Recei ed: 9 July 2025
Re ised: 5 Sep embe 2025
Accep ed: 7 Sep embe 2025
Published: 10 Sep embe 2025
Ci a ion: Yöney, B.; Oboˇ ilo á, R.;
Lacina, K.; Fa ka, Z.; Skládal, P.
Pa hogen-on-a-Chip: Impedance-
Based De ec ion o Bio ilm Fo ma ion
o S aphylococcus au eus and
S aphylococcus epide midis.Biosenso s
2025,15, 596. h ps://doi.o g/
10.3390/bios15090596
Copy igh : © 2025 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license
(h ps://c ea i ecommons.o g/
licenses/by/4.0/).
A icle
Pa hogen-on-a-Chip: Impedance-Based De ec ion o Bio ilm
Fo ma ion o S aphylococcus au eus and S aphylococcus epide midis
Bengisu Yöney 1, Radka Oboˇ ilo á 1,2 , Ka el Lacina 1,2, Zdenˇek Fa ka 1and Pe Skládal 1,*
1Depa men o Biochemis y, Facul y o Science, Masa yk Uni e si y, Kamenice 5,
625 00 B no, Czech Republic; [email p o ec ed] (B.Y.); [email p o ec ed] (R.O.);
[email p o ec ed] (K.L.); [email p o ec ed] (Z.F.)
2Cen al Eu opean Ins i u e o Technology, Masa yk Uni e si y, Kamenice 5, 625 00 B no, Czech Republic
*Co espondence: [email p o ec ed]
Abs ac
Bac e ial bio ilms a e complex mic obial communi ies ha con ibu e o he pa hogene-
sis o ch onic in ec ions. The e o e, i is c ucial o de ec bio ilm-associa ed in ec ions in
ea ly s ages as hei delayed ea men becomes mo e complica ed. He ein, we desc ibe a
label- ee elec ochemical impedance spec oscopy (EIS) me hod o de ec ing bio ilm o -
ma ion by S aphylococcus au eus and S aphylococcus epide midis. P in ed ci cui boa d-based
biampe ome ic gold elec odes we e modi ied wi h poly-L-lysine o enhance bac e ial
a achmen o he senso su ace. Fo ma ion and inhibi ion o bio ilms we e e alua ed
based on changes in cha ge ans e esis ance (R
c
). The con ol R
c
alue inc eased by
~90 k
Ω
o S. epide midis bio ilm and by ~60 k
Ω
o S. au eus bio ilms. An ibio ic- ea ed
samples exhibi ed simila alues o hose using he con ol. In addi ion, bio ilm o ma ion
was e alua ed h ough op ical mic oscopy using sa anin s aining, and he mic og aphs
sugges signi ican biomass on he elec odes, whe eas he con ol appea ed clea . A omic
o ce mic oscopy was used o isualize he bio ilm on he elec ode su ace, ob ain c oss-
sec ional p o iles, and e alua e i s oughness. The oughness pa ame e s indica e ha
S. au eus o ms a oughe bio ilm han S. epide midis, while S. epide midis o ms a mo e
compac bio ilm. These indings sugges ha he op imized EIS-based me hod e ec i ely
moni o s changes ela ed o bio ilms and se es as a p omising ool o e alua ion o new
an i-bio ilm agen s, such as an ibio ics, phages o an ibodies.
Keywo ds: mic obial bio ilm; elec ochemical impedance spec oscopy; a omic o ce mi-
c oscopy; disposable p in ed ci cui boa d gold elec ode
1. In oduc ion
In he mic obial wo ld, o e 99% o mic oo ganisms na u ally exis as agg ega ed
popula ions wi hin sel -p oduced biopolyme s known as bio ilms, which p omo e collabo-
a ion h ough a complex ne wo k [
1
]. Bio ilms a e o med by mic obial cells ha adhe e
igh ly o a su ace and p oduce an ex acellula ma ix (ECM) con aining ex acellula
polyme ic subs ances (EPS) ha mainly composed o complex biomolecules, such as ex-
opolysaccha ides, ex acellula DNA, p o eins, and lipids [
2
,
3
]. Bio ilms can o m on a wide
ange o abio ic and bio ic su aces whe e bac e ia adhe e h ough a ious physicochemical
in e ac ions [
4
,
5
]. These in e ac ions include Li shi z- an de Waals o ces, elec os a ic
double-laye con ibu ions, and acid-base in e ac ions [
6
]. The ini ial a achmen o bac e-
ia o he su ace is in luenced by mul iple ac o s, including su ace condi ioning, mass
Biosenso s 2025,15, 596 h ps://doi.o g/10.3390/bios15090596
Biosenso s 2025,15, 596 2 o 14
anspo , su ace cha ge, hyd ophobici y, and oughness [
7
]. All hese ac o s con ibu e o
ini ia ion o bio ilm o ma ion, which p o ides a p o ec i e en i onmen o bac e ia.
