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ELECTROCHEMICAL BIOSENSOR BASED ON MODIFIED REDUCED GRAPHENE OXIDE
WITH SILVER NANOPARTICLES FOR DETECTION OF METHYLATED DNA
1,2
Eliska SEDLACKOVA,
1
Eliska BIRGUSOVA,
1,2
Zuzana BYTESNIKOVA,
1
Pa el SVEC
,
1,2
Lukas RICHTERA,
1,2
Voj ech ADAM
1
Depa men o Chemis y and Biochemis y, Mendel Uni e si y in B no, Czech Republic, EU
2
Cen al Eu opean Ins i u e o Technology, B no Uni e si y o Technology, B no,
Czech Republic, EU
Abs ac
DNA me hyla ion is one o he mos s udied and basic epigene ic p ocess ela ed o se e al diseases such as
diabe es, neu odegene a i e o ca dio ascula diseases and e en cance . Me hyla ed DNA p esen s a new
gene a ion o bioma ke s which can be used o poin -o -ca e de ec ion. Elec ochemical biosenso s p o ide
simple, as , cos -e ec i e, easy- o-use, eliable and e icien de ec ion in con as wi h con en ional diagnos ic
me hods. These biosenso s can be used o ea ly diagnosis o men ioned diseases and inc ease pa ien
eco e y by ea ly clinical in e en ions. In his s udy, me hyla ed DNA was de ec ed elec ochemically by he
de eloped biosenso . The ba e gold elec ode was modi ied by d op-cas ing educed g aphene oxide wi h
sil e nanopa icles which enhance elec ochemical signal due o hei s ong a ini y o hiol modi ied DNA
p obe ia a disul ide b idge. A e wa ds, he sensi i i y and selec i i y o he nanocomposi e we e examined.
The a adic elec ochemical impedance spec oscopy was used o de e mina ion o he hyb idiza ion o he
DNA p obe wi h a me hyla ed DNA sequence. The ab ica ed biosenso shows p omising analy ical ea u es
wi h a wide de ec ion o he linea ange.
Keywo ds: Reduced g aphene oxide, sil e nanopa icles, nanoma e ials, me hyla ed DNA, elec ochemical
biosenso
1. INTRODUCTION
Epigene ics deals wi h modi ica ions ha inhe en ly a ec gene exp ession wi hou al e ing he p ima y gene ic
in o ma ion, especially he nucleo ide sequence in he DNA. These modi ica ions include DNA me hyla ion,
his one modi ica ion, ch oma in emodelling and pos ansc ip ional gene egula ion by miRNA. DNA
me hyla ion plays a c ucial ole in he egula ion o se e al biological p ocesses. Mo eo e , me hyla ed DNA
is associa ed wi h se e al diseases including cance [1]. Ea ly ea men o cance equi es p e en i e and
accu a e diagnosis, which is e y expensi e, ime-consuming, equi es specialized s a and complica ed
ins umen a ion and only can be pe o med in he hospi als [2]. E e y yea in he Czech Republic, nea ly
100,000 o he wo ld popula ion su e om malignan ca cinoma, and las yea nea ly 30,000 people died
because o cance . Howe e , due o he lack o apid diagnos ics, hese numbe s a e apidly inc easing. The
numbe o newly diagnosed pa ien s is expec ed o inc ease apidly in he coming yea s. Recen s udies [3, 4,
5] show ha me hyla ed DNA is a new gene a ion o bioma ke s, which a e a p omising al e na i e o use in
bo h clinical diagnos ics and he apy [6]. Ne e heless, classical me hods a e expensi e, ime-consuming and
equi e skilled s a . On he o he hand, elec ochemical biosenso s a e p omising o clinical diagnosis and
can be used as analy ical ools in he poin -o -ca e applica ion.
The p oposed wo k was aimed o c ea e a simple and uni e sal biosenso o he de ec ion o DNA me hyla ion
and M. SssI ac i i y assay. To achie e he be e sensi i i y and selec i i y was used he nanocomposi e
h ps://doi.o g/10.37904/nanocon.2019.8610
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consis ed om educed g aphene oxide wi h sil e nanopa icles ( GO-AgNPs), because o he good chemical
s abili y and abili y o acili a e elec on ans e be ween elec odes and biomolecules [7].
