Suppo ing In o ma ion
o Small, DOI 10.1002/smll.202302274
Vapo -Phase Syn hesis o Molecula ly Imp in ed Polyme s on Nanos uc u ed Ma e ials a
Room-Tempe a u e
Elisabe a Mazzo a*, Tiziano Di Giulio, S e ano Ma iani, Ma ina Co si, Cosimino Mali es a
and Giuseppe Ba illa o*
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Suppo ing In o ma ion
Syn hesis o Molecula ly Imp in ed Polyme s on Nanos uc u ed Ma e ials ia Room-
Tempe a u e Vapo -Phase Polyme iza ion o P o ein Sensing
Elisabe a Mazzo a*, Tiziano Di Giulio, S e ano Ma iani, Ma ina Co si, Cosimino Mali es a
and Giuseppe Ba illa o*
P o . Elisabe a Mazzo a, D . Tiziano Di Giulio, P o . Cosimino Mali es a
Labo a o y o Analy ical Chemis y, Depa men o Biological and En i onmen al Sciences
and Technologies (Di.S.Te.B.A.), Uni e si y o Salen o, ia Mon e oni, 73100 Lecce (I aly)
D . S e ano Ma iani, Ma ina Co si, P o . Giuseppe Ba illa o
In o ma ion Enginee ing Depa men , Uni e si y o Pisa, ia G. Ca uso 16, 56122 Pisa (I aly)
E-mail: elisabe a.mazzo a@unisalen o.i , g[email p o ec ed]
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Me hods
Ma e ials and chemicals
All chemicals we e o analy ical g ade and we e used as ecei ed. Ul a-pu e wa e
(conduc i i y <0.1μS/cm) was used.
The chemical eagen s used included (3-aminop opyl) ie hoxysilane (APTES), 99%,
glu a aldehyde solu ion II g ade, 25%, human hemoglobin (HHb), lysozyme om chicken
egg whi e lyophilized powde (Lyz), human se um albumin (HSA), cy och ome C (Cy C),
ob ained om Sigma-Ald ich (S . Louis, MO, USA). I on chlo ide, sodium hyd oxide,
monosodium phospha e (MSP), NaH2PO4, and disodium phospha e (DSP), Na2HPO4, we e
p o ided om Honeywell Fluka (College Pa k, GA, USA).
All solu ions (excep APTES) we e p epa ed in ul a-pu e wa e . Phospha e bu e saline
(PBS) solu ions (50 mM, pH 7.4), we e p epa ed by dissolu ion o he comme cial MSP and
DSP in app op ia e p opo ions, adding NaOH 5 M o adjus he inal pH.
2% APTES solu ions ( / ) we e p epa ed in oluene. S ock solu ion o HHb (8 mg mL-1) we e
eshly p epa ed in PBS and we e dilu ed o ob aining HHb s anda d solu ion a di e en
concen a ions ( om 0.1 o 8 mg mL-1) o ebinding expe imen s. Solu ions o Lyz, HSA and
Cy C, we e eshly p epa ed in PBS in he same way, be o e hei use. The a i icial se um
sample used o HHb de ec ion es s was p epa ed acco ding o a p o ocol epo ed in
li e a u e1. I consis ed o 111 mM NaCl, 29 mM NaHCO3, 2.2 mM K2HPO4, 0.8 mM
MgCl2•6H2O, 2.5 mM u ea and 4.7 mM glucose. Plasma om human was pu chased om
Sigma Ald ich.
Silicon bo on-doped wa e s (p++ ype) wi h esis i i y o 0.8 - 1.2 mΩ×cm, o ien a ion
<100>, we e pu chased om Sil onix Silicon Technologies (F ance). Aqueous hyd o luo ic
acid (HF, 48%), sodium hyd oxide (NaOH, 98%) we e pu chased om Sigma Ald ich
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(Ge many). Absolu e e hanol (E OH, 99.9%), and die hyl e he (E 2O, > 99%), we e
pu chased om Ca lo E ba Reagen s (I aly).
