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Click and De ec : Ve sa ile Ampicillin Ap asenso Enabled
by Click Chemis y on a G aphene–Alkyne De i a i e
José M. R. Flauzino, Ma in-Alex Nalepa, Deme ios D. Ch onopoulos, Ve onika Šedajo á,
Da id Panáček, Pe Jakubec, Pe a Küh o á, Ma in Pykal, Pa el Banáš, Aleš Panáček,
A is ides Bakand i sos, and Michal O yepka*
DOI: 10.1002/smll.202207216
he indisc imina e exposu e o mic o-
o ganisms o an ibio ics. This equi es
con olling an ibio ic use and b oad moni-
o ing o hei le els in complex samples,
such as ood, ap and was ewa e , u ine,
and blood.[3] Ch oma og aphy and he
enzyme-linked immunoso ben assay a e
conside ed he gold s anda d me hods
o an ibio ic de ec ion. Howe e , hey a e
ime-consuming and equi e expensi e
ins umen a ion and ained pe sonnel.[4]
Thus, he de elopmen o poin -o -in e es
an ibio ic de ec ion me hods ha a e as ,
cheap, e ec i e, sensi i e, and do no
equi e sophis ica ed labo a o y equip-
men o expe pe sonnel is c ucial. To his
end, p ope ly designed nano-biosenso s
mee all he abo e equi emen s, com-
bining selec i e and apid bio ecogni ion
o a ge analy es wi h a concen a ion-
dependen signal esponse o quan i a i e
ead-ou .[5]
The mos e ec i e biosenso s ely on
selec i e ecogni ion o an analy e in a com-
plex sample ia speci ic binding on a high-a ini y bio ecep o ,
which adi ionally comp ises an enzyme o an ibody immobi-
lized on an app op ia e suppo . Howe e , owing o he high
cos and di icul handling o such delica e biomolecules, syn-
he ic nucleic acids, such as ap ame s, may be be e al e na i es
because hey a e mo e s able and simple o syn hesize.[6] An
ap ame ’s nucleo ide sequence can be ailo ed o selec i ely bind
Tackling he cu en p oblem o an imic obial esis ance (AMR) equi es as ,
inexpensi e, and e ec i e me hods o con olling and de ec ing an ibio ics in
di e se samples a he poin o in e es . Cos -e ec i e, disposable, poin -o -
ca e elec ochemical biosenso s a e a pa icula ly a ac i e op ion. Howe e ,
he e is a need o conduc i e and e sa ile ca bon-based ma e ials and inks
ha enable e ec i e bioconjuga ion unde mild condi ions o he de elop-
men o obus , sensi i e, and selec i e de ices. This wo k desc ibes a simple
and as me hodology o cons uc an ap asenso based on a no el g aphene
de i a i e equipped wi h alkyne g oups p epa ed ia luo og aphene chem-
is y. Using click chemis y, an ap ame is immobilized and used as a suc-
cess ul pla o m o he selec i e de e mina ion o ampicillin in eal samples
in he p esence o in e e ing molecules. The elec ochemical ap asenso
displayed a de ec ion limi o 1.36nM, high selec i i y among o he an ibi-
o ics, he s o age s abili y o 4 weeks, and is e ec i e in eal samples. Addi-
ionally, s uc u al and docking simula ions o he ap ame shed ligh on he
ampicillin binding mechanism. The e sa ili y o his pla o m opens up wide
possibili ies o cons uc ing a new class o ap asenso based on disposable
sc een-p in ed ca bon elec odes usable in poin -o -ca e de ices.
ReseaRch aR icle
J. M. R. Flauzino, M.-A. Nalepa, D. D. Ch onopoulos, V. Šedajo á,
D. Panáček, P. Jakubec, P. Küh o á, M. Pykal, P. Banáš,
A. Bakand i sos, M. O yepka
Regional Cen e o Ad anced Technologies and Ma e ials
Czech Ad anced Technology and Resea ch Ins i u e (CATRIN)
Palacký Uni e si y Olomouc
Šlech i elů 27, Olomouc 783 71, Czech Republic
E-mail: [email p o ec ed]
The ORCID iden i ica ion numbe (s) o he au ho (s) o his a icle
can be ound unde h ps://doi.o g/10.1002/smll.202207216.
© 2023 The Au ho s. Small published by Wiley-VCH GmbH. This is an
open access a icle unde he e ms o he C ea i e Commons A ibu-
ion License, which pe mi s use, dis ibu ion and ep oduc ion in any
medium, p o ided he o iginal wo k is p ope ly ci ed.
A. Panáček
Depa men o Physical Chemis y
Facul y o Science
Palacký Uni e si y
Olomouc 771 46, Czech Republic
A. Bakand i sos
Nano echnology Cen e
Cen e o Ene gy and En i onmen al Technologies
VSB – Technical Uni e si y o Os a a
17. lis opadu 2172/15 708 00, Os a a-Po uba, Czech Republic
M. O yepka
IT4Inno a ions
VSB – Technical Uni e si y o Os a a
17. lis opadu 2172/15, Os a a-Po uba 708 00, Czech Republic
1. In oduc ion
AMR de elopmen is becoming one o he mos se e e h ea s
o public heal h his cen u y. Wo ldwide, 4.95 million dea hs
we e associa ed wi h AMR in 2019,[1,2] and he dea h oll is
expec ed o ise o mo e han 10 million by 2050.[2] One o he
key measu es o es ic he de elopmen o AMR is o p e en
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a a ge using he sys ema ic e olu ion o ligands by exponen ial
en ichmen (SELEX) echnique.[7] Cu en ly, many ap asenso s
ely on complica ed de ec ion me hods based on nanopa icle
agg ega ion o equi e a ge ampli ica ion.[8,9] Elec ochemical
ap asenso s can simpli y he de ec ion o a ge molecules based
on a cu en esponse ela ed o a con o ma ional change in he
ap ame , which is conjuga ed o a edox p obe, a e a ge mol-
ecule binding.[10] Howe e , hei use is so a limi ed o gold and
glassy ca bon elec odes,[11] and low-cos , p in ed elec odes ha e
no ye been explo ed due o hei nonuni o m su aces. Thus,
new ca bon-based ansduce s a e highly desi ed.