2. MATERIAL AND METHODS
2.1. Reagen s
In his s udy we e used syn he ics oligonucleo ides DNA_1: HS-(CH
2
)
6
-5'-CCT CGT GCG GGA TCA TTG TTA
TTA GGCA-3' and DNA_2: 3'-GGA GCA CGC CCT AGT AAC AAT AAT CCGT-5', enzymes: CpG MTase
M.SssI is supplied wi h 10× NEBu e 2, 200× S-adenosylme hionine (SAM, 32 mM) and es ic ion
endonuclease HpaII wi h 10× Cu Sma bu e (The mo Fishe ), me hylene blue (MB) chemicals o bu e
p epa a ion: sodium hyd oxide (NaOH), po assium sul a e (K
2
SO
4
), po assium hexacyano e a e(III)
(K
3
[Fe(CN)
6
]), po assium hexacyano e a e(II) (K
4
[Fe(CN)
6
]), sodium dihyd ogen phospha e (NaH
2
PO
4
) and
sodium phospha e dibasic (Na
2
HPO
4
). Reagen s o he syn hesis o GO and GO-AgNPs: sil e ni a e
solu ion (AgNO
3
), 37% HCl, po assium pe mangana e (KMnO
4
), sodium bo ohyd ide (NaBH
4
) and H
2
O
2
. All
used chemicals we e ob ained om Sigma-Ald ich (S . Louis, MO, USA) unless o he wise s a ed. High pu i y
deionized wa e (Milli-Q Millipo e 18.2 MΩcm
-1
, Bed o d, MA, USA) was used h oughou he s udy.
2.2. Syn hesis o GO and GO-AgNPs
GO was p epa ed by chemical oxida ion o 5 g g aphi e lakes (100 mesh, ≥75 % min) in a mix u e o
concen a ed H
2
SO
4
(670 ml) and 30 g KMnO
4
acco ding o he simpli ied Humme ’s me hod [8]. The eac ion
mix u e was s i ed igo ously. A e 4 days, he oxida ion o g aphi e was e mina ed by he addi ion o H
2
O
2
solu ion (250 ml, 30 w %). Fo med GO was washed 3 imes wi h 1 M HCl (37 w %) and se e al imes wi h
Milli-Q wa e ( o al olume used 10 l) un il cons an pH alue (3-4) was achie ed. A e wa ds, he 1 ml o GO
solu ion (5 gl
-1
) was added d opwise in o he 50 ml o AgNO
3
(10 mM) unde igo ous s i ing. A e ha 40
mg o sodium bo ohyd ide (NaBH
4
) was added slowly o he eac ion mix u e and he esul ing mix u e was
s i ed in ensi ely o 24 h o allow educ ion. The inal composi e was washed wi h Milli-Q wa e se e al imes.
2.3. The p ocedu e o elec ode su ace p epa a ion
Fi s ly, he ba e gold elec ode (GE) wi h a 2 mm adius (Ch Ins umen s Inc., Aus in, USA) was mechanically
and elec ochemically polished. The h ee ba e GEs we e i s ly sonica ed in ul asonic cleane Elmasonic
P60H (Singen am Hohen wiel, Ge many) o 15 mins in e hanol (E OH) and hen mechanically polished on a
polishing pad (Buehle ) wi h wa e -based diamond suspension and an alumina slu y. As he las s ep we e
elec odes cleaned elec ochemically in 0.5 M H
2
SO
4
by 50 scans o CV by po en ial scanning be ween −0.05 V
and +1.1 V. The GEs we e insed wi h Milli-Q wa e .
2.4. Biosenso de elopmen
The p oposed biosenso was ab ica ed s ep by s ep as i desc ibed in Scheme 1. Fi s ly, he 5 µl o GO-
AgNPs was d op-cas ed on o he su ace and le i d y. Then he 1 µM DNA_1 was immobilised on o GO-
AgNPs and le i incuba e o e nigh in he idge (4 °C). A e incuba ion, he DNA_1 hyb idized wi h 100 nM
DNA_2. While he hyb idiza ion was success ul, he elec ode was ea ed by enzyme M. SssI
me hyl ans e ase, which added he me hyl g oups (‒CH
3
) o he i h ca bon o cy osine. A leas he es ic ion
HpaII, which blocked he clea ing a a speci ic si e. To e alua e he sensi i i y o de eloped biosenso , he M.
SssI ac i i y was es ed. The modi ied GE was ea ed by a di e en concen a ions o M. SssI (5; 25; 50; 100;
200; 300; 400 and 500 Uml
-1
) and measu ed in 20 µM MB solu ion.
