Electrochemical aptasensor for the selective detection of vancomycin based on nanostructured "in‐lab" printed electrodes

Vol.:(0123456789)
Mic ochimica Ac a (2025) 192:107
h ps://doi.o g/10.1007/s00604-025-06952-1
RESEARCH
Elec ochemical ap asenso o  heselec i e de ec ion o  ancomycin
based onnanos uc u ed “in‑lab” p in ed elec odes
MalekBibani1,2 · MagdolnaCasian1 · BogdanFeie 1 · DianaBogdan3 · OanaHosu‑S ancioiu1 ·
NadiaK a i2 · Ra ikKal a 2 · CeciliaC is ea1
Recei ed: 20 Oc obe 2024 / Accep ed: 2 Janua y 2025 / Published online: 25 Janua y 2025
© The Au ho (s) 2025
Abs ac
Alabel- ee, lexible, and disposable ap asenso was designed o he apid on-si e de ec ion o ancomycin (VAN) le els.
The elec ochemical senso was based on lab-p in ed ca bon elec odes (C-PE) en iched wi h cauli lowe -shaped gold
nanos uc u es (AuNSs), on which VAN-speci ic ap ame s we e immobilized as bio ecogni ion elemen s and sho -chain
hiols as blocking agen s. The AuNSs, cha ac e ized by scanning elec on mic oscopy (SEM) and a omic o ce mic oscopy
(AFM), enhanced he elec ochemical p ope ies o he pla o m and he ap ame immobiliza ion ac i e si es. The de eloped
disposable ap asenso allowed label- ee de ec ion o VAN ia elec ochemical impedance spec oscopy (EIS) ac oss a wide
ange o concen a ions (50–1000 nM), wi h a limi o de ec ion (LOD) o 1.721 nM. The ap asenso p esen ed good selec-
i i y agains some commonly ound in e e ences in human se um and milk and was success ully applied o he analysis
o hese samples.
Keywo ds Vancomycin de ec ion· Elec ochemical ap asenso · EIS· Gold nanos uc u e· Cauli lowe -shaped gold
nanos uc u es· An ibio ic esis ance
In oduc ion
Glycopep ide an ibio ics ha e been a key weapon in he
igh agains bac e ial in ec ions o o e hal a cen u y and
a e he mos equen ly used an ibio ics o he ea men
o se e e in ec ions, such as endoca di is, pneumonia, and
meningi is [1, 2]. Vancomycin (VAN) is an impo an an i-
bio ic, used as a i s -line ea men o in ec ions caused by
an ibio ic- esis an G am-posi i e pa hogens, pa icula ly
me hicillin- esis an S aphylococcus au eus (MRSA) and
En e ococcus sp. [3]. As one o he “eme gency” an ibio -
ics, VAN is used in bo h human and e e ina y medicine o
ea se e e in ec ions, adminis e ed in a enously h ough
in usion and o ally o local e ec . I is cha ac e ized by a
na ow he apeu ic index and is associa ed wi h a ela i ely
high occu ence o neph o oxici y and o o oxici y. I used
a sub- he apeu ic concen a ions, VAN helps he sp ead o
an imic obial esis ance (AMR). The e o e, he apeu ic d ug
moni o ing (TDM) o VAN is ecommended [4]. Recen ly,
a co ela ion was obse ed be ween VAN swea concen a-
ion and se um concen a ion [5], hus, a nonin asi e TDM
could be achie ed using wea able senso s.
Malek Bibani and Magdolna Casian con ibu ed equally o his
wo k..
Highligh s
• Fab ica ion o lexible in-lab p in ed ca bon elec odes o he
de elopmen o disposable senso s
• Vancomycin-speci ic ap ame cha ac e iza ion s udy using
iso he mal calo ime y and su ace plasmon esonance
echniques
• Cons uc ion o cauli lowe -like gold nanos uc u es and
ancomycin-speci ic ap ame -based sensing pla o m
• Label- ee elec ochemical de ec ion o ancomycin in he low
nanomola ange
• Success ul de elopmen o a disposable on-si e apid es ing
de ice o an ibio ic concen a ion moni o ing om eal samples.
* Bogdan Feie
eie .geo [email p o ec ed]; eie _bogdan_geo [email protected]
1 Depa men o Analy ical Chemis y, Facul y o Pha macy,
“Iuliu Hațieganu” Uni e si y o Medicine andPha macy, 4
Pas eu S ee , 400349Cluj-Napoca, Romania
2 Labo a oi e Ma é iaux, T ai emen E Analyse, INRAP,
Bio echPole Sidi-Thabe , 2020A iana, Tunisia
3 Na ional Ins i u e o Resea ch andDe elopmen
o Iso opic andMolecula Technologies, 67-103 Dona S .,
400293Cluj-Napoca, Romania
Mic ochim Ac a (2025) 192:107107 Page 2 o 15
Al hough he use o VAN in animals is limi ed in o de
o impai he sp ead o AMR [6], i s excellen an ibac e-
ial ac i i y encou ages he use o VAN as a eed addi i e
o e e ina y d ug o ea bac e ial in ec ions, especially
cow mas i is. VAN abuse may lead o po en ial esidues in
milk o in he en i onmen , hus a o ing he AMR [7]. To
comba his conce n and ensu e ha milk en e ing he ood
supply adhe es o egula o y s anda ds, he Eu opean Union
has se maximum esidue limi s (MRLs) o an ibio ics ha
a e s ill allowed o be adminis e ed o animals, along wi h
he addi ional equi emen o implemen ing a wi hd awal
pe iod o an ibio ics in animal ood p oduc s o a oid ood
sa e y p oblems [8].