Bio ilms con ain pe sis en cells, a do man subpopula ion o bac e ia ha exhibi
high ole ance o an ibio ics, con ibu ing o he ch onic and ecu en na u e o bio ilm-
associa ed in ec ions [
8
–
10
]. Bac e ial bio ilms lead o signi ican complica ions due o
he complexi y o ea men , as bio ilm o ma ion con ibu es o an ibio ic esis ance and
uncon olled pe sis ence [11,12]. Once a (bio)ma e ial o a de ice has been implan ed o a
body, bac e ia compe e wi h hos issue cells o adhe e o i s su ace and o m a bio ilm [
13
].
S aphylococcus au eus and S aphylococcus epide midis a e among he species well-known
o o ming bio ilm on medical de ices, causing in ec ions, such as p os he ic hea al e
in ec ion, ca he e bio ilm in ec ions, and blood s eam in ec ions [14–18].
S anda d me hods o obse ing bio ilm o ma ion include mic oscopic echniques:
ligh mic oscopy, phase-con as mic oscopy, con ocal lase scanning mic oscopy, a omic
o ce mic oscopy (AFM), scanning elec on mic oscopy, and ansmission elec on mi-
c oscopy. Sui able biological app oaches include quan i a i e polyme ase chain eac ion,
c ys al iole assay, Congo ed aga assay and de e mina ion o colony- o ming uni s;
howe e , hese op ions a e conside ed expensi e, edious, and ime-consuming [19–21].
To add ess hese limi a ions, impedance mic obiology has been applied as a powe ul
echnique o de ec and quan i y bac e ia, and impedance-based me hods ha e been imple-
men ed as a ansduc ion mechanism [
22
]. Elec ochemical impedance spec oscopy (EIS)
has been used o unde s and bio ilm phenomena due o i s sensi i i y, non-des uc i e na-
u e, and he abili y o moni o bio ilm dynamics in eal ime [
22
,
23
]. EIS p o ides insigh s
in o bio ilm beha io by examining capaci i e (Z
C
), induc i e (Z
I
), and Wa bu g (Z
w
) com-
ponen s o impedance, solu ion esis ance (R
s
), and cha ge ans e esis ance (R
c
) [
24
,
25
].
Al hough EIS o e s eal- ime bio ilm moni o ing, con inuous impedance measu emen
in he p esence o a edox p obe may al e bac e ial g ow h dynamics [
26
]. Compa ed o
op ical sys ems, elec ical impedance biosenso s p o ide quan i a i e da a while enabling
minia u iza ion, mul iplexing, au oma ion, and in eg a ion in lab-on-chip de ices, along
wi h he unique abili y o cha ac e ize elec ophysiological p ope ies o di e en bac e ial
pheno ypes [
27
]. Owing o hese ad an ages, EIS has been adop ed o bio ilm de ec ion on
a ious elec ode ypes, such as gold, indium- in-oxide, and pla inum [
28
–
31
]. Howe e ,
many o hese pla o ms ely on mic o luidic sys ems o cus om-made low cells, which
can be echnically demanding and cos in ensi e.
This wo k p oposes a wo-elec ode sys em u ilizing gold elec odes based on he
economic p in ed ci cui boa d p ocess; a poly-L-lysine (PLL) coa ing se ed o su ace
modi ica ion and be e adhesion o bio ilms. Al oge he , his simpli ies he se up, educes
cos s, and suppo s minia u iza ion. We de eloped a low-cos , simple, and label- ee
impedance-based biosenso o de ec bio ilm o ma ion wi h equi ed sensi i i y. Gold
elec odes we e modi ied wi h PLL o p omo e bac e ial a achmen , and he o ma ion
o bio ilm by ei he S aphylococcus au eus o S aphylococcus epide midis was moni o ed. In
addi ion o EIS, op ical mic oscopy and AFM we e used o isually con i m p esence o
bio ilm and changes on he su ace. The bio ilm- o ming beha io o he wo s ains was
compa ed using elec ochemical and mic oscopic da a. Bio ilm inhibi ion was also ini ially
in es iga ed by applying amoxicillin (AMO) as an an ibio ic.
