s ained). Acco ding o he bes o ou knowledge, he e is no such
TGQD ha can s ain only bac e ia and no he human cells. This expe imen al da a was u he
alida ed by con ol cell imaging (Figu e S6) whe e TGQD s ained VRSA cells showed high
luo escence unde a luo escence mic oscope, bu he PNT1A cells we e uns ained and no
luo escence was obse ed.
Figu e 4. The mic oscopic images. (a) VRSA in p esence o PNT1A cells; (b) VRSA in p esence o
PNT1A cells s ained wi h TGQDs and imaged unde UV il e ; (c) VRSA mixed wi h PNT1A cells
s ained wi h SYTO9 and imaged unde luo escence mic oscope h ough 485/498 il e ; (d) Me ged
image o (a) and (b); (e) Me ged image o (a) and (c); ( ) Me ged image o (b) and (c).
3.3. An ibac e ial Ac i i ies
The an ibac e ial ac i i y o TGQDs was ini ia ed by he de e mina ion o he MIC o TGQD,
which was pe o med by he b o h mic o-dilu ion me hod agains VRSA, MRSA, and S. au eus. The
Figu e 4.
The mic oscopic images. (
a
) VRSA in p esence o PNT1A cells; (
b
) VRSA in p esence o
PNT1A cells s ained wi h TGQDs and imaged unde UV il e ; (
c
) VRSA mixed wi h PNT1A cells
s ained wi h SYTO9 and imaged unde luo escence mic oscope h ough 485/498 il e ; (
d
) Me ged
image o (a) and (b); (e) Me ged image o (a) and (c); ( ) Me ged image o (b) and (c).
Nanoma e ials 2020,10, 778 16 o 18
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