T adi ional an ibio ic de ec ion echniques include me h-
ods based on ch oma og aphy coupled wi h mass spec om-
e y [9, 10], mic obiological assays [11, 12], and enzyme-
linked immunoso ben assays [13, 14], which a e expensi e,
ime-consuming, and in ol e labo ious p e ea men p o-
ocols, long assay ime, and lack o po abili y. Al hough
ELISA ki s ha e been made a ailable o easie public use,
he e a e s ill conce ns ha need o be add essed, includ-
ing enzyme inac i a ion, c oss- eac i i y, and long de ec-
ion imes, which a e unsui able o an ibio ic moni o ing in
eal-wo ld si ua ions [15].
In he a ea o animal heal h managemen , biosenso s ha e
come in o he spo ligh as e sa ile and inno a i e de ices,
gaining ecogni ion in he global ma ke . Cu en ly, p eci-
sion li es ock a ming echniques a e applied in swea and
sali a y sensing, se odiagnosis, and animal p oduc sa e y
moni o ing [16].
Elec ochemical senso s o e dis inc ad an ages o e
con en ional me hods o he de ec ion o an ibio ics, mak-
ing hem a supe io choice o ce ain applica ions [17]. One
key ad an age is hei ema kable sensi i i y, which enables
he de ec ion o analy es a low concen a ions. The eal-
ime na u e o he elec ochemical measu emen s allows
apid and dynamic moni o ing, acili a ing imely esponses
insi ua ions whe e apid analysis is c ucial. In addi ion,
elec ochemical senso s o en exhibi excellen selec i i y,
minimizing he in e e ences om complex samples. Thei
ela i e simplici y, po abili y, wea abili y, lexibili y, and
cos -e ec i eness make hem pa icula ly a ac i e o on-
si e and poin -o -ca e applica ions, allowing he analysis o
di e en ypes o samples (human se um, swea , ood, and
en i onmen al samples). Fu he mo e, elec ochemical sen-
so s can be easily minia u ized (allowing hei in eg a ion
in o a ious au oma ed de ices o con inuous moni o ing)
and modi ied wi h nanoma e ials and (bio) ecogni ion ele-
men s (leading o g ea sensi i i y and selec i i y). Va ious
gold nanos uc u es (AuNSs) can be (elec o)chemically
syn hesized, enhancing he ca aly ic and elec ical p ope -
ies, he ac i e su ace o ap ame immobiliza ion h ough
Au–S bonds and he in e ac ion o he ap ame wi h he
a ge . O e all, all hese a ibu es make elec ochemical sen-
so s ad an ageous ools o e icien and eliable an ibio ic
de ec ion in di e se se ings [18, 19].
Ap ame s, o en e e ed o as “chemical an ibodies,” a e
sho , single-s anded DNA o RNA sequences ha exhibi
high a ini y and speci ici y o a speci ic a ge molecule.
In elec ochemical senso s, ap ame s play a c ucial ole
because o hei unique ad an ages. One key ad an age is
hei abili y o ecognize speci ic a ge s, including small
molecules, p o eins, and whole cells, making hem e sa ile
o a ious sensing applica ions [20, 21]. Ap ame s o e
a cos -e ec i e and mo e s able al e na i e o adi ional
an ibodies, wi h he added bene i o easie syn hesis and
unc ionaliza ion, wi h ew ba ch- o-ba ch a ia ions. Thei
s able and well-de ined h ee-dimensional s uc u es and
a ge -induced con o ma ional changes con ibu e o hei
enhanced sensi i i y and selec i i y in elec ochemical sens-
ing, making hem ideal candida es o use as bio ecogni ion
elemen s [22]. Mo eo e , ap ame s can be easily immobi-
lized on elec ode su aces, he eby p o iding a s able and
ep oducible sensing pla o m. This immobiliza ion p ocess
acili a es he de elopmen o obus and eusable elec o-
chemical senso s, con ibu ing o he cos -e ec i eness and
sus ainabili y o sensing echnologies [23]. O e all, he
unique p ope ies o ap ame s make hem aluable com-
ponen s o he design and de elopmen o highly e icien
elec ochemical senso s o a wide a ay o applica ions in
ields such as medical diagnosis, en i onmen al moni o ing,
and ood sa e y.
This s udy de eloped an inno a i e, sensi i e, and dis-
posable ap asenso o on-si e VAN de ec ion. The elec-
ochemical senso was based on an in-lab p in ed ca bon
elec ode (C-PE) unc ionalized wi h gold nanos uc u es
(AuNSs), on which a VAN-speci ic ap ame was g a ed
as a bio ecogni ion elemen . Building on he s abili y and
ep oducibili y o ap ame s, he in-lab p in ed ca bon elec-
odes (C-PEs) ep esen an impo an aspec o his s udy,
signi ican ly enhancing he e sa ili y and cos -e ec i eness
o he sensing pla o m. C-PEs educe dependence on expen-
si e comme cial op ions and p o ide g ea e accessibili y
and cus omiza ion, allowing esea che s o design and ai-
lo hem o speci ic expe imen al needs wi h p ecise con ol
o e dimensions, ma e ials, and con igu a ions. The lexible
suppo o C-PEs u he acili a es hei use in wea able
senso s, o e ing a con enien and a o dable s a ing poin .
Thus, in-lab C-PEs align wi h he g owing emphasis on sus-
ainable and esou ce-e icien me hodologies.