G aphene ul ills many equi emen s o an e ec i e ans-
duce due o i s high conduc i i y, high su ace a ea, and low
cos . Howe e , he low chemical eac i i y and hyd ophobic
na u e o p is ine g aphene hampe s i s unc ionaliza ion[12,13]
and a achmen o bio ecogni ion uni s,[14,15] as well as i s
e ec i e handling and in eg a ion in de ices. The low densi y
o elec ochemically ac i e de ec s in p is ine g aphene also
limi s i s pe o mance in elec ochemical sensing,[16] while
in oducing de ec s and unc ionali ies ia oxida ion owa d
g aphene oxide (GO) u ns g aphene in o an insula o [17] and
o en equi es gold co-deposi ion o inc ease he conduc i i y.[16]
Reduc ion s eps can eco e pa o i s conduc i i y bu a he
expense o diminishing he densi y o chemical unc ionali ies.
Ano he limi a ion in using GO s ems om he la ge di e si y
o oxygen unc ionali ies (e.g., hyd oxyl, ca boxyl, e he , epoxy,
and ke o g oups),[18] making i s ep oducible, e ec i e and
selec i e unc ionaliza ion e y challenging.
The chemis y o luo og aphene[20] bypasses such sho com-
ings by p o iding a la ge po olio o selec i ely and densely
su ace- unc ionalized conduc i e g aphene de i a i es.[21–23]
G aphene acid (GA),[24] wi h i s high dispe sibili y in aqueous
media, la ge numbe o su ace-exposed ca boxylic g oups,
elec ical conduc i i y, and biocompa ibili y, can be consid-
e ed an ideal sca old o he ab ica ion o biosenso s. GA has
al eady been used o elec ochemical sensing H2O2,[25] and i s
ca boxylic g oups ha e been employed o co alen conjuga ion
o biomolecules, such as enzymes.[26] Recen ly, i was also u i-
lized o conjuga ion wi h an DNA p obe speci ic o he pig
mi ochond ial genome o p epa e a label- ee elec ochemical
genosenso .[27] The conjuga ion elied on pep ide bond o ma-
ion be ween ca boxyl g oups o GA and amines o he bio-
molecule ia ca bodiimide chemis y. Howe e , conjuga ion o
ap ame s equi es speci ic chemis y conjuga ion me hods o
a oid ap ame damage, especially app oaches ha do no ely
on amino g oups because o hei abundance in biomolecules
and abili y o o m nonspeci ic c osslinks.
In his espec , click chemis y ep esen s an ideal bioconju-
ga ion me hod. The e m was coined by he ecen Nobel P ize
awa dee K. Ba y Sha pless in 2001 o desc ibe eac ions wi h
high yields, wide scope, and no o ew byp oduc s.[27] One clas-
sical click eac ion is coppe -ca alyzed azide-alkyne cycloaddi-
ion (CuAAC), in which an azide eac s wi h an e minal alkyne
o o m a i e-membe ed iazole ing unde ambien condi-
ions.[28] Howe e , he eac ions condi ions mus be well- uned,
as i s success depends on innume ous ac o s such as sol en ,
ca alys concen a ion, and numbe o ac i e si es and he use
o biomolecules equi es bu e solu ions and speci ic condi-
ions. The use o CuAAC o cons uc ap asenso s is s ill a an
ea ly s age,[29,30] and i s po en ial on g aphene subs a es has
no ye been explo ed.
In his wo k, we p esen a new alkyne- e mina ed g aphene
de i a i e (GA-NH-YN, Figu e 1) capable o unde going acile
conjuga ion wi h bio ecogni ion uni s ia click chemis y. We
demons a e s aigh o wa d CuAAC conjuga ion wi h a edox-
p obe-modi ied DNA ap ame ha selec i ely binds ampicillin.
The de eloped elec ochemical ap asenso displayed high selec-
i i y and sensi i i y o ampicillin e en in complex samples,
such as ap wa e , sali a, and milk. The de eloped g aphene
de i a i e p o ides a click-chemis y- eady sys em o conjuga-
ion wi h comme cially a ailable azide-modi ied bio ecogni ion
uni s as a e sa ile pla o m o he design o a wide po olio
o biosenso s. Fu he mo e, GA-NH-YN allows he ab ica ion
o lexible p in ed elec odes on disposable subs a es o use
as low-cos , poin -o -in e es elec ochemical senso s ha can
be ope a ed ia a mobile phone. Impo an ly, hese ea u es
a e combined wi h high selec i i y, e en in p esence o s uc-
u ally simila an ibio ics, and long shel -li e, while o e ing a
de ec ion limi o ampicillin ha is eigh - old lowe han he
maximum esidue limi o ampicillin in milk.
