Lase -T ea ed Sc een-P in ed Ca bon Elec odes o
Elec ochemiluminescence imaging
Claudio Ignazio San o, Guille mo Conejo-Cue as, F ancesco Paolucci, F ancisco Ja ie Del Campo,*
and Gio anni Valen i*
Ci e This: Chem. Biomed. Imaging 2024, 2, 835−841
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ABSTRACT: Elec ochemiluminescence (ECL) is nowadays a powe ul
echnique widely used in biosensing and imaging, o e ing high sensi i i y
and speci ici y o de ec ing and mapping biomolecules. Sc een-p in ed
elec odes (SPEs) o e a e sa ile and cos -e ec i e pla o m o ECL
applica ions due o hei ease o ab ica ion, disposabili y, and sui abili y o
la ge-scale p oduc ion. This esea ch in oduces a no el me hod o
imp o ing he ECL cha ac e is ics o sc een-p in ed ca bon elec odes
(SPCEs) h ough he applica ion o CO2lase ea men ollowing
ab ica ion. Using ad anced ECL mic oscopy, we analyze h ee dis inc
ca bon pas e-based elec odes and show ha low-ene gy lase exposu e
( anging om 7 o 12 mJ·cm−2) enhances he elec ochemical pe o mance
o he elec odes. This enhancemen esul s om he selec i e emo al o
su ace binde s and con aminan s achie ed by he lase ea men . We employed ECL mic oscopy o cha ac e ize he ECL emission
using a bead-based sys em inco po a ing magne ic mic obeads, like hose used in comme cial pla o ms. This app oach enabled high-
esolu ion spa ial mapping o he elec ode su ace, o e ing aluable insigh s in o i s elec ochemical pe o mance. Th ough
quan i a i e assessmen using a pho omul iplie ube (PMT), i was obse ed ha GST elec odes could de ec bioma ke s wi h high
sensi i i y, achie ing an app oxima e de ec ion limi (LOD) o 11 an ibodies pe μm2. These indings emphasize he po en ial o
lase -modi ied GST elec odes in enabling highly sensi i e elec ochemiluminescen immunoassays and a ious biosensing
applica ions.
KEYWORDS: elec ochemiluminescence, sc een-p in ed ca bon elec ode, lase ea men , beads-based biosenso , ECL mic oscopy,
an ibody de ec ion
1. INTRODUCTION
Elec ochemiluminescence is a sensi i e and e sa ile me hod-
ology, ecognized o i s nume ous bene i s, which ha e
es ablished i as a aluable ool ac oss a ious analy ical
applica ions, especially in he ield o biosensing.
1,2
ECL me ges he ad an ages o elec ochemical and
luminescen me hods, p o iding excellen sensi i i y and
selec i i y, s aigh o wa d spa ial and empo al con ol, and
a simpli ied op ical a angemen . The gene a ion o ligh
igge ed by an elec ochemical exci a ion b ings o he ECL a
supe io signal- o-noise a io han he common pho ochemical
me hods. The de elopmen o he ECL co eac an mechanism
p omo ed by Ba d and co-wo ke s
3
opened a new ield o ECL
applica ions and in ecen yea s o he ECL imaging. This new
ou e o ECL gene a ion in ol es he use o a co eac an ; an
auxilia y eagen ha , upon elec ochemical eac ion a he
elec ode, assis s in popula ing he luminopho e’s exci ed
s a e.
4
Mo eo e , his new mechanism inds a pe ec
applica ion in aqueous media, ne e pe o med be o e o
he p oblem o a es ic ed wa e po en ial window o p oduce
ECL h ough he annihila ion pa hway. Since ha disco e y,
ECL has been widely u ilized o de ec ing a ious subs ances,
om biological molecules o clinical indica o s, due o i s high
sensi i i y, low backg ound noise, and con ollabili y.
5
Today, [Ru(bpy)3]2+ and T ip opylamine (TP A), a e he
mos widely used luminopho e and co eac an , espec i ely, in
comme cially a ailable ECL biosenso s. They ope a e h ough
ahe e ogeneous ECL co eac an mechanism, whe e he
luminopho e is posi ioned away om he elec ode su ace
o p e en di ec elec on ans e . This con igu a ion is limi ed
by he di usion o co eac an adicals gene a ed a he
elec ode.
6,7
This is commonly achie ed by immobilizing he
luminopho e- agged bio ecogni ion componen on o mic o-
sphe es o mic opla e wells. Such a s a egy is employed in
sys ems like Elecsys and Meso Scale Disco e y analyze s.