The AuNSs elec osyn hesis was op imized and he
AuNSs we e cha ac e ized by scanning elec on mic oscopy
(SEM) and a omic o ce mic oscopy (AFM). The ap ame -
VAN a ini y was de e mined using iso he mal i a ion calo-
ime y (ITC) and su ace plasmon esonance (SPR). Each
s ep in he ap asenso ab ica ion and op imiza ion p ocess
Mic ochim Ac a (2025) 192:107 Page 3 o 15 107
was cha ac e ized elec ochemically by cyclic ol amme y
(CV), di e en ial pulse ol amme y (DPV), and elec o-
chemical impedance spec oscopy (EIS).
Based on he ac ha he binding o VAN o he ap ame
induces changes a he elec ode su ace ha al e he elec-
on ans e o he e o/ e icyanide edox p obe, which is
hen measu ed using EIS, he quan i ica ion o VAN was
assessed using he EIS spec a in he p esence o 5 mM
e o/ e icyanide as edox p obe, ob aining an ap asenso
wi h LOD in he nanomola ange. The de eloped ap asen-
so p esen ed good selec i i y agains some commonly
ound in e e ences in se um and milk and was success ully
applied o he analysis o hese samples. To he bes o ou
knowledge, his is he i s elec ochemical senso ha com-
bines he ad an ages o in-lab C-PEs, AuNSs, and ap ame s
o VAN de ec ion om milk and human se um.
VAN is cu en ly he an ibio ic o choice in se e e cases
o cow mas i is and he occu ence o an ibio ic esidues in
animal p oduc s can p oduce signi ican heal h haza ds o
consume s, oge he wi h po able po en ios a s, his pla -
o m could be a g ea s a ing poin o he ab ica ion o
easy- o-use, disposable, on-si e apid es ing de ices o he
assessmen o possible con amina ions ha could occu in
milk and de i a es.
Fo he apeu ic d ug moni o ing, gi en he he apeu ic
ange o his an ibio ic (10–20 mg/L o ~ 7–14 µM), ou
senso ma ches he c i e ia equi ed o sa e y and e icacy
assessmen . Mo eo e , gi en he ecen ad ances in pe son-
alized medicine using wea able nonin asi e senso s, along
wi h he co ela ion ound be ween VAN concen a ions
in plasma and swea [16], he lexible design o ou senso
could be a s a ing poin o he de elopmen o a nonin-
asi e wea able nex -gene a ion poin -o -ca e moni o ing
sys em ha could p o ide swea -based eal- ime VAN ea -
men moni o ing.
Ma e ials andme hods
Ma e ials
All chemicals we e o analy ical g ade and used wi hou u -
he pu i ica ion. Sul u ic acid (H2SO4), hyd ochlo ic acid
(HCl), sodium hyd oxide (NaOH), sodium chlo ide (NaCl),
magnesium chlo ide hexahyd a e (MgCl2·6H2O), po assium
chlo ide (KCl), sodium ca bona e (Na2CO3), sodium hyd o-
gen phospha e dodecahyd a e (Na2HPO4·12H2O), sodium
dihyd ogen phospha e monohyd a e (NaH2PO4·H2O), e -
achlo oau ic (III) acid (HAuCl4), is(hyd oxyme hyl)
aminome hane (TRIS), is(2-ca boxye hyl)phosphine
hyd ochlo ide (TCEP), po assium hexacyano e a e (II)
ihyd a e K4[Fe(CN)6]·3H2O), po assium hexacyano e -
a e (III) (K3[Fe(CN)6]), 6-me cap o-1-hexanol (MCH),
his idine, lac ose (Lac), glucose (GLU), and human se um
we e pu chased om Sigma-Ald ich (Ge many).
Vancomycin hyd ochlo ide (VAN) was ob ained om
Lina is Biologische P oduk e GmB (F ankenweg, Ge -
many), and gen amicin sul a e (Gen) om Biowo ld (Louis
Pa k, MN, USA). The h ee di e en ypes o milk used in
his s udy (low- a , 1.5%, and 3% a ) we e pu chased om
a local supe ma ke .
The VAN-speci ic ap ame (Ap ) used in his s udy was
p e iously selec ed [24] and syn hesized by Eu ogen ec
(Belgium) wi h a cus omized hiol g oup a he 5′ end and a
e ocene g oup a he 3′ end, ha ing he ollowing sequence:
5′-SH-(CH2)6-CGA GGG TAC CGC AAT AGT ACT TAT TGT
TCG CCT ATT GTG GGT CGG-Fe ocene-3′.
The suppo ing elec oly e solu ions used in his s udy
included a 0.1 M KCl solu ion and a TRIS bu e (pH 7.4)
con aining 10 mM TRIS, 100 mM NaCl, 100 mM KCl, and
10 mM MgCl2·6H2O. All solu ions we e p epa ed using
Ul aPu e™ DNase/RNase- ee dis illed wa e (The mo
Fishe , USA). The lyophilized Ap was esuspended in 10
mM TRIS bu e pH 7.4 o a s ock solu ion o 100 µM, ali-
quo ed, and s o ed a –20°C. The HAuCl4 and his idine
solu ion mix u e was p epa ed in 0.5M H2SO4. Fo MCH,
a 10mM ini ial s ock was p epa ed in TRIS bu e con-
aining 20% e hanol, a e which u he dilu ions we e pe -
o med using TRIS bu e alone. Fo VAN analysis, esh
concen a ions o VAN we e p epa ed in TRIS bu e . Fo
he analysis o eal samples, human se um (Sigma-Ald ich)
and milk ( om a local supe ma ke ) we e used wi hou u -
he il a ion.
Me hods andins umen s
All elec ochemical expe imen s we e pe o med using an
Au olab PGSTAT302N po en ios a (Me ohm Au olab, The
Ne he lands) equipped wi h No a 1.11 so wa e. The hea -
ing o he Ap was pe o med using an Eppendo The mo-
mixe ® C. The pH measu emen s we e ca ied ou using a
mic opH me e (Hanna Ins umen s).