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Figu e 1. A) Reac ion scheme depic ing click chemis y be ween GA-NH-YN
and a DNA ap ame bea ing an azide moie y. B) FTIR spec a, and C) p o-
posed s uc u es o he s a ing nanoma e ial (g aphene acid – GA),
a e conjuga ion wi h p opa gylamine (GA-NH-YN) and click-chemis y
eac ion wi h he ap ame (GA-T iazole-DNA). D) Elemen al composi-
ions o species in ol ed in he click-chemis y eac ion by XPS analysis
(see Figu e S1, Suppo ing In o ma ion o su ey XPS spec a).
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2. Resul s and Discussion
2.1. Nanoma e ial Syn hesis and Cha ac e iza ion
The ca boxylic g oups o GA we e eac ed wi h he p ima y
amine uni s o p opa gylamine ia ca bodiimide chemis y,
yielding he co esponding alkyne- e mina ed g aphene de i a-
i e (GA-NH-YN, Figu e 1). Subsequen ly, GA-NH-YN was
conjuga ed ia click chemis y o he DNA ap ame bea ing an
azide moie y a e he o ma ion o a 5-membe ed iazole ing
(GA-T iazole-DNA) ia 1,3-dipola CuAAC.[29]
Chemical changes du ing his de i a iza ion o GA we e
moni o ed by Fou ie - ans o m in a ed (FTIR) spec oscopy
(Figu e1B). The FTIR spec um o GA-NH-YN displayed a peak
a 2190 cm−1, asc ibed o he e minal alkyne g oup, con i ming
he success ul coupling o GA wi h p opa gylamine. Mo eo e ,
he in ensi y o he ca boxyl g oup band in he s a ing GA,
cen e ed a 1720 cm−1, was signi ican ly educed in compa ison
o he o he GA bands due o amide bond o ma ion.[24] A e
he click eac ion o he alkyne g aphene de i a i e wi h he
azide-bea ing ap ame , he band o he alkyne g oup anished
because o i s con e sion o a 1,2,3- iazole ing.[32] The suc-
cess ul co alen conjuga ion o he DNA ap ame on o GA-NH-
YN was indica ed by he appea ance o h ee sha p bands a
3400, 3360, and 3200 cm−1 a ibu able o N-H s e ching modes
o he guanine bases o he ap ame .[33]
X- ay pho oelec on spec oscopy (XPS) u he p o ed he
ap ame ’s success ul a achmen on GA-NH-YN owing o he
eme gence o a high a omic phospho us con en (0.7 a .%,
Figu e1D, Figu e S1, Table S1, Suppo ing In o ma ion) in he
GA-T iazole-DNA p oduc . In compa ison, he p oduc ob ained
by simple physical mixing o GA-NH-YN and he ap ame
(wi hou coppe and he asco ba e ca alys ) showed lowe P con-
en due o nonspeci ic so p ion, e i ying indi ec ly co alen
g a ing o he DNA ap ame in he GA-T iazole-DNA p oduc .
In o ma ion abou he mo phology o GA-T iazole-DNA was
collec ed by high- esolu ion ansmission elec on mic oscopy
(HR-TEM), ene gy dispe si e spec oscopy (EDS) elemen al
mapping, a omic o ce mic oscopy (AFM), and classical mole-
cula dynamics (MD) simula ions (Figu e 2). A ep esen a i e
HR-TEM image showed ha GA-T iazole-DNA consis ed o
e y ew laye s o g aphene (Figu e2A). EDS elemen al map-
ping con i med he p esence and homogeneous dis ibu ion o
he DNA ap ame ia de ec ion o phospho ous in he sample
(Figu e2F). Acco ding o AFM measu emen s, he hicknesses
o GA-NH-YN and GA-T iazole-DNA we e 1.6nm (o 3.1nm o
double-laye s uc u e) and 1.9nm, espec i ely (Figu e S4, Sup-
po ing In o ma ion and Figu e2G). The heigh o GA-NH-YN
de i ed om MD simula ions was 1.7 ± 0.01nm, co esponding
well o he AFM da a. MD calcula ions o GA-T iazole-DNA ca -
ied ou in wa e sugges ed ha he DNA co alen ly ancho ed
o he alkyne- e mina ed g aphene occu ed in ei he a hai pin-
like o iplex s uc u e (Figu e 2H, see also Me hodology
sec ion and empe a u e eplica-exchange MD simula ions
(T-REMD) discussed la e o u he de ails). The simula ions
indica ed ha he heigh o GA-T iazole-DNA in wa e eached
3.6 ± 0.2 and 4.8 ± 0.3nm o he hai pin-like and iplex old
s uc u es, espec i ely. These alues a e highe han hose
ob ained by AFM, pe haps because in he AFM expe imen ,
sample d ying caused he DNA ap ame o lose i s na i e old
and s ack mo e on he g aphene su ace.