2. Ma e ials and Me hods
2.1. Chemicals and Reagen s
PLL and AMO we e pu chased om Sigma-Ald ich (S . Louis, MO, USA). T yp one
soya b o h (TSB) was ob ained om Oxoid (UK). Po assium hexacyano e a e(II) was om
Pen a (P ague, Czech Republic). Po assium hexacyano e a e(III) was ob ained om Ro h
Biosenso s 2025,15, 596 3 o 14
(P ague, Czech Republic). Alumina slu y (0.05
µ
m) was om Buehle (Lake Blu , IL, USA).
Sa anin was pu chased om Lachema (B no, Czech Republic). All o he chemicals we e
ob ained om Sigma-Ald ich (USA) o Pen a (Czech Republic). T is-bu e ed saline (50 mM
T is, 150 mM NaCl, pH 7.5), and phospha e-bu e ed saline (10 mM NaH
2
PO
4
/Na
2
HPO
4
,
150 mM NaCl, pH 7.4; abb e ia ed as PBS) we e p epa ed. Milli-Q wa e (Pu elab Ul a,
ELGA, High Wycombe, Buckinghamshi e, UK) was used in his s udy.
2.2. Bac e ial S ains and Cell Cul u e Condi ions
The bac e ial s ains used in his s udy included S aphylococcus au eus RN4220
∆
a M
dele ion mu an (S. au eus) and S aphylococcus epide midis s ain 1457-1000 (S. epide midis),
ob ained om he Depa men o Gene ics and Molecula Biology (Masa yk Uni e si y).
The bac e ial cul u es we e s o ed in he eeze a
−
30
◦
C. They we e inocula ed in o
yp ic soy b o h and incuba ed o e nigh a 37
◦
C in an incuba o ; he ob ained op ical
densi ies a 600 nm we e ~0.890 and ~0.870 o S. au eus and S. epide midis, espec i ely. The
cells we e collec ed by cen i uga ion a 4600
×
g o 10 min and subsequen ly esuspended
in TSB o u he expe imen s.
2.3. Elec ode Su ace Modi ica ion
The p in ed ci cui boa d elec odes we e manu ac u ed acco ding o ou design by he
company P in ed (www.p in ed.cz, accessed on 5 Janua y 2025; Mˇelník, Czech Republic).
EIS expe imen s we e conduc ed using a wo-elec ode biampe ome ic sys em consis ing o
wo simila gold elec odes (0.5 mm diame e ). The elec odes we e polished wi h
0.05 µm
alumina slu y on a Mic oClo h pad o emo e impu i ies, insed wi h deionized wa e ,
ollowed by a 20 min incuba ion in basic Pi anha solu ion (500 mM KOH, 3% H
2
O
2
) and
ho ough insing wi h deionized wa e ; all s eps we e ca ied ou a oom empe a u e [
32
].
The elec odes we e subsequen ly s e ilized h ough ul a iole (UV) ligh exposu e o a
du a ion o 30 min o p e en con amina ion. Finally, he elec odes we e incuba ed wi h
30 µL o PLL (10 µg/mL) o 30 min and insed wi h deionized wa e [33].
2.4. S. au eus and S. epide midis Bio ilm Fo ma ion and I s Inhibi ion
S. au eus and S. epide midis we e immobilized by incuba ing he PLL-coa ed elec odes
in each bac e ial s ain o ei he 1 h o 10 min. A e wa ds, he elec odes we e incuba ed
a 37
◦
C o 24 h in TSB. Fo he an ibio ic expe imen s, only he 10 min p e-incuba ion
op ion was used; 5 mg/L AMO was added o TSB. Fo blank con ols, he PLL-coa ed
elec odes we e di ec ly ans e ed in o ei he TSB o TSB con aining AMO.
All expe imen s we e pe o med in biological and echnical iplica es o bo h bac e-
ial s ains. S e ile condi ions we e main ained h oughou all p ocedu es, and elec odes
we e handled using s e ile o ceps. A e he incuba ion s eps, he elec odes we e collec ed
and insed wi h dis illed wa e and d ied wi h comp essed ai o u he analyses.
2.5. Elec ochemical Impedance Spec oscopy Measu emen s
Elec ochemical impedance spec oscopy was pe o med using he PalmSens4 po en-
ios a and PST ace e . 5.11 so wa e sys em (Palm Ins umen s, Hou en, The Ne he lands).