Iso he mal i a ion calo ime y (ITC) was pe o med
using an A ini y ITC mic ocalo ime e (TA Ins umen s,
New Cas le, USA) con olled by ITCRun .3.8.4.24000 and
Nano&A ini y ITC Da a Collec ion so wa e. Fo degassing
he solu ions, he compa ible Degassing S a ion (TA Ins u-
men s, New Cas le, USA) was used. The he modynamic
pa ame e s we e ob ained by i ing he i a ion cu es
agains he buil -in independen si es using NanoAnalyze
.3.12.5 so wa e.
The su ace plasmon esonance (SPR) expe imen s
we e pe o med on a h ee-channel Biosensing BI-2500
ins umen using he compa ible ba e gold chips (Biosens-
ing Ins umen Inc., Tempe, AZ, USA). To ex ac he
SPR kine ic pa ame e s, he kine ic da a we e analyzed
Mic ochim Ac a (2025) 192:107107 Page 4 o 15
in he amewo k o he Langmui iso he m 1:1 bind-
ing model using BI-so wa e e sion 2.4.4 (Biosensing
Ins umen Inc., Tempe, AZ, USA), which includes he
so wa e Sc ubbe (BioLogic So wa e P y L d., Camp-
bell, Aus alia).
In his s udy, in-lab p in ed elec odes wi h a ca bon-
based wo king su ace (C-PE) we e used. Fo he elec-
ode p in ing p ocess, he ca bon conduc i e ink Elec-
odag 423SS was pu chased om Henkel (Dusseldo ,
Ge many), and Ag/AgCl ink Elec odag PF-410 was
pu chased om Acheson (Delawa e, USA). An au ocla e
(Memme GmbH, Schwabach, Ge many) was used o
he successi e and con olled d ying o he conduc i e
ink du ing he p in ing p ocess.
Mo phological and opog aphical su ace cha ac e iza-
ion o he de eloped senso was pe o med using a omic
o ce mic oscopy (AFM). The measu emen s we e con-
duc ed on a Cyphe S mic oscope (Asylum Resea ch-
Ox o d Ins umen s, San a Ba ba a, CA) in apping mode,
in ai , unde ambien condi ions, wi h silicon p obes
(AC240TS-R3, Olympus, Japan), e lex side aluminum
coa ed, ip adius ypical 7 nm, wi h a sp ing cons an
o 2 N/m (0.6–3.5 N/m) and a esonance equency o
70(± 20) kHz. Da a acquisi ion and image analysis we e
pe o med using he in eg a ed Asylum Resea ch so -
wa e (AR 16.33.234, Asylum Resea ch) w i en wi hin
he Igo P o so wa e package (Igo P o 6.38B01, Wa -
eMe ics, Inc., Lake Oswego, OR, USA). Se e al a eas
o he sample su ace we e analyzed, wi h 512 pixels/
line and wi h a scan a e o less han 1 Hz. Scanning
elec on mic oscopy (SEM) images we e ob ained using
a Hi achi SU8230 SEM (Tokyo, Japan) a 30 kV, 10 µA,
and a wo king dis ance o 15 mm, using Az ec so wa e
om Ox o d Analy ics.
Elec ochemical echniques
The elec ochemical echniques used in his s udy we e
cyclic ol amme y (CV), di e en ial pulse ol amme-
y (DPV), and elec ochemical impedance spec oscopy
(EIS). CV ( he po en ial was cycled wice be ween – 0.2
V and + 1.2 V, wi h a scan a e o 100 mV/s), DPV ( he
po en ial was scanned om – 0.2 and + 0.5 V, wi h a mod-
ula ion ampli ude o 50 mV, modula ion ime o 40 ms
and a scan a e o 10 mV/s), and EIS ( he equency ange
was be ween 0.01 and 100,000 Hz, wi h an ampli ude
o 0.01 V and DC po en ial de e mined by Open Ci cui
Po en ials) measu emen s we e pe o med in he p esence
o 5 mM [Fe(CN)6]3–/4– edox p obe p epa ed in 0.1 M
KCl solu ion. The measu emen s using e ocene-labeled
ap ame we e pe o med in a 0.1 M KCl solu ion.
Ap ame a ini y e alua ion
Iso he mal i a ion calo ime y (ITC) The ITC analyses we e
conduc ed acco ding o he ollowing p o ocol. P io o each
measu emen , he Ap solu ion was subjec ed o he mal
ea men and each solu ion was degassed o 10 min unde
acuum (25 inHg) a 25 °C using he Degassing S a ion.
Fo he Ap solu ion, 300 µL o 10 µM Ap in TRIS bu e
was p epa ed, loaded in o he ITC sample cell, and i a ed
wi h 150 µM VAN solu ion p epa ed in he same bu e .
The e e ence cell o he calo ime e was illed wi h 300
µL nuclease- ee wa e . Fo i a ion, 23 i a ion s eps we e
pe o med o each injec ion using 5 µL o VAN solu ion,
excep o he i s injec ion, o which 2 µL was used. The
o he i a ion pa ame e s we e as ollows: ini ial baseline
(300 s), injec ion in e al (150 s), s i ing a e (100 pm),
and empe a u e se poin (25 °C).