UV-Vis spec oscopy (Figu e S6, Suppo ing In o ma ion)
also sugges ed DNA a achmen o GA-NH-YN based on an
inc ease in he signal a ≈260 nm a e he click-chemis y
eac ion. Ze a po en ial measu emen s (Table S2, Suppo ing
In o ma ion) e ealed a highe nega i e po en ial o GA-
T iazole-DNA (−20.9 mV) in compa ison wi h ba e GA-NH-
YN (−10.7 mV) and he con ol sample ob ained om simple
mixing wi h DNA wi hou ca alys (−13.8mV), indica ing ha
he nega i ely cha ged DNA ap ame was success ully a ached
o he nanoma e ial. The Raman spec a (Figu e S7, Suppo ing
In o ma ion) show dis inc i e D band (a ≈1330 cm−1) and G
band (≈1595 cm−1) bands, bo h e lec ing he sp2 hyb idized
ca bon (D band wi h nea by sp3 hyb idized ca bon, ypically a
de ec o acancy and G band a oma ic sp2 a ea).[33,34] The ID/IG
a io o all h ee samples emained almos unchanged, wi h he
alue anging only 1.28–1.24. Howe e , sha p bands ≈2800 cm−1
o he GA-T iazole-DNA sample can be asc ibed o he p es-
ence o he C–H ib a ions coming om he alipha ic linke s o
he conjuga ed DNA.[35,36]
2.2. Biosenso Design and Op imiza ion
To ine- une he click-chemis y eac ion o op imize he elec-
ochemical esponse, CuAAC eac ion pa ame e s need o be
s udied on o he solid su ace o he elec ode. The a io be ween
coppe and educing agen and he eac ion ime a e key pa am-
e e s and hus we e e alua ed in his wo k. A e 60min o eac-
ion, he me hylene blue (MB) educ ion cu en peak ( he edox
epo e on he DNA) emained cons an (Figu e 3A), indica ing
ha he su ace was sa u a ed wi h DNA. Thus, a eac ion ime
o 60min was selec ed o u he s eps. The op imum asco -
ba e concen a ion was ound o be 20mM (Figu e3B): lowe
concen a ions esul ed in low eac ion yields and elec ochem-
ical signal, whe eas highe concen a ions appea ed o hinde
he eac ion o e en damage he ap ame .[38]
De ec ion o ampicillin by squa e wa e ol amme y (SWV)
was mo e e icien a a high equency (100Hz) and low ampli-
ude (10 mV), esul ing in a signal inc ease o 90% in com-
pa ison o ha o he blank, i.e., in he absence o ampicillin
(Figu e3C,D). Thus, he ap asenso exhibi ed “signal-on” ype
beha io due o he cu en esponse inc ease upon a ge
binding owing o con o ma ional changes o he ap ame and
consequen app oxima ion o he edox p obe on he elec ode/
g aphene su ace. A low equencies (25 Hz), we obse ed
he opposi e beha io , as he signal gain was nega i e, i.e., he
esponse o he a ge was lowe han in he blank, indica ing a
“signal-o ” esponse. P e ious s udies ha e indica ed ha such
beha io is dependen on he equency applied du ing SWV.[38]
In ou wo k, a equency o 100 Hz was chosen o he nex
s eps due o he highe signal gain.
Elec ochemical expe imen s wi h he [Fe(CN)6]3-/4− edox
p obe p o ided aluable in o ma ion abou he es ed ma e ials
and con i med success ul ap ame binding. Cyclic ol amme y
(CV) (Figu e3E) demons a ed ha he elec ode modi ied wi h
alkyne-g aphene had signi ican ly imp o ed elec ochemical
pe o mance since i showed highe cu en signals compa ed
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o ba e, p econdi ioned, and ap ame -modi ied elec odes. Elec-
ochemical impedance spec oscopy (EIS) analysis and i ing
wi h an equi alen Randles ci cui (Figu e3F) e ealed ha he
ba e sc een-p in ed ca bon elec ode (SPCE) was e y esis i e
wi h a high cha ge- ans e esis ance (RCT) o 3620 ± 145 Ω,
bu he acidic p econdi ioning imp o ed he conduc i i y, low-
e ing RCT o 1640 ± 90 Ω. Modi ica ion o he SPCE wi h he
nanoma e ial lowe ed his alue u he (814 ± 58 Ω), high-
ligh ing he conduc i e p ope ies o he g aphene de i a i e.
A e he click-chemis y eac ion wi h he ap ame , he e was
an inc ease in RCT (1420 ± 72 Ω). This may be due o he ne
nega i e cha ge o DNA, which epels he nega i ely cha ged
[Fe(CN)6]3-/4− ions, combined wi h g ea e s e ic hind ance a
he elec ode su ace.
2.3. Ampicillin De ec ion
The cu en esponse o he ap asenso a ied depending on
he concen a ion o ampicillin (Figu e 4A). As he ap ame
became sa u a ed wi h he a ge , highe concen a ions
showed a simila esponse, esul ing in a loga i hmic-shaped
cu e (Figu e S8, Suppo ing In o ma ion) ha could be line-
a ized by applying a log unc ion o yield he ollowing equa ion
ip= (2.2±0.1) * log cAMP+ (6.8 ±0.6) ( = 0.991, n= 21), whe e
ip is he peak cu en o MB educ ion (in µA) and cAMP is he
ampicillin concen a ion (in nM, see Figu e 4B). The limi o
de ec ion was calcula ed as 3.3*σ/S, whe e σ is he s anda d
de ia ion o he blank and S is he slope o he cu e, esul ing
in 1.36 nM (o ≈0.5 µg L−1). This limi is highe han ha o
o he epo ed biosenso s o ampicillin (Table S3, Suppo ing
In o ma ion). Howe e , i is eigh - old lowe han he Eu opean
maximum esidue limi s in milk (4µg L−1),[40,41] sa is ying he
egula o y and ma ke needs. Mo eo e , he p esen ed pla o m
is he only one epo ed o da e ha is based on disposable
ca bon elec odes, o e ing supe io cos bene i s, and hus a
highly compe i i e solu ion o ex ensi e sample sc eening.