The measu emen s we e conduc ed in 5 mL o 5 mM po assium hexacyano e a e(II) and
(III) solu ion in PBS. A equency scan ype was applied wi h 20 equencies anging om
100 kHz o 2 Hz, and he AC ampli ude was se o 10 mV peak- o-peak, he DC componen
was 0 V [
34
]. Be o e all measu emen s, elec odes we e igo ously washed wi h T is bu e
o emo e he loosely bound plank onic bac e ia om bio ilms.
The acqui ed EIS da a we e analyzed using an equi alen ci cui model consis ing o a
cons an phase elemen CPE, R
s
,R
c
, and Z
W
. In his s udy R
c
and R
s
we e e alua ed. The
i ing and analysis we e pe o med using he so wa e PST ace.
Biosenso s 2025,15, 596 4 o 14
2.6. Bio ilm E alua ion Using Ligh Mic oscopy
Bio ilm o ma ion on gold elec ode su aces was examined using an BX41 ligh
mic oscope (Olympus, Tokyo, Japan) wi h a Deep Sky As o Came a 1.7 MP (Explo e
Scien i ic, Sp ingdale, AR, USA). Magni ica ions and exposu e imes we e 20
×
/450 ms,
and 40
×
/1000 ms. Following incuba ion, he elec odes we e gen ly washed wi h dis illed
wa e o emo e plank onic cells wi hou dis up ing he bio ilm a ached o he su ace.
Subsequen ly, he samples we e s ained wi h sa anin (0.5% w/ ) a oom empe a u e o
15 min o acili a e bio ilm isualiza ion [
35
]. Excessi e s ain was emo ed by insing he
samples wi h PBS, ollowed by ai -d ying a oom empe a u e.
2.7. AFM Cha ac e iza ion o Bio ilms on Elec odes
Be o e AFM measu emen s, he bio ilm on elec odes was insed wi h deionized wa e
and ai -d ied. AFM was pe o med using he Dimension Icon (B uke , Bille ica, MA, USA)
in PeakFo ce Tapping mode using he ScanAsys -Ai p obe (B uke ) wi h a silicon ip on a
ni ide le e wi h a sp ing cons an o 0.4 N/m. A omic o ce mic og aphs
(10 µm×10 µm
,
esolu ion 512
×
512 pixels) we e p ocessed using he Gwyddion e . 2.69 so wa e (Czech
Me ology Ins i u e, B no, Czech Republic) [
36
]. Fo each mic og aph, oughness was
measu ed in h ee di e en egions, each wi h a line o 9 µm.
2.8. S a is ical Da a Analysis
T iplica es we e conduc ed in all expe imen s in ol ing biological samples and mea-
su emen s. Da a analysis was conduc ed using O igin 2023 (O iginLab, No hamp on,
MA, USA). A sca e in e al cha was used o ep esen he mean alues along wi h
s anda d e o s (SE) o isualize he a iabili y be ween he eplica es. S a is ical analysis
was conduc ed using G aphPad P ism e . 10 (G aphPad So wa e, Bos on, MA, USA).
S a is ical signi icance was e alua ed and is ep esen ed by as e isks: **** o p< 0.0001,
*** o p< 0.001, ** o p< 0.01, and * o p< 0.05.
3. Resul s and Discussion
3.1. Elec ochemical E alua ion o Bio ilms
The elec ode su ace was i s ly cleaned using an alkaline Pi anha solu ion o emo e
impu i ies and o ganic subs ances and o in oduce hyd oxyl g oups on he su ace. Su -
ace hyd oxyla ion wi h i s pa ial nega i e cha ge plays a c ucial ole in enhancing he
a achmen o PLL, he posi i e cha ges o which in u n help cap u ing bo h G am-posi i e
and G am-nega i e bac e ia ia elec os a ic in e ac ions [
37
]. In his s udy, S. au eus and S.
epide midis, bo h G am-posi i e bac e ia, we e success ully cap u ed using his app oach.
Simila ly, Ma ka e al. employed PLL-coa ed gold elec odes o p omo e cell a achmen
p io o impedance measu emen s which demons a ed he e icacy o PLL in acili a ing
ini ial cell adhesion on su aces be o e u he o ma ion o bio ilms [38].