Su ace plasmon esonance (SPR) Fo SPR analysis, he ba e
Au chip was ini ially washed wi h e hanol, d ied unde a
ni ogen s eam, and hyd a ed wi h 300 µL TRIS bu e o
1 h a oom empe a u e. The Au chip was hen co e ed wi h
300 µL o 1 µM Ap solu ion p epa ed in TRIS bu e (p e-
iously educed and he mally ac i a ed) and le o e nigh
a 4 °C in a wa e -sa u a ed a mosphe e. Subsequen ly, he
su ace was insed wi h 300 µL o TRIS bu e and incuba ed
again o 30 min a oom empe a u e wi h 300 µL o 100
µM MCH solu ion p epa ed in he same bu e . A e a inal
wash wi h 300 µL o TRIS bu e , he modi ied Au chip was
moun ed on he SPR p ism.
The unning bu e o he a ini y e alua ion was TRIS
bu e , main aining a cons an low a e o 60 µL/min. P io
o analysis, he bu e was il e ed h ough a 0.2 µm po e
diame e il e and degassed o 1 h unde acuum (25 inHg)
a 25 °C using he Degassing S a ion. A e achie ing a s a-
ble baseline, inc easing concen a ions (0.05–10 µM) o
VAN solu ions p epa ed in TRIS bu e we e injec ed. Fo
each concen a ion, 300 µL o he solu ion was injec ed o
300 s (associa ion phase), ollowed by 300 s o bu e un-
ning h ough he sys em be o e a new injec ion (dissocia-
ion phase and new baseline s abiliza ion). Be o e he i s
injec ion o VAN, 300 µL o TRIS bu e was injec ed o
blank co ec ion.
In‑lab elec ode p in ing p ocess
The componen s o he elec ochemical cell we e p in ed on
a hin lexible polyme ic subs a e by op imizing he p in -
ing p ocedu e desc ibed in wo p e ious s udies [25, 26].
B ie ly, he s encil was placed on op o he polyme ic sub-
s a e, a laye o conduc i e ink was applied, and a ubbe
squeegee was d agged along he s encil o dis ibu e he ink
Mic ochim Ac a (2025) 192:107 Page 5 o 15 107
uni o mly in he ape u e. Ag/AgCl ink was p in ed i s o
p o ide he e e ence elec ode (RE), a e which he wo k-
ing (WE) and coun e (CE) elec odes we e p in ed using
ca bon conduc i e ink. A e each p in ing s ep, he poly-
me ic subs a e was au ocla ed o 15 min a 50 °C. To mini-
mize sho cu s on he connec ions, insula o ape was used o
co e he in e connec ions, a e which he esul ing C-PEs
we e le o d y ou comple ely a 50 °C o e nigh . In addi-
ion, se pen ine connec ions we e p in ed using Ag/AgCl
conduc i e ink. Finally, sil e wi es we e a ached o he
se pen ine connec ion o acili a e he connec ion be ween
C-PE and he po en ios a .
C‑PE condi ioning andgold nanos uc u ed pla o m
ab ica ion
The i s s ep in he ap asenso de elopmen consis ed o
he ac i a ion o C-PEs wi h 1 M Na2CO3 solu ion using
an ampe ome ic p ocedu e by applying a cons an po en-
ial o + 1.2 V ( s. Ag/AgCl) o 600 s. A e ac i a ion, he
C-PEs we e modi ied wi h gold nanos uc u es (AuNSs) in
he op imal condi ions: 10 mM HAuCl4 solu ion in 0.5 M
H2SO4 con aining 150 mM his idine by ch onopo en iome y
(CP)-assis ed elec odeposi ion (− 100 µA cu en applied
o 600s). A e modi ica ion, he elec ode was insed h ee
imes wi h 100 µL o nuclease- ee wa e .
Ap asenso de elopmen
The ob ained gold nanos uc u ed pla o m (C-PE/AuNSs)
was u he used o he immobiliza ion o he VAN-spe-
ci ic ap ame h ough a gold- hiol co alen bond. Two di -
e en immobiliza ion me hods we e es ed: (i) o e nigh
incuba ion a 4 °C and (ii) mul ipulse pulse ampe ome y
(MPA). Fo MPA, he applied po en ial was swi ched
be ween + 0.5V and –0.2V ( s. Ag/AgCl) wi h a 10ms
pulse du a ion o 300s.
P io o use, he possible disul ide bonds o med du -
ing Ap s o age we e educed wi h TCEP, by incuba ing a
oom empe a u e o 1h in he da k, a 1:1 ( / ) mix u e o
100µM Ap solu ion wi h 20mM TCEP, bo h p epa ed in
TRIS bu e . The educed ap ame was hen dilu ed wi h
TRIS bu e o a inal concen a ion o 1µM and subjec ed o
he mal ea men by hea ing o 95 °C o 5 min, ollowed by
cooling o –20°C o 5min. A e Ap immobiliza ion, he
elec ode was insed h ee imes wi h 100 µL o TRIS bu e .
To p e en non-speci ic adso p ion on he su ace o he
elec ode, he emaining gold ac i e si es we e blocked wi h
20 µL o 100µM MCH solu ion p epa ed in TRIS bu e
by incuba ion o 30min a oom empe a u e. A e block-
ing, he elec ode was insed h ee imes wi h 100 µL o
TRIS bu e . The elec ode su ace was consis en ly co e ed
du ing ap asenso de elopmen wi h a d op o TRIS bu e
o p e en dehyd a ion and spa ial degene a ion o he Ap .
The elec ode su ace was elec ochemically cha ac e -
ized a e each modi ica ion s ep by EIS and DPV, using a 5
mM [Fe(CN)6]3−/4− solu ion.