To e alua e he ap asenso selec i i y, ou o he an ibio ics
we e es ed. The ap asenso has an inhe en esponse (back-
g ound cu en ) o 10µA, which co esponds o he me hylene
blue educ ion cu en in bu e solu ion wi hou any binding
molecule in he ap ame . The binding wi h he speci ic a ge
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Figu e 2. HR-TEM/EDS o he GA-T iazole-DNA: A) b igh ield, B) da k ield, C) o e lap o he elemen al mapping o D) ca bon, E) ni ogen, and F)
phospho ous a oms. G) AFM analysis o GA-T iazole-DNA. H) Snapsho s om ee 100ns long MD simula ions showing he iplex and hai pin loop
s uc u e o he ancho ed DNA-ap ame on g aphene acid. Bo h mo i s showed good s uc u al compa ibili y wi h he subs a e. The me hylene blue
molecule (connec ed o he 5’ end o he ap ame ) is shown in cyan. Wa e molecules and ions a e omi ed o cla i y.
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(ampicillin) makes he cu en esponse double due o con o -
ma ional changes. As he esponse o he ap asenso in con-
ac wi h o he an ibio ics, including wo s uc u al analogs
penicillin G and amoxicillin, was he same as he absence o
a ge (blank), he ap asenso is highly speci ic (Figu e4C). The
esponse in he p esence o azi h omycin and e acycline was
also equi alen o ha in he blank solu ion (PBS-MgCl2). A e
4 weeks o s o age, he ap asenso cu en esponse o ampi-
cillin inc eased by 5%, p obably due o changes in he con o -
ma ion o he ap ame , in which he edox p obe can be close
o he elec ode su ace (Figu e4D). Ne e heless, his indica es
excellen s abili y o e ime and demons a es i s po en ial as a
p ac ical and long shel -li e p oduc .
Nex , we e alua ed he po able se up based on a mic opo-
en ios a connec ed o a sma phone (Figu e 5A) o poin -o -
ca e analysis and ound ha i ga e only a small shi in peak
po en ial (Figu e5B), which did no a ec he senso ’s pe o -
mance. Fu he , he analysis in eal samples demons a ed he
abili y o he ap asenso o selec i e de ec ion e en in complex
media. The cu en esponse o he senso in eal samples
Small 2023, 2207216
Figu e 3. Click-chemis y in-si u op imiza ion. Peak cu en o me hylene blue educ ion as a unc ion o A) ime, and B) asco ba e concen a ion.
C) Pe cen age signal gain o he a ge and blank o di e en SWV pa ame e s. D) Squa e wa e ol ammog ams o me hylene blue educ ion wi h
op imized pa ame e s (100Hz, 10mV) wi h/wi hou ampicillin and in he absence o ap ame . E) Cyclic ol ammog ams, and F) impedance spec a
we e eco ded a a ious s ages o he biosenso cons uc ion wi h edox p obe (5mM [Fe(CN)6]3-/4− in PBS solu ion 10mM, pH = 7.4). All po en ials
a e e sus me allic sil e .
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spiked wi h ampicillin was close o he signal eco ded in he
bu e (Figu e5C). Spiked ap wa e showed a eco e y a e o
104%, while sali a and milk had 90% and 88% eco e y a es,
espec i ely, e en hough hey a e highly complex samples con-
aining many po en ial in e e ing species, such as p o eins,
ca bohyd a es, and lipids (Table 1). Such supe b pe o mance
and ope a ion wi h he mic opo en ios a connec ed o a sma -
phone opens he possibili y o using he se up o eal-li e and
a o dable poin -o -ca e analysis.
2.4. Mechanism o Ac ion
To da e, he 3D s uc u e o he apo- and holo- o ms o he
ampicillin-binding DNA ap ame and he con o ma ion o he
complex emain unknown. To gain some p elimina y insigh s
in o he mechanism o he ampicillin-binding DNA ap ame ,
we used compu a ional chemis y ools ha can desc ibe
he biomolecula sys ems wi h unp eceden ed empo al and
spa ial esolu ion.[42,43] We used se e al se s o classical and
Small 2023, 2207216
Figu e 4. Ampicillin de ec ion. A) Rep esen a i e ol ammog ams o he esponse o he ap asenso in he p esence o di e en ampicillin concen a-
ions. B) Calib a ion cu e o he ap asenso . C) Nega i e con ol es s, wi h he s uc u es o he ampicillin analogues. D) S abili y o e ime s udy.
All po en ials a e e sus me allic sil e .
Figu e 5. Real sample de ec ion. A) Se up comp ises a sma phone connec ed o a mic opo en ios a holding a sc een-p in ed ca bon elec ode. B)
Vol ammog ams we e eco ded in di e en samples. C) Ba cha o he cu en esponse in di e en samples.