Fab ica ion o PSi sca olds
Po ous silicon (PSi) sca olds we e p epa ed by anodic e ching o p- ype silicon squa e wa e
samples (1.5 × 1.5 cm2) using 2 mL o solu ion HF:E OH (3:1 / ). A cus om-made Te lon
cell (ci cula wo king a ea o 0.567 cm2) wi h pla inum wi e ca hode and aluminium la
anode was used o elec ochemical anodiza ion o he silicon subs a e. A sou ce
measu emen uni (SMU, Kei hley 2602A) was used o se he e ching cu en and measu e
he ol age be ween anode and ca hode.
A sac i icial PSi laye was e ched a 250 mA cm-2 cu en densi y o 15 s, insed wi h E OH
o 120 s o emo e esidual HF, dissol ed in a solu ion NaOH(1M):E OH (9:1 / ) o 120 s
o achie e he comple e emo al o PSi lea ing an inc eased su ace oughness able o
inc ease he po es size2, and inally insed wi h DIW and E OH o emo e he dissol ing
solu ion. The nPSi in e e ome e was hen e ched wi h a cons an cu en densi y o 250 mA
cm-2 o 40 s, o ob ain a po ous laye wi h ~4.1 μm hickness and ~75 % po osi y.
E en ually, he nPSi sample was insed wi h E OH o 120 s and E 2O o 60 s o ob ain a
c ack- ee nPSi laye . The mal oxida ion o he PSi in e e ome e was ca ied ou in a mu le
u nace (ZB 1, ASAL, I aly) a 750 °C o 1 h ( amp-up/ amp-down 12 °C min−1) in oom
a mosphe e.
Op ical cha ac e iza ion o PSi sca olds
Re lec ance spec a o he PSi in e e ome e s we e acqui ed in ai (bo h be o e and a e
oxida ion) in he wa eleng h ange [400−1000 nm] using an op ical se up consis ing o a
UV−VIS spec ome e (SM242 SP) p o ided by Spec al p oduc s, a bi u ca ed ibe -op ic
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p obe (QR200−7-VIS-BX), and a halogen lamp sou ce (HL-2000) pu chased om Ocean
Op ics (USA). Ligh exi ing he halogen lamp sou ce is ed o hogonally on o he PSi su ace
h ough one a m o he ibe -op ic p obe; he ligh e lec ed om he PSi laye is collec ed
in o a UV−VIS spec ome e h ough he o he a m o he ibe -op ic p obe. Acquisi ion
pa ame e s o e lec ion spec a we e: in eg a ion ime 15 ms, a e age scan numbe 10,
boxca wid h 5, wi h he spec ome e wo king in pho on coun s mode. Po osi y o as-
p epa ed PSi in e e ome e s was es ima ed by bes - i ing o he e lec ance spec a o PSi
laye s acqui ed be o e oxida ion, using a home-made so wa e (Ma lab, Ma hWo ks, USA)3.
FFT e lec ance spec oscopy
FFT o he e lec ance spec a o PSi in e e ome e s was pe o med o calcula e he EOT
alues, namely, 2nL, whe e n = e ec i e e ac i e index and L = hickness o he PSi laye ,
using a home-made so wa e (Ma lab, Ma hWo ks, USA). The wa eleng h axis o he
e lec ance spec um was i s in e ed (x axis changed om wa eleng h o 1/wa eleng h) o
ob ain a wa enumbe axis. A cubic-spline in e pola ion o e lec ance da a was hen ca ied
ou o ob ain a da ase ( e lec ion, wa enumbe ) spaced e enly (sample- o-sample dis ance
8.57 × 10−7 nm−1). A Hanning window was applied o he e lec ance spec um, which was
ze o padded o 224. E en ually, applica ion o he FFT algo i hm o he ze o- padded
e lec ance spec um yielded he Fou ie ans o m ampli ude and phase (y axis in he Fou ie
ans o m domain) as a unc ion o 1/wa enumbe (x axis in he Fou ie ans o m domain),
wi h spa ial esolu ion o abou 0.07 nm. The EOT alue is ob ained as he alue o he
1/wa enumbe axis (x axis) in he Fou ie ans o m domain o which he main peak in he
Fou ie ans o m ampli ude (y axis) occu s.