Vancomycin quan i ica ion p ocedu e
Fo VAN quan i ica ion, he ob ained sensing pla o m
(C-PE/AuNSs/Ap /MCH) was incuba ed wi h 50 µL o a i-
ous concen a ions o VAN p epa ed in TRIS bu e o 45
min a oom empe a u e in a wa e -sa u a ed a mosphe e.
A e incuba ion, he elec ode was insed h ee imes wi h
100 µL o TRIS bu e o elimina e he possibili y o non-
speci ic adso p ion. Two di e en VAN quan i ica ion p o-
cedu es we e es ed, using he changes be o e and a e VAN
incuba ion in he elec ochemical signal o he (i) Ap e -
ocene label o (ii) a 5 mM [Fe(CN)6]3−/4− solu ion.
Fo he i s p ocedu e, a 50 µL 0.1M KCl solu ion was
applied on he elec ode and he elec ochemical signal o
he Ap e ocene label was eco ded. The analy ical signal
was de e mined based on he anodic cu en in ensi y (I)
alues be o e and a e VAN incuba ion.
Fo he second quan i ica ion p ocedu e, he analy i-
cal signal o he edox p obe was de e mined based on he
cha ge- ans e esis ance (Rc ) and anodic cu en in ensi y
(I) alues be o e and a e a ge incuba ion. The EIS and
DPV signals we e calcula ed as a pe cen age o educ ion
in Rc , espec i ely inc ease in I a e incuba ion wi h VAN,
acco ding o he ollowing Eqs. (1) and (2):
In e e ence s udies
Fo he ea men o se ious MRSA in ec ions, VAN is o en
combined wi h a second an ibio ic, mos o en gen amicin.
In he ap asenso de elopmen p ocess, he po en ial in e e -
en s we e es ablished depending on he inal applicabili y on
eal sample analysis (human se um and milk). The e o e, he
selec i i y o he de eloped ap asenso in complex ma ices
was e alua ed agains gen amicin, glucose, and lac ose. The
assessmen in ol ed examining he EIS signal esponses o
indi idual in e e en solu ions (1.0 µM) and 1:1 mix u e
wi h VAN (1.0 µM each), ollowing he p o ocol desc ibed
o he VAN quan i ica ion p ocedu e (2.3.5).
(1)
S
incuba ion =
(
Rc MCH −Rc VAN
)
×
100
R
c MCH
(2)
S
incuba ion =
|
|IMCH −IVAN
|
|×
100
I
MCH

Mic ochim Ac a (2025) 192:107107 Page 6 o 15
Real sample analysis
To e alua e he pe o mance and applicabili y o he de el-
oped ap asenso o he quan i ica ion o VAN in eal samples,
a i icial human se um and milk wi h di e en a con en s
(0.1%, 1.5%, and 3.5%) we e used o analysis. All samples
we e dilu ed 10 imes wi h TRIS bu e o ensu e he elec-
oly ic composi ion and ionic s eng h necessa y o p ope
ecogni ion and binding o he ap ame . The esul ing VAN
solu ions we e hen incuba ed o 45 min a oom empe a-
u e in he same manne as desc ibed o VAN quan i ica-
ion. The applicabili y o he de eloped sensing me hod was
hen e alua ed by spiking he dilu ed samples wi h a known
concen a ion o VAN (1 µM) and calcula ing he eco e y
a es as ollows: eco e y (%) = Cs/C × 100, whe e Cs is he
mean VAN concen a ion o he spiked sample and C is he
expec ed heo e ical concen a ion o he sample a e spiking.
Resul s anddiscussion
Ap ame a ini y s udies
Since hei disco e y in he 90s, many ap ame s ha e been
epo ed in he li e a u e o nume ous applica ions. To be
used as a bio ecogni ion elemen in elec ochemical sensing,
an ap ame mus unde go a binding-induced con o ma ional
change ha p oduces a signi ican change in he elec on
ans e o he edox p obe. The selec ed VAN-speci ic
ap ame was designed o exhibi his p ope y by des abiliz-
ing he pa en DNA sequence wi h he emo al o ou base
pai s om i s s em (4 unc), leading o a binding-induced
con o ma ional change obse ed ia ci cula dich oism [27].
The 4 unc ap ame wi h he mos s able p edic ed seconda y
con o ma ion, cha ac e ized by he lowes Gibbs ee ene gy
(ΔG = − 7.03 kcal/mol), is shown in Fig.1A. Seconda y
s uc u e p edic ion was ca ied ou a 25 °C, wi h a 0.15
M Na+ and 0.01 M Mg2+ concen a ion, using he Nupack
Web se e [28]. The sequence was linked o a hiol unc-
ional g oup ia a C6 linke , o enable su icien spacing o
ap ame olding and a ge binding.
When selec ing an ap ame om li e a u e, in addi ion
o he cha ac e iza ion p esen ed in he selec ion pape , i is
essen ial o es whe he he ap ame is eliable and wo ks in
he designed se ings. The e o e, he a ini y o he selec ed
Ap sequence, modi ied wi h hiol g oup, was e alua ed
using wo di e en and complemen a y a ini y e alua ion
echniques, ITC, and SPR.
ITC is a highly e ec i e echnique o s udying binding
in e ac ions, along wi h he de e mina ion o a ini y and
he modynamic cons an s, in a label- ee manne . ITC expe -
imen s showed ha he binding eac ion be ween he ap ame
used in his s udy and i s a ge molecule was exo he mic
(Fig.1B). The a ini y and he modynamic pa ame e s we e
ob ained by u he in eg a ing he esul ing hea p o iles
(Fig.1C): dissocia ion cons an (KD = 13.35 ± 2.74µM),
binding en halpy (ΔH = –40.27 ± 0.73kJ/mol), and en opy
(ΔS = –42.92 ± 1.06J/mol·K), s oichiome y o he eac ion
(n = 1.10 ± 0.05).