Fo EIS expe imen s, PLL-coa ed elec odes we e i s incuba ed wi h S. au eus,S.
epide midis, o TSB (con ol) o 1 h, ollowed by hei incuba ion in TSB nu ien medium
a 37
◦
C o 24 h. Unde such condi ions, he o ma ion o bio ilms was expec ed [
39
].
The EIS p ocedu e was employed o e i y bio ilm o ma ion on bo h elec odes ha we e
used in he elec ochemical cell, and o compa e wo bac e ial s ains on he modi ied
su aces. In e ms o bio ilm de ec ion, bo h R
s
and R
c
we e e alua ed and compa ed
(Figu e 1). The Nyquis (imagina y impedance (Z
′′
) e sus eal impedance (Z
′
)) plo s o
[Fe(CN)
6
]
3−/4−
a S. au eus- and S. epide midis-immobilized gold elec odes and he con ol
elec ode in PBS (7.4) a e displayed in Figu e 1A. In hese Nyquis plo s, he diame e o
he semici cula ea u e in he high equency egion is used o es ima e R
c
, while he o
se on he eal impedance axis om he o igin co esponds o R
s
. Based on he equi alen
Biosenso s 2025,15, 596 5 o 14
ci cui displayed in he inse o Figu e 1A, simula ed Nyquis plo s we e also gene a ed,
and hey a e ep esen ed by dashed aces in Figu e 1A. A plo o he R
s
alues (Figu e 1B)
o he h ee elec ode sys ems in Figu e 1B shows a s a is ically signi ican di e ence in
R
s
(p< 0.01) be ween he con ol and S. epide midis bio ilm. This is expec ed as R
s
mos ly
co esponds o he p ope ies o he su ounding medium. On he o he hand, he R
c
alues (Figu e 1C), ep esen ing he cha ge ans e esis ance o [Fe(CN)
6
]
3−/4−
a he
bio ilm-coa ed elec odes, we e able o clea ly dis inguish be ween bio ilms and con ol
g oups. The o he pa ame e s a e p esen ed in Table 1, CPE is capaci ance a ising om he
bio ilm laye , he Helmhol z double laye and su ace oughness o he elec odes, and Z
W
is Wa bu g impedance o di usion-limi in e acial esis ance.
Figu e 1. (A) Nyquis plo s o [Fe(CN)
6
]
3−/4−
a gold elec odes in PBS as suppo ing elec oly e
a e being incuba ed unde di e en condi ions o 24 h. The solid aces ep esen illus a i e
expe imen al da a, while he dashed aces co espond o he i ed alues ob ained using he Randles
equi alen ci cui model consis ing o solu ion esis ance (R
s
), cons an phase elemen (CPE, o Q),
cha ge ans e esis ance (R
c
), and Wa bu g elemen (W). The esul s o i ing wi h al e na i e
equi alen ci cui s a e summa ized in Table 1. (B) Sca e plo wi h e o ba s ep esen ing he
dis ibu ion o R
s
alues ob ained om equi alen ci cui i ing o gold elec odes incuba ed in
TSB. (C)R
c
alues ob ained om equi alen ci cui i ing o gold elec odes incuba ed in TSB. Each
poin ep esen s an independen measu emen , and ho izon al ba s indica e mean
±
s anda d e o .
S a is ical signi icance is indica ed by as e isks: **** o p< 0.0001, *** o p< 0.001, ** o p< 0.01.
In he case o con ols, he luc ua ions in bo h R
s
and R
c
combine a iabili y o
he elec ode sizes and p e ea men p ocedu es, plus some con ibu ion o adso bed
species om he cul i a ion medium used also in he blank expe imen s. Addi ionally, in
se e al cases p esen ed in Figu e 1B, he R
s
alues o he S. au eus bio ilms did no change
signi ican ly compa ed o he con ols. This can be expec ed as he su ounding solu ion
was he same. In ac , he uppe su ace o bio ilms is a he dynamic wi hou p ecisely
de ined bounda y; he e a e some loosely bound bac e ia which can be eleased du ing he
EIS p ocedu e. On he o he hand, he R
c
alues (Figu e 1C) clea ly dis inguished be ween
bio ilm and con ol g oups. The R
c
alues e ealed ha S. epide midis exhibi ed ~120 k
Ω
inc eased alue (an a e age) compa ed o he blank con ol, whe eas S. au eus showed
~90 kΩ
change wi h wide dis ibu ion ange in R
c
alues. Mo eo e , S. epide midis bio ilm
o ma ion showed highe s a is ical signi icance compa ed o he con ol (p< 0.0001) han
S. au eus (p< 0.001).
Se e al o he ci cui s we e also conside ed o i ing he expe imen al aces om
Figu e 1C, he compa ison o i s based on equi alen ci cui pa ame e s and he Chi-
squa ed (χ2) sum o esiduals is p o ided in Table 1.