Liquid-phase deposi ion o PPy in PSiO2 sca olds
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Liquid-phase deposi ion o Polypy ole (PPy) on PSiO2 subs a e was ca ied ou sligh ly
modi ying a p o ocol epo ed in li e a u e4,5. PSiO2 samples we e imme sed in 0.1 M FeCl3
solu ion o 30 min o allow oxidizing agen pe mea ion in o he nanos uc u ed laye o PSi.
Then, he samples we e imme sed in a solu ion o Py 0.1 M, o di e en ime in e als (1, 2,
5, 8 h).
Vapo -phase PPy deposi ion o PPy in PSiO2 sca olds
Vapo -phase polyme iza ion o PPy was ca ied ou sligh ly modi ying a p o ocol epo ed in
li e a u e6,7. The PSiO2 samples we e imme sed in a FeCl3-e hanol solu ion (0.5% w / ) o 20
min. A e aking he samples ou om he solu ion and gen ly d ying hem wi h a N2 low,
he PSiO2 samples we e placed in a closed chambe sa u a ed wi h py ole apo s o di e en
ime in e als (1, 2, 5, 8 h), a oom empe a u e and a mosphe ic p essu e. The samples we e
epea edly insed wi h wa e (5 min) o emo e un eac ed monome , hen wi h e hanol, and
d ied unde a N2 low.
Mo phological and composi ional cha ac e iza ion o PSi and PSiO2 sca olds be o e
and a e PPy polyme iza ion
Mo phological and composi ional cha ac e iza ion o PSi and PSiO2 sca olds be o e and a e
PPy deposi ion we e ca ied ou using a scanning elec on mic oscope (FE-SEM, FEI Quan a
450 ESEM FEG) equipped wi h an ene gy-dispe si e mic oanaly ic sys em (EDX, B uke ,
QUANTAX XFlash De ec o 6|10 o EDX analysis). SEM and EDX analysis we e
pe o med on c oss-sec ions o samples wi h a 10 kV accele a ion ol age a a ious
magni ica ions. Dis ibu ion o he po e diame e s was ob ained om he analysis o op- iew
SEM images wi h Gwyddion so wa e.
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XPS cha ac e iza ion
XPS measu emen s we e eco ded wi h an AXIS ULTRA DLD (K a os Analy ical)
pho oelec on spec ome e using a monoch oma ic AlKα sou ce (1486.6 eV) ope a ed a 150
W (10 kV, 15 mA). Base p essu e in he analysis chambe was 5.3 x 10-9 o . Su ey scan
spec a we e eco ded using a pass ene gy o 160 eV and a 1 eV s ep. High esolu ion spec a
we e acqui ed using a pass ene gy o 20 eV and a 0.1 eV s ep. In each case he a ea o
analysis was abou 700 μm x 300 μm. Du ing he da a acquisi ion a sys em o neu aliza ion
o he cha ge has been used. P ocessing o he spec a was accomplished by CasaXPS Release
2.3.16 so wa e. The binding ene gy (BE) scale was e e enced o he Au 4 7/2 peak a 84.0
eV. Fo he analysis o high esolu ion spec a all peaks we e i ed using Shi ley backg ound
and GL(30) lineshape (a combina ion o Gaussian 70% and Lo en zian 30%). Fo quan i a i e
analysis, he ela i e sensi i i y ac o s p esen in he lib a y o CasaXPS o he a eas o he
signals we e used. Su ace cha ging was co ec ed conside ing ad en i ious C 1s (binding
ene gies (BE) = 285 eV).
HHb ancho ing
The p o ein immobiliza ion on PSiO2 sca olds was based on he co alen ancho ing o HHb
p o ein and in ol es a p elimina y unc ionaliza ion o silicon su ace wi h a sui able linke .
Speci ically, PSiO2 samples we e imme sed in a solu ion o APTES p epa ed in oluene (2%,
/ ) o 30 min a 55°C 8–10, hen washed wi h MeOH o 5 min and insed wi h wa e and
E OH. Nex , he samples we e imme sed in 5% ( / ) glu a aldehyde solu ions p epa ed in
PBS, pH 7.4, o 2 h11,12. P o ein ancho ing was ca ied ou by d op cas ing 100 L o a
solu ion o HHb (1 mg mL-1) p epa ed in PBS, pH 7.4 on o he unc ionalized PSiO2
sca olds, hen le incuba ing o 2 hou s a oom empe a u e.