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enhanced-sampling all-a om MD simula ions o s udy he
con o ma ional beha io o DNA ap ame (see Table S4, Sup-
po ing In o ma ion o a comple e lis o simula ions). We
pe o med T-REMD simula ions s a ing om he un olded
s a e o he DNA single-s anded ap ame in he p esence o
absence o ampicillin. Al hough he simula ions we e no well
con e ged, hey p o ided aluable insigh s in o he s uc u al
p e e ences o he ap ame sequence and binding con o ma-
ion o ampicillin o he DNA nucleo ides. Clus e ing and base
pai ing analyses (Figu e S9–11, Suppo ing In o ma ion) iden-
i ied he loca ions whe e he sys em ended o o m s ems,
and con e sely, in combina ion wi h he s acking analysis, hey
iden i ied posi ions in he sequence ha we e p one o bending
(Figu e S12–14, Suppo ing In o ma ion) and o ma ion o
loops. All T-REMD simula ions, wi h o wi hou ampicillin,
showed ha he ap ame had a endency o o m wo loops
posi ioned a ou dis inc si es in he sequence (Figu es S9 and
S10, Suppo ing In o ma ion), he eby o ming a iplex-like
s uc u e.
In he T-REMD simula ions wi h ampicillin, we ocused
on he beha io o he an ibio ic i sel , i.e., how and whe e i
is bound. The simula ions sugges ed ha he DNA bound o
ampicillin in i s ze o-ne -cha ge zwi e ionic o m. The e was
almos no ampicillin-DNA binding in he T-REMD simula ions
o dep o ona ed nega i ely cha ged ampicillin (wi h a neu al
N18 amino g oup). In he zwi e ionic o m, ampicillin bound
o DNA nucleo ides p edominan ly ia he N18 ammonium
g oup (Figu e 6). This g oup s ongly a o ed binding o hy-
mine nucleo ides, and showed modes binding o guanine and
cy osine and no endency o in e ac wi h adenine. In addi-
ion, we obse ed ha he R3 phenyl ing o ampicillin ended
o s ack o he nucleobases. This migh sugges ha hyd ogen
bonding o he N18 ammonium g oup oge he wi h s acking
o he R3 phenyl ing a e in ol ed in he ecogni ion o ampi-
cillin by he ap ame , which would explain he selec i i y o he
ap ame o ampicillin o e penicillin G. Howe e , he sam-
pling o ampicillin binding was oo limi ed o be su icien ly
p edic i e.
3. Conclusions
We syn hesized a new conduc i e g aphene de i a i e bea ing
alkyne g oups ha can unde go click-chemis y eac ion
wi h biomolecules unde mild condi ions. Using he clas-
sical CuAAC click eac ion, we de eloped a simple, as , and
s aigh o wa d p o ocol o ap ame conjuga ion on o dispos-
able, sc een-p in ed elec odes ha had been p emodi ied wi h
he alkyne-g aphene de i a i e. The de eloped ap asenso was
shown o be sensi i e and selec i e, as demons a ed o he
case o he an ibio ic ampicillin. The design o he pla o m is
e sa ile, as he p o ocol can be used wi h di e en ap ame s o
de ec a wide a ie y o a ge analy es, hus enabling low-cos
and disposable sc een-p in ed ca bon elec odes o be used o
he cons uc ion o sensi i e, selec i e, and s able poin -o -ca e
elec ochemical senso s.
4. Expe imen al Sec ion
Ma e ials: All eagen s we e used as ecei ed wi hou u he
pu i ica ion. P opa gylamine, anhyd ous e ahyd o u an (THF), and N-(3-
dime hylaminop opyl)-N′-e hylca bodiimide hyd ochlo ide (EDC-HCl)
we e pu chased om Sigma-Ald ich. E hyl isoni osocyanoace a e
(Oxyma) was pu chased om Al a Aesa . Ampicillin, amoxicillin,
penicillin G, azi h omycin, e acycline, phospha e bu e ed saline (PBS
10 mM, pH = 7.4), po assium e ocyanide, po assium e icyanide,
magnesium chlo ide, coppe sul a e, sodium asco ba e, and sul u ic
acid we e pu chased om Sigma-Ald ich/Me ck. Deionized wa e
(ρ= 18.2 MΩ cm−1) was used o p epa e all solu ions.
The ap ame o ampicillin was selec ed by Song and collabo a o s,[44]
which pe o med op ical es s and chose AMP17 sequence as he bes
op ion o binding o ampicillin. I was pu chased om Biome s, Ge many,
wi h modi ica ions a bo h ends (Figu e S2, Suppo ing In o ma ion)
Small 2023, 2207216
Table 1. The eco e y a e o he ap asenso in spiked eal samples.
Sample cAMP spiked cAMP measu ed Reco e y a e
Tap wa e 1 mM 1.040 mM 104%
50% Sali a 1 mM 0.904 mM 90.4%
50% Milk 1 mM 0.882 mM 88.2%
Figu e 6. The popula ion o H-bonds in e ac ions and s acking in e ac ion be ween ampicillin and DNA ap ame in he e e ence 298 K eplica calcu-
la ed om he las mic osecond o T-REMD simula ion o holo o m (zwi e ionic elec os a ically neu al AMP(0)) o he d(CGGGCGGTTGTATAGCGG)
sequence. The blue boxes ep esen H-bonds be ween ampicillin and he backbone, g een boxes be ween ampicillin and DNA bases, and o ange boxes
ep esen s acking in e ac ions be ween phenyl ing o ampicillin and DNA bases.
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as ollows: 5′-(me hylene blue)-GCGGGCGGTTGTATAGCGG-(azido-
p oline)-3’. The ap ame was esuspended in deionized wa e a 1mM.
Fo op imal binding, he ap ame was olded in o i s e ia y s uc u e.