Biosenso s 2025,15, 596 6 o 14
Table 1. Fi ing o he selec ed equi alen ci cui s o he expe imen al EIS scans o con ol and
bio ilm-modi ied elec odes. The Chi-squa ed (
χ2
, sum o squa es o esiduals om he i s) alues
cha ac e ize he igh ness o indi idual i s. The alues o i ed pa ame e s a e ollowed by hei
ela i e s anda d de ia ions (%, he pa en hesised alues). The uni s o indi idual pa ame e s we e
k
Ω
o all R, nF o all capaci ance alues C, k
σ
o all W, and nT o Q alues (based on he Equi alen
Ci cui Analysis module o he PST ace so wa e).
Equi alen Ci cui Con ol Bio ilm 1 Bio ilm 2
Rs(Rc C)χ2= 0.014 χ2= 0.013 χ2= 0.014
Rs= 1.04 (5.5)
Rc = 67.0 (4.1)
C= 14.3 (4.3)
Rs= 0.996 (5.4)
Rc = 109 (4.5)
C= 18.5 (4.1)
Rs= 0.995 (5.5)
Rc = 88.8 (4.3)
C= 16.6 (4.2)
Rs([Rc W]C)χ2= 0.0040 χ2= 0.33 χ2= 0.37
Rs= 1.03 (3.0)
Rc = 60.1 (3.0)
C= 14.0 (2.4)
W= 60.3 (16)
Rs= 0.93 (na)
Rc = 10−6(in )
C= 6.7 (60)
W= 477 (22)
Rs= 0.91 (na)
Rc = 10−6(in )
C= 5.2 (87)
W= 408 (22)
Rs(R1C1)(Rc C)χ2= 0.0054 χ2= 0.0054 χ2= 0.0059
Rs= 1.04 (3.5)
R1= 21.9 (21)
C1= 1250 (38)
Rc = 60.4 (4.1)
C= 14.3 (2.8)
Rs= 0.98 (3.4)
R1= 105 (4.6)
C1= 25.7 (11)
Rc = 8.32 (51)
C= 47.9 (3.6)
Rs= 0.99 (3.6)
R1= 41.8 (23)
C1= 127 (54)
Rc = 56.0 (18)
C= 18 (7.7)
Rs([Rc W]Q)χ2= 0.00030 χ2= 0.00010 χ2= 0.00050
(Randles)
Rs= 0.99 (0.90)
Rc = 63.9 (0.90)
W= 47.8 (5.9)
Q= 21.8 (3.1)
n= 0.95 (0.3)
Rs= 0.94 (0.60)
Rc = 108 (0.70)
W= 49.9 (6.6)
Q= 30.4 (1.9)
n= 0.94 (0.20)
Rs= 0.94 (1.2)
Rc = 86.4 (1.4)
W= 52.5 (10)
Q= 26.3 (3.9)
n= 0.94 (0.40)
Abb e ia ions: na no a ailable; in in ini y.
All he selec ed models p o ided e y simila R
s
alues a ound 1 k
Ω
. The R
s
([R
c
W]C)
model ( he second line in Table 1) ailed o e alua e he R
c
alues o he elec odes wi h
bio ilms, he C esul s (high pa ame e de ia ions) unexpec edly dec eased, hough he
inc ease in Wa bu g pa ame e s Wwas easonable. The R
s
(R
1
C
1
)(R
c
C) model ( he hi d
line in Table 1) migh seem app op ia e, as we expec wo laye s—PLL and bio ilm on
he elec odes. Howe e , he esul ing Rand Cse s o esul s look a he luc ua ing
p obably due o he ailu e o he i ing algo i hm. The simpli ied R
s
(R
c
C) and classic
R
s
([R
c
W]Q) Randles models ( he i s and las lines in Table 1, espec i ely) pe o med
a he well and p o ided simila changes and e en he absolu e alues o R
c
. The ends
in Cand Q alues we e also simila hough a he small inc eases o he bio ilm-coa ed
elec odes we e ob ained. This indica es ha bio ilm is no p o iding su icien isola ion o
he elec ode o modi y i s capaci ance cha ac e is ics. Thus, elaying on he R
c
pa ame e
seems mo e easonable o e alua ing bio ilm o ma ion and i s changes. Some mino
inc eases we e iden i ied also o he Wa bu g alues, bu wi h subs an ially highe ela i e
de ia ions compa ed o R
c
. The inally adop ed classic Randles ci cui ( he las line in
Table 1) p o ided he smalles sums o esiduals, and i was used h oughou o i ing he
ollowing EIS aces.