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MIP syn hesis by PPy apo -phase deposi ion
MIP ilms o HHb ( e e ed o as “d y apo -phase MIP”) we e p epa ed by PPy apo -phase
deposi ion o 8 hou s on PSiO2 sca olds p elimina ily unc ionalized wi h HHb, as de ailed
in sec ion “Vapo -phase PPy deposi ion o PPy in PSiO2 sca olds”, and subsequen emo al
o HHb molecules om he polyme ma ix by washing (1 h, s i ing) he PPy-coa ed
sca olds in 50 mM HCl solu ion p epa ed in wa e . The washing s ep was epea ed, i
necessa y, un il a s able e lec ance spec um o he MIP-coa ed PSiO2 sca olds was
achie ed.
Fu he MIP ilms o HHb ( e e ed o as “we apo -phase MIP”) we e achie ed by apo -
phase deposi ion (a oom empe a u e and a mosphe ic p essu e) o PPy on PSiO2 sca olds
p elimina ily unc ionalized wi h HHb in he p esence o a small wa e olume in he
e apo a ion chambe oge he wi h py ole, o ensu e sa u a ion o he e apo a ion chambe
wi h wa e and py ole apo s. A e polyme iza ion, he PPy-coa ed sca olds we e washed
as desc ibed abo e o HHb emo al.
No imp in ed polyme (NIP) was p epa ed by 8 hou s PPy deposi ion on PSiO2 subs a e
p elimina ily unc ionalized wi h APTES and glu a aldehyde only.
HHb ebinding es s
MIP- unc ionalized PSiO2 samples we e es ed wi h inc easing concen a ions o HHb (0.1- 8
mg mL-1). F eshly p epa ed HHb solu ions (100 L) p epa ed in PBS a pH 7.4 we e d op cas
on he MIP- unc ionalized PSiO2 sca olds and incuba ed o 1 h. A e incuba ion, he
samples we e insed wi h wa e (5 min) unde s i ing (200 pm), wi h E OH, and d ied unde
a N2 low. Then, e lec ance spec a we e eco ded on he so-p ocessed PSiO2 sca olds o
each HHb concen a ion es ed. All es s we e pe o med in iplica e. MIP and NIP sensi i i y
alues we e es ima ed om he slope o he calib a ion cu es.
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Rebinding es s we e also pe o med using complex eal ma ices, namely, plasma om
human and syn he ic se um, o e alua e he po en ial use o he MIP-PSiO2 senso o clinical
pu poses. HHb s ock solu ion was spiked in plasma and se um samples o ebinding
expe imen s, pe o med unde he same condi ions used o calib a ion expe imen s wi h
bu e solu ion.
De i a ion o he MIP associa ion cons an using he Langmui -F eundlich iso he m
model
The MIP associa ion cons an (K0) was es ima ed using Langmui -F eundlich iso he m model
acco ding o he ollowing equa ions:
log 𝑆
𝑆𝑚𝑎𝑥−𝑆 = log 𝑎 + 𝑚 log 𝐶 (1)
𝐾0= 𝑎1
𝑚 (2)
whe e S is he senso ou pu signal, Smax is he maximum alue o he senso ou pu , a is
ela ed o he median binding a ini y, m is he he e ogenei y index, C is he analy e
concen a ion 13,14. The i ing pa ame e s Smax, a, m we e ex ac ed by bes - i ing he
expe imen al calib a ion cu e eco ded on MIP senso s in he ange 0.1 o 8 mg mL-1 o HHb
using Eq. (1).
Selec i i y, epea abili y and s abili y expe imen s
Selec i i y o he MIP was e alua ed by es ing he MIP-PSiO2 senso esponses o di e en
in e e ing molecules, namely, human se um albumin (HSA), cy oc ome C (Cy C), Lysozyme
(Lyz), unde he same condi ions used o HHb, using a eshly p epa ed senso o each
expe imen .
Repea abili y was e alua ed by es ing he MIP-PSiO2 senso o HHb de ec ion in iplica e,
consecu i ely pe o med on he same senso . Be o e a new expe imen , he MIP was