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Scheme 1 S ep by s ep o he biosenso ab ica ion
2.5. Elec ochemical measu emen s
Syn hesised GO-AgNPs was e alua ed ia scanning elec ochemical mic oscopy (SEM). P ocess o
biosenso de elopmen was cha ac e ised ia elec ochemical impedance spec oscopy (EIS) in Fa adaic
mode, wi h a equency ange om 100 kHz o 100 MHz. All he elec ochemical measu emen s we e
pe o med in a s anda d h ee-elec ode sys em. Sensi i i y and eliabili y we e cha ac e ised by di e en ial
pulse ol amme y (DPV). EIS and DPV we e pe o med in 5 mM Fe(CN)
64−
/ Fe(CN)
63−
dilu ed in 100 mM
phospha e bu e (PB, pH 7.0). Pa ame e s o DPV we e ollowing: po en ial ange om 0.0 V o −0.6 V,
ampli ude 0.02 V and scan- a e 0.015 Vs
-1
.
3. RESULTS AND DISCUSSION
3.1. Syn hesis o GO and GO-AgNPs
AgNPs we e s abilized on he GO su ace by using
NaBH
4
as a educing agen . The esul ing ma e ial is
GO-Ag nanocomposi e wi h uni o mly dis ibu ed AgNPs
on o GO. The mo phology o nanocomposi e GO-Ag
was obse ed using SEM (Figu e 1).
Figu e 1 SEM pic u e o he GO-AgNPs
nanocomposi e
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3.2. Elec ochemical pe o mance o he de eloped biosenso
The modi ied su ace o GE by GO-AgNPs nanocomposi e was cha ac e ized by Elec ochemical Impedance
Spec oscopy (EIS). Reco ded Nyquis plo s (Figu e 3) show inc easing cha ge ans e alue due o he
change be ween edox po en ial and he GE su ace. Mo eo e , he sensi i i y o he p oposed biosenso was
ob ained by di e en ial pulse ol amme y (DPV). The linea equa ion was ob ained om calib a ion cu e I(µA)
= −0.0005c (U/ml) − 0.1003; (R
2
= 0.9922) and has shown a b oad linea ange om
5 U/ml o 500 U/ml o M. SssI. The e o ba s we e calcula ed as a s anda d de ia ion om measu emen
(n = 3).
Figu e 2 A) Nyquis plo s ob ained om EIS measu emen in 5 mM Fe(CN)
64−
/ Fe(CN)
63−
in 100 mM PB. (a)
The ba e GE, (b) GE/ GO-AgNPs, (c) GE/ GO-AgNPs/DNA_1, (d) GE/ GO-AgNPs/p obe DNA_1/ DNA_2,
(e) GE/ GO-AgNPs/DNA_1/ DNA_2/ M. SssI MTase (500 U/ml)/ HpaII (20 Uml
-1
) and ( ) GE/ GO-AuNPs/
DNA_1/ DNA_2/ M. SssI MTase (500 Uml
-1
). B) DPV eco ds ob ained om a ious concen a ions
measu emen . The DNA was me hyla ed by 5; 25; 50; 100; 200; 300; 400 and 500 U/ml M. SssI. C)
Calib a ion cu e esponds o he accumula ed MB in he helix s uc u e. E o ba s we e calcula ed in
dependence on s anda d de ia ions om he measu emen .
4. CONCLUSION
He ein, a simple and sensi i e biosenso o speci ic de ec ion o DNA me hyla ion and M. SssI MTase was
ab ica ed. The biosenso modi ied wi h GO-AgNPs nanocomposi e has shown he de e mina ion o M. SssI
wi h a b oad linea ange om 5 Uml
-1
o 500 Uml
-1
. Ou biosenso p o ides an easy measu emen o DNA
me hyla ion. The main ad an age o his is, ha i does no equi e expensi e ins umen s, PCR ampli ica ion
and mul iple-s ep p ocedu es. F om he p elimina y da a is ob ious, ha ou biosenso can be easily
ans e ed o lab-on-a-chip and be pa o sma de ice, which will be c ea ed in he u u e.
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ACKNOWLEDGEMENTS
The esea ch was inancially suppo ed by he IGA MENDELU AF-IGA2019-IP059 and ca ied
ou unde he p ojec CEITEC 2020 (LQ1601) wi h inancial suppo om he Minis y o Educa ion,
You h and Spo s o he Czech Republic unde he Na ional Sus ainabili y P og amme II.
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