P e ious s udies con i m ou esul s ega ding R
c ,
which can be sensi i ely moni o ed
using EIS. Fo example, Oli e e al. in es iga ed he moni o ing o impedance du ing
bio ilm o ma ion by S. epide midis RP62A on gold elec odes and epo ed an inc ease in
R
c
o e ime [
40
]. This sugges s ha R
c
is a sensi i e indica o o de ec ion o bio ilm on
he elec ode su ace.
Biosenso s 2025,15, 596 7 o 14
3.2. Valida ion o Bio ilm Fo ma ion on Elec odes
A e he impedance measu emen s sugges ed he p esence o bio ilms, op ical mi-
c oscopy was employed o isualize bio ilm on he elec odes. In his s udy sa anin dye
was u ilized, as i is o en used o s aining cell nuclei in his ology [
41
]. Howe e , Ommen
e al. epo ed ha sa anin can also se e as a eliable s aining agen o bio ilm biomass
quan i a ion [
42
]. Figu e 2p esen s mic og aphs o sa anin-s ained elec odes incuba ed
wi h only TSB (con ol), S. au eus, and S. epide midis a 20
×
and 40
×
magni ica ions. As
expec ed, he con ol sample con ains no bac e ia and hus no isible dye e en ion; how-
e e , some su ace impu i ies p obably caused by he polishing s ep can be obse ed. The
mic og aphs ob ained om bac e ium-incuba ed elec odes in Figu e 2C–F show speci ic
ed colo a ion o sa anin, indica ing he p esence o a bio ilm on he elec odes. Howe e ,
i is di icul o dis inguish be ween he wo s ains. Due o he limi a ions o op ical
mic oscopy, i is essen ial o use a complemen a y echnique, such as AFM.
Figu e 2. Op ical mic og aphs o gold elec odes s ained wi h sa anin o isualize bio ilm o ma ion.
(A,B) TSB con ol elec odes a 20
×
(A) and 40
×
(B) magni ica ions. (C,D) Elec odes incuba ed
wi h S. au eus, magni ica ion 20
×
(C) and 40
×
(D). (E,F) Elec odes incuba ed wi h S. epide midis,
magni ica ions 20×(E) and 40×(F). Scale ba s: 100 µm.
AFM can be used o s udy whole bio ilms o hei componen s, such as he ECM,
o e ing high esolu ion [
43
]. I also enables he obse a ion o changes in he size o
bac e ial cells du ing g ow h and bio ilm o ma ion. In addi ion, heigh p o iles and
oughness pa ame e s ob ained om AFM p o ide insigh s in o homogenei y and su ace
mo phology o bio ilms. Based on hese bene i s, AFM was employed in his s udy o
in es iga e he bio ilm s uc u e in g ea e de ail.
Biosenso s 2025,15, 596 8 o 14
Figu e 3A illus a es he a omic o ce mic og aphs o he con ol and bio ilm-coa ed
elec odes along wi h he co esponding c oss-sec ional p o ile lines (Figu e 3B) o each
sample. The a omic o ce mic og aphs based on heigh p o iles demons a e no signi ican
su ace di e ences be ween he PLL-coa ed elec ode and he elec ode a e incuba ion
in TSB a 37C o 24 h. The c oss-sec ional p o ile o bac e ia lacking ex acellula ma ix
e eals a smoo h and con inuous con ou , co esponding o he sphe ical mo phology
o indi idual cells as in S. au eus. Howe e , when ex acellula ma ix is p esen , he
su ace becomes ma kedly oughe and mo e i egula , displaying inc eased opog aphical
complexi y likely associa ed wi h bio ilm-inco po a ed s uc u al componen s as in S.
epide midis. He e, c oss-sec ional heigh p o ile is de e mined along 7
µ
m long lines o
p o ide de ailed mo phologies o he samples.