Ano he echnique in ensi ely applied in he s udy o
ap ame binding kine ics is SPR, a su ace-sensi i e op ical
echnique ha measu es he e ac i e index changes due o
analy e binding. The SPR expe imen al se up consis ed o
he immobiliza ion o he Ap on he su ace o a gold chip,
blocking o he emaining gold si es wi h MCH, ollowed
by a se ies o injec ions using inc easing concen a ions o
VAN (0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10µM). The in e ac ion o
he immobilized Ap wi h di e en concen a ions o VAN in
solu ion was moni o ed, and subsequen ly p ocessed and ana-
lyzed o ob aining bo h kine ics and equilib ium pa ame e s.
The no malized, blank-sub ac ed sensog ams we e i o a
1:1 kine ic binding model (Fig.1D) and a s eady-s a e a ini y
model (Fig.1E). The a e cons an s ob ained we e as ollows:
kon 3.44 ± 1.38 × 103µM−1 s−1, ko 0.064 ± 0.03 × 103 s−1.
Acco ding o he exp ession KD = ko /kon, he dissocia ion
cons an was calcula ed o be 18.31 ± 1.15µM.
The ob ained KDs h ough he wo di e en a ini y
e alua ion me hods (ITC and SPR) we e simila , as o
he KD p e iously epo ed in he ap ame selec ion pape
(45.5 ± 2.2µM) [24], he di e ence can be a ibu ed o he
di e en media used o he a ini y e alua ion (bloods eam
s. TRIS bu e ) and he di e en me hod applied (calib a-
ion- ee E-AB senso s. ITC/SPR).
Pla o m design andsensing p inciple
The ap ame -based elec ochemical sensing pla o m
used in his s udy was designed s a ing om an in-lab-
ab ica ed lexible C-PE unc ionalized wi h cauli lowe -
like AuNSs, on which hiola ed VAN-speci ic Ap was
g a ed ia Au–S co alen bonds. To p e en nonspeci ic
adso p ion, he emaining a ailable Au si es we e blocked
using a sho -chain hiol (MCH). The wo king p inciple
o he de eloped ap asenso was based on he selec ion
s a egy o he chosen ap ame ha p esen ed a key s em-
loop closing con igu a ion, meaning ha he ap ame was
selec ed o speci ically espond o he p esence o VAN
by de aching he complemen a y pa and opening he
s em-loop [24, 27]. In he case o ou sensing pla o m,
be o e VAN binding he s em-loop con igu a ion o he
ap ame p esumably hinde s he elec on ans e a e,
whe eas when VAN is p esen he s em-loop is opened,
hus exhibi ing a binding-induced change in he elec on
ans e a e. Mo eo e , VAN is posi i ely cha ged a pH
7.4 [29], which could u he acili a e he elec on ans e
a e om he nega i ely cha ged edox p obe [30]. The
Mic ochim Ac a (2025) 192:107 Page 7 o 15 107
schema ic illus a ion o he C-PE p in ing and ap asenso
ab ica ion p ocess, along wi h he “signal-on” sensing
p inciple is shown in Fig.2.
Fo a p elimina y cha ac e iza ion o he ob ained
C-PEs, a simple CV ol ammog am was eco ded in 5
mM [Fe(CN)6]3−/4− in 0.1M KCl. As shown in Fig.S1A,
Fig. 1 A Seconda y s uc u e p edic ion o he modi ied VAN Ap .
The analysis was ca ied ou a 25 °C, wi h a 0.15 M Na+ and 0.01
M Mg+2 concen a ion, using he Nupack web se e [28]. B ITC
measu emen s: ITC da a ob ained o he exo he mic eac ion du ing
i a ion o 10µM Ap (g een) and 0µM Ap (black) wi h 150µM
Van. C In eg a ed hea a es o each injec ion plo ed agains he
Van Ap mole a io in he ITC cell; inse : mean he modynamic and
a ini y pa ame e alues ob ained om h ee di e en measu emen s.
D SPR analysis: Sensog ams ep esen ing he kine ic binding p o-
ile o VAN-Ap ( ep esen a i e no malized, blank sub ac ed SPR
sensog ams o eigh concen a ions o VAN (0.05, 0.1, 0.2, 0.5, 1,
2, 5, and 10µM); he colo ed mo e anspa en lines ep esen he i
o expe imen al da a o a 1:1 kine ic binding model). E Co espond-
ing equilib ium binding cu e ( i ed o a s eady-s a e a ini y model
(n = 3)). Expe imen al condi ions: 60 µL/min low a e, injec ions o
300s, 100s dissocia ion ime
Mic ochim Ac a (2025) 192:107107 Page 8 o 15
he ca bon su ace did no exhibi clea edox peaks o he
[Fe(CN)6]3−/4− edox couple; he e o e, o enhance and s and-
a dize he elec ochemical signal, a simple ch onoampe o-
me ic su ace ea men was applied using a 1M Na2CO3
solu ion. A e p e ea men , he peak po en ial sepa a ion
(ΔEp) changed om 0.64 o 0.15V, highligh ing a lowe ba -
ie o elec on ans e and elec ochemical e e sibili y.