P io o use, an aliquo o 10µL he ap ame solu ion was dilu ed o
0.1mM in PBS con aining 2mM MgCl2 (PBS-MgCl2), and hen hea ed
o 5min a 70 °C and cooled o oom empe a u e (RT).
Nanoma e ial Syn hesis: GA was syn hesized acco ding o a p e iously
published epo .[24] Fo he conjuga ion o GA wi h p opa gylamine
o o m he GA-NH-YN, a suspension o GA (30 mg) was p epa ed
in anhyd ous THF (10 mL) in a h ee-neck ound-bo om glass lask,
hen EDC-HCl (444mg, 2.32mmol) and e hyl isoni osocyanoace a e
(330mg, 2.32mmol) we e added and he esul ing mix u e was s i ed
o 30 min a RT unde a ni ogen a mosphe e. A e wa d, 440 µL
(6.87mmol) o p opa gylamine was added d opwise o he suspension
and he eac ion mix u e was s i ed o 120h a RT. Nex , he mix u e
was dilu ed in e hanol and he p ecipi a e was collec ed by cen i uga ion.
The inal p oduc was isola ed by consecu i e cen i ugal washing s eps
using N,N-dime hyl o mamide, wa e , e hanol, and dichlo ome hane.
Click Chemis y in Solu ion: Fo FTIR-ATR, XPS, SEM-EDS, TEM, AFM,
ze a po en ial measu emen s, and UV- is and Raman spec oscopy
analysis, he click-chemis y eac ion be ween GA-NH-YN and he
ap ame was pe o med in solu ion as ollows: 700 µL o a 1mg mL−1
suspension o GA-NH-YN was mixed wi h 100 µL o 0.1 mM azide-
ap ame solu ion, 100 µL o 1 mM CuSO4 solu ion and 100 µL o
3 mM asco ba e solu ion. The eac ion was le o p oceed o 48 h
unde agi a ion (1000 pm) and a oom empe a u e (25 °C). To a oid
damaging he biomolecule, sonica ion was no used. The nanoma e ial
was pu i ied by cen i uga ion a 14 000 pm o 10 min and hen
esuspended in 500 µL deionized wa e . This cen i ugal washing was
epea ed wice mo e. GA-NH-YN physical mix u es wi h only ap ame o
coppe and asco ba e we e used as con ols.
Biosenso Cons uc ion (in si u Click Chemis y): Comme cially a ailable
SPCE we e used as a disposable sensing pla o m (DRP-110, Me ohm
D op Sens, Spain). They comp ised a ca bon disk wo king elec ode
(4mm diame e ), ca bon auxilia y elec ode, and sil e pseudo- e e ence
elec ode. Be o e use, p econdi ioning was ca ied ou in 0.1M H2SO4
solu ion by CV (−1.5–+1.5 V, 5 cycles, 100 mV s−1) o emo e o ganic
c osslinks om he ca bon ink, inc easing he SPCE unc ionali y and
ep oducibili y. An Au olab po en ios a (model PGSTAT128N) was used
o all elec ochemical analyses unless o he wise speci ied.
The alkyne-g aphene was deposi ed on o he wo king elec ode by
d op-cas ing acco ding o a p e iously es ablished s a egy,[27] in which
he nanoma e ial suspension was cen i uged o 10min a 10 000 pm
and 20 µL o he supe na an was deposi ed on o he wo king elec ode
su ace. A e d ying a oom empe a u e, he CuAAC eac ion was
pe o med in si u a oom empe a u e (25 °C). A 100µM solu ion o
he ap ame was mixed wi h 1mM CuSO4 and a ious concen a ions o
sodium asco ba e solu ion in a 2:1:1 p opo ion, espec i ely. Di e en
sodium asco ba e concen a ions (3, 5, 10, 20, 30, 40, and 50mM) and
eac ion imes (15, 30, 45, 60, and 90min) we e es ed o op imize he
eac ion. A e wa d, he elec ode su ace was washed wi h PBS-MgCl2.
Immobilized ap ame was de ec ed by moni o ing he educ ion peak o
MB by SWV using di e en equencies (10, 25, 50, and 100 Hz) and
ampli udes (10, 25, 50, and 100mV) in PBS-MgCl2. All measu emen s
we e made in iplica e and p esen ed as mean ± s anda d de ia ion.
Elec ochemical s udies o he biosenso cons uc ion s eps we e
pe o med using 5 mM [Fe(CN)6]3-/4- edox p obe in PBS solu ion
(10mM, pH = 7.4). CV was measu ed be ween −0.4V and + 0.7V a scan
a e o 50mV s−1, EIS was measu ed a E1/2= 0.115V wi h a equency
ange o 0.1Hz o 10kHz and ampli ude o 10mV.
Sample De ec ion: PBS-MgCl2. con aining di e en ampicillin
concen a ions (10, 1, 100, 10, 1, 100, and 10nM) was added d opwise
o he su ace o he modi ied wo king elec ode and le o incuba e o
30min. SWV was pe o med o obse e he MB peak (po en ial ange:
0–−0.6V, equency: 100Hz, ampli ude: 10mV). Solu ions o PBS-MgCl2
con aining di e en an ibio ics (amoxicillin, penicillin G, azi h omycin,
e acycline) a 1mM concen a ion we e used as nega i e con ols. The
s abili y o he ap ame o e ime was e alua ed by keeping he di e en
p epa ed elec odes a 4 °C and measu ing he esponse in a solu ion o
1mM ampicillin in PBS-MgCl2 in a pe iod o 4 weeks.