Figu e 3. A omic o ce mic oscopy analysis o elec ode su aces in he absence and in he p esence o
bio ilms. (A) Heigh senso mic oscopic analysis o : PLL-coa ed ba e elec ode, PLL-coa ed elec ode
incuba ed in TSB, PLL-coa ed elec ode wi h S. au eus bio ilm, and PLL-coa ed elec ode wi h S.
epide midis bio ilm. Su ace opog aphy e eals biomass accumula ion in he p esence o bac e ial
bio ilms compa ed o con ol elec odes. (B) Rep esen a i e heigh p o iles co esponding o each
condi ion along he ma ked 7 µm lines. Scale ba : 2 µm.
The obse ed end likely e lec s undamen al di e ences in bio ilm a chi ec u e
and ex acellula ma ix composi ion be ween he wo species. S. epide midis is known o
p oduce a dense and mo e cohesi e bio ilm, which can inc ease bo h he solu ion esis ance
(due o limi ed ionic mobili y in he ma ix) and he R
c
(due o hinde ed access o he
elec ode su ace). This in e p e a ion is suppo ed by he a omic o ce mic og aphs, whe e
S. epide midis o med mo e uni o m and con luen laye s wi h a g ea e su ace co e age (in
he case o 1 h immobiliza ion), in con as o he pa chie and i egula s uc u e obse ed
o S. au eus. The EIS i ing u he suppo s his explana ion, as he highe R
c
alues
co ela e wi h mo e compac bio ilms impeding elec on ans e a he in e ace.
3.3. Inhibi ion o Bio ilm Fo ma ion
In his pa , he PLL-coa ed elec odes we e incuba ed wi h ei he S. au eus o S.
epide midis o only 10 min p io o bio ilm o ma ion. This sho e incuba ion pe iod
was chosen o educe he o e all expe imen al ime. We aimed o inhibi he bio ilm
de elopmen by applying 5 mg/L o he an ibio ic AMO) and moni o ing he esul ing
Biosenso s 2025,15, 596 9 o 14
changes ia EIS. The elec odes we e incuba ed in a mix u e o AMO and TSB medium, and
a e 24 h, EIS measu emen s we e pe o med as p e iously, ollowed by AFM imaging.
Figu e 4shows he a ia ion o R
s
,R
c
om EIS expe imen s, and heigh p o iles o elec-
odes coa ed by S. au eus and S. epide midis om a omic o ce
mic oscopic in es iga ions.
Figu e 4. Compa ison o sho - e m bac e ial immobiliza ion and bio ilm inhibi ion on gold elec odes.
(A) Sca e plo o R
s
alues o bio ilm- o ming elec odes, an ibio ic- ea ed elec odes, TSB wi h
an ibio ics (TSB AMO), and TSB (con ol). (B) Sca e plo o R
c
alues o bio ilm- o ming elec odes,
an ibio ic- ea ed elec odes, TSB wi h an ibio ics (TSB AMO), and TSB (con ol). (C) AFM heigh
senso images o elec odes incuba ed wi h S. au eus and S. epide midis in TSB (bio ilm o ma ion)
and in TSB con aining an ibio ics (bio ilm inhibi ion). Scale ba : 2
µ
m. Ho izon al lines indica e
he e alua ed heigh p o iles. (D) Heigh p o iles along 3
µ
m long lines on each elec ode su ace,
showing di e ences in e ical ea u es be ween bio ilm- o ming and bio ilm-inhibi ed condi ions.
The e ical axes ex en s co espond o 1
µ
m. Biological and echnical eplica es a e ep esen ed,
and ho izon al ba s indica e mean
±
s anda d e o . S a is ical signi icance is indica ed by as e isks:
**** o p< 0.0001, *** o p< 0.001, ** o p< 0.01.
To assess bio ilm inhibi ion, bo h R
s
and R
c
alues we e compa ed, as shown in
Figu e 4A. TSB con ol exhibi ed a s a is ically signi ican di e ence compa ed o TSB
wi h AMO, S. epide midis in AMO and TSB, and S. au eus in AMO and TSB. This may
sugges ha he p esence o AMO in he medium o 24 h can sligh ly in luence he solu ion
esis ance. On he o he hand, bio ilm o ma ion i sel did no esul in a no able di e ence
in Rs alues.
EIS esul s ob ained by i ing he da a wi h he equi alen ci cui model, along wi h
he co esponding R
c
alues, a e p esen ed in Figu e 4B. S. au eus exhibi ed ~100 k
Ω
and
mo e a iable R
c
alues compa ed o S. epide midis (~120 k
Ω
), consis en wi h he p e ious