The size o he in-lab- ab ica ed C-PEs was 1.5 × 1 cm
(leng h × wid h), wi h a geome ic su ace o he WE o
0.196 cm2. The su ace a ea a ailable o elec on ans e
o he species in solu ion was calcula ed using he Ran-
dles–Se cik Eq.(3):
whe e Ip is he peak cu en (A), n is he numbe o ans-
e ed elec ons, A is he elec oac i e a ea (cm2), D is he
di usion coe icien o he oxidized species (cm2 s−1), is
he scan a e (V s−1), and C is he analy e concen a ion (mol
L−1). The CV measu emen s we e pe o med on he ba e
C-PEs in 5mM [Fe(CN)6]3−/4− in 0.1M KCl by a ying
he scan a e om 25 o 300mV s–1 (Fig.S1B). F om he
slope o he Randles–Se cik plo (peak cu en s s. squa e
oo o he scan a e; Fig.S1C), he elec oac i e a ea was
calcula ed o be 0.226 cm2.
AuNSs elec odeposi ion andsu ace
cha ac e iza ion
In he ield o biosenso s, gold nanopla o ms no only allow
easy g a ing o speci ic bio ecogni ion elemen s ia Au–S
(3)
Ip
=
(
2.69 ×10
5)
×n
3∕2
×A×D
1∕2
×
1∕2
×
C
co alen bonds bu also enhance in e acial molecula ec-
ogni ion by accele a ing molecula di usion and educing
s e ic hind ance [31].
By examining he Au elec odeposi ion p ocess, wo
essen ial phases can be dis inguished, nuclea ion and g ow h,
which a e bo h in luenced by he eac ion condi ions [32].
Thus, op imiza ion o he elec odeposi ion condi ions, such
as he elec ochemical echnique, elec odeposi ion po en ial,
HAuCl4 concen a ion, and deposi ion ime, in luences he
egula ion o nuclea ion and di ec ional agg ega ion. By se -
ing he app op ia e condi ions, a non-equilib ium sys em can
be c ea ed, p omo ing he o ma ion o AuNSs wi h dis inc
a chi ec u es.
Ou app oach included he op imiza ion o a p e iously
epo ed AuNSs o ma ion s a egy in ol ing he inco po a-
ion o amino acids in o a HAuCl4 solu ion o con olled Au
nanos uc u e o ma ion on he su ace o C-PE. The gal a-
nos a ic deposi ion o AuNSs on sc een-p in ed elec odes
by combining HAuCl4 wi h amino acids, such as cys eine
[33] and his idine [31], has been p e iously explo ed by ou
g oup. Those ini ial condi ions se ed as a s a ing poin
o his s udy and we e sys ema ically op imized. Hence,
he augmen a ion o he elec odeposi ion p ocess o he
o ma ion o AuNSs on he sensing su ace o C-PE in ol ed
he op imiza ion o h ee key pa ame e s: (i) his idine con-
cen a ion, (ii) HAuCl4 solu ion concen a ion, and (iii)
elec odeposi ion ime. Unde di e en expe imen al condi-
ions, AuNSs wi h a ious shapes we e ob ained ia one-s ep
elec ochemical deposi ion. Two di e en nega i e cu en s
we e applied o he elec ode (− 100 µA and − 200 µA) in he
p esence o absence o His o ob ain he h ee-dimensional
Fig. 2 Schema ic ep esen a-
ion o he ap asenso ab ica-
ion p ocess and i s wo king
p inciple
Mic ochim Ac a (2025) 192:107 Page 9 o 15 107
s uc u es. The mo phologies o he esul ing AuNSs we e
cha ac e ized by SEM and AFM (Fig.3).
The i s goal o he op imiza ion s udy was o assess
whe he he p esence o His exe ed a c ucial in luence
on he shape o size o he AuNSs. The SEM images
(Fig.3A–C) e ealed cap i a ing AuNSs, wi h dis inc mo -
phological di e ences based on he p o ocols employed. As
can be obse ed in Fig.3A, he elec odeposi ion o Au om
a 10 mM HAuCl4 a − 100 µA, in he absence o His esul ed
in he o ma ion o his le-like s uc u es. Con e sely, he
addi ion o 150 mM o His o he elec odeposi ion solu ion
esul ed in he o ma ion o cauli lowe -like s uc u es, as
shown in Fig.3B. A mo e nega i e cu en (− 200 µA),
c own-like nanos uc u es o med on he C-PE su ace
(Fig.3C).
The AFM images o he ob ained h ee pla o ms a e
p esen ed in Fig.3A’–C’. The su ace ea u es iden i ied by
AFM co esponded o he su ace cha ac e is ics obse ed
by SEM. Fo he his le-like s uc u es, he heigh anged
be ween 0.9 and 1.7 µm, he c own-like s uc u es had a
heigh ange o 0.2–1.0 µm, whe eas he cauli lowe -like
nanos uc u es eached heigh s o 0.5–1.0 µm.
The esul ing C-PE/AuNSs pla o ms we e ana-
lyzed elec ochemically by CV measu emen s in 5 mM
[Fe(CN)6]3−/4− in 0.1M KCl. The pa ame e alues and
hei in luence on he anodic cu en peak a e shown in
Fig.4A-C. As shown, using 150mM o his idine in a
10mM HAuCl4 solu ion and an elec odeposi ion pe iod o
600s led o he mos a o able and ep oducible pla o m,
wi h he highes cha ge ans e o [Fe(CN)₆]3⁻/4⁻ edox
Fig. 3 SEM images o he ob ained C-PE/AuNSs pla o ms: his le-
like (ob ained using 10 mM HAuCl4, 0 mM His, CP –100 µA) (A),
cauli lowe -like (ob ained using 10 mM HAuCl4, 150 mM His, CP
–100 µA) (B), and c own-like (ob ained using 10 mM HAuCl4, 150
mM His, CP –200 µA) (C) s uc u es a di e en magni ica ions and
he co esponding AFM images (A’, B’, and C’, scan size 10 µm,
scale ba 2 µm)