Fo es eal sample de ec ion, ap wa e , 50% dilu ed eshly collec ed
sali a, and 50% dilu ed milk (1.5% a ) spiked wi h 1mM ampicillin we e
used and he eco e y pe cen age was calcula ed. A se up consis ing
o a mic opo en ios a (Sensi Sma , Palm Sens) connec ed o a
sma phone was u ilized.
The baseline o all squa e wa e ol ammog ams was sub ac ed
and he peak cu en was calcula ed using NOVA 2.1 and PST ace 5.9
so wa e. To aid he p esen a ion, he ol ammog ams we e in e ed and
he mos ep esen a i e example was p esen ed. O igin 2018 so wa e
was u ilized o plo all da a.
Simula ion Se up: The un olded s a ing s uc u e o he DNA
ap ame used in REMD olding and classical g aphene-g a ed
simula ions wi h sequence d(GCGGGCGGTTGTATAGCGG) was
p epa ed using he Nucleic Acid Builde ool in AMBER.[44] Addi ionally,
o he g aphene-g a ed model, he seconda y s uc u e o he ap ame
was p edic ed using he M old se e ,[45] con e ed in o a 3D s uc u e
using he RNACompose se e ,[46] and ancho ed in he middle o he
g aphi ic su ace. GA ( unc ionaliza ion deg ee 13%, o which 60%
was addi ionally conjuga ed wi h he amino alkyne, and he emaining
unmodi ied ca boxylic g oups we e simula ed in hei dep o ona ed
o m) was modeled as a pe iodic shee in he xy plane wi h dimensions
≈99 × 101 Å, and un unc ionalized g aphi ic ca bons we e ea ed
as uncha ged Lenna d-Jones sphe es. The pa ame e s o ampicillin
in di e en p o ona ion s a es, MB and azido-p oline (N3-P o) we e
adop ed om he GAFF o ce- ield. Pa ial cha ges o he alkyne-
e mina ed GA (modeled on a small, unc ionalized py ene model), N3-
P o linke s, MB and an ibio ics we e assessed using he RESP me hod
a he HF/6-31G* le el o heo y[47] using he Gaussian 16 p og am.[48]
Fo each se o simula ions (see Table SX1, Suppo ing In o ma ion), all
ele an molecules we e subsequen ly assembled in o a single sys em
and he AMBER18[49] o GROMACS 5.0 (in he case o a sys em wi h
pe iodic g aphene)[50] packages we e used o gene a e he s a ing
opology and coo dina es and un simula ions.T-REMD[51] and classical
MD simula ions o he ap ame we e pe o med wi h he AMBER
DNA o ce- ield OL21[52] including he pa mbsc0[53–55] o ce ield wi h
χOL4[56]+ εζOL1[57]+ βOL1[58]+ α/γ[52] modi ica ions. The simula ions we e
pe o med by combining he SPC/E[59] explici wa e model and ∼10mM
KCl sal excess using Joung-Chea ham ion pa ame e s.[60] P io o he
simula ions, he p epa ed sys ems we e minimized and equilib a ed
using he s anda d equilib a ion p o ocol desc ibed in he Suppo ing
In o ma ion. The inal p oduc ion un o he ap ame -g aphene sys em
was ca ied ou in he NVT ensemble o 100ns. T-REMD simula ions
we e un using 64 eplicas a empe a u es anging om 278 o 461 K.
The 64 di e en con o ma ions we e chosen as s a ing s uc u es o
ensu e be e con e gence. The simula ion ime o he T-REMD
simula ions was a leas 4 µs (Table S4, Suppo ing In o ma ion).
Suppo ing In o ma ion
Suppo ing In o ma ion is a ailable om he Wiley Online Lib a y o
om he au ho .
Acknowledgemen s
The au ho s g a e ully acknowledged suppo om he Technology
Agency o he Czech Republic, P og am TREND (GEFSEM, FW01010183),
and he ERDF/ESF p ojec “Nano4Fu u e” (No. CZ.02.1.01/0.0/0.0/16
_019/0000754). The au ho s acknowledged he assis ance p o ided by
he Resea ch In as uc u e NanoEn iCz, suppo ed by he Minis y o
Educa ion, You h and Spo s o he Czech Republic unde P ojec No.
LM2018124. This wo k was suppo ed by he Minis y o Educa ion,
You h and Spo s o he Czech Republic h ough he e-INFRA CZ
(ID: 90140). This p ojec has ecei ed unding om he Eu opean
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Small 2023, 2207216
Union’s Ho izon Eu ope esea ch and inno a ion p og am unde
g an ag eemen No 101059266. MO g a e ully acknowledged an ERC
consolida o g an (683024) om Ho izon 2020. AB acknowledges
unding om he Czech Science Founda ion (p ojec No 19–27454X).
The au ho s hank Jiří Hošek, Ondřej Tomanec, and Klá a Čépe o
conduc ing he SEM, AFM, and CPEM measu emen s.
Con lic o In e es
The au ho s decla e no con lic o in e es .
Da a A ailabili y S a emen
The da a ha suppo he indings o his s udy a e a ailable a ZENODO
(h ps://doi.o g/10.5281/zenodo.7509720).
Keywo ds
an ibio ic de ec ion, ap ame s, biosenso s, g aphene acid, sc een
p in ed ca bon elec odes
Recei ed: No embe 20, 2022
Re ised: Decembe 20, 2022
Published online:
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