2,8,9
Recei ed: Sep embe 20, 2024
Re ised: No embe 13, 2024
Accep ed: No embe 18, 2024
Published: No embe 22, 2024
A icle
pubs.acs.o g/ChemBioImaging
© 2024 The Au ho s. Co-published by
Nanjing Uni e si y and Ame ican
Chemical Socie y 835
h ps://doi.o g/10.1021/cbmi.4c00070
Chem. Biomed. Imaging 2024, 2, 835−841
ACS Pa ne Jou nal
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ECL immunoassays o e a powe ul pla o m o he accu a e
and sensi i e de ec ion o biological indica o s in diagnos ic
applica ions.
The ECL signal is gene a ed ia a low-po en ial mechanism,
ex ensi ely desc ibed p e iously,
3,4
whe ein TP A is di ec ly
oxidized a he elec ode su ace (1−2). A ac ion o he
oxidized TP A is dep o ona ed (3), o ming a s able adical
in e media e. This adical species educes he [Ru(bpy)3]2+
luminopho e o i s exci ed s a e, [Ru(bpy)3]+(4). Con-
cu en ly, he con inuous oxida ion o TP A a he elec ode
gene a es oxidized co eac an species, which can eac wi h
[Ru(bpy)3]+ o o m he exci ed s a e, [Ru(bpy)3]2+*(5). The
subsequen elaxa ion o [Ru(bpy)3]2+* o i s g ound s a e
emi s ECL adia ion (6).
+
+ +
VTP AH TP A H
(1)
•+
VTP A e TP A
(2)
+
•+ • +
VTP A TP A H
(3)
+ [ ] + [ ]
• + + +
VTP A Ru(bpy) Im Ru(bpy)
323
(4)
+ [ ] + [ ]
•+ + +*
VTP A Ru(bpy) TP A Ru(bpy)
3 3 2
(5)
[ ] [ ] +
+* + hRu(bpy) Ru(bpy)
3232
(6)
whe e Im+(iminium ion) esul s om he oxida ion o TP A•.
The in eg a ion o elec ochemiluminescence wi h mic os-
copy (ECLM) has signi ican ly enhanced he op imiza ion o
his echnique, pa icula ly by enabling he isualiza ion o he
physicochemical p ope ies o he elec ode su ace.
10−13
This
me hod, known as ECL mic oscopy, imp o es he spa ial
esolu ion o ECL, acili a ing he cha ac e iza ion o elec ode
su aces, isualiza ion o indi idual objec s, cells and assess-
men o hei ECL ac i i y.
14,15
Enhancing he ECL mechanism’s e iciency o op imize i s
gene a ion is a majo goal o ECL esea ch. An essen ial
componen in he c ea ion o ECL is elec ode ma e ial. Fo
e ec i e ECL signal p oduc ion, op imal elec ical conduc i i y
and app op ia e op ical p ope ies mus be ensu ed by high-
quali y elec ode ma e ials.
16
Noble me als such as gold and pla inum p o ide op imal
conduc i i y and a e well-sui ed o ECL p oduc ion,
emaining p e alen in many comme cial applica ions.
Howe e , hei high cos and ela i ely na ow po en ial
windows es ic hei use o speci ic ields, and hey equi e
mul iple ea men s o main ain good ep oducibili y. In his
con ex , ca bon-based elec ode ma e ials o e an ideal
comp omise be ween excellen elec ochemical p ope ies
and a o dabili y. Mo eo e , inco po a ing ca bon mic o- and
nanoma e ials signi ican ly enhances he elec ochemical
p ope ies o he elec ode. This syne gy le e ages he inhe en
ad an ages o ca bon wi h he bene i s o nanoscale
dimensions, such as a la ge su ace a ea, excellen elec ical
conduc i i y, low cos and abundance, chemical and
mechanical s abili y, and ease o ab ica ion.
17−23
Ca bon-based ma e ials like g aphi e, g aphene, and nano-
ubes can be eadily ans o med in o pas es and inks, enabling
cos -e ec i e, easy- o- ab ica e small and disposable elec odes.
Sc een-p in ed ca bon-based elec odes p esen high su ace
a eas, excellen conduc i i y, and du abili y agains chemical
and mechanical s ess, which a e undamen al p ope ies o
disposable elec odes. Wi h a wide po en ial window sui able
o di e se elec ochemical applica ions, ca bon-based elec o-
des a e e sa ile ools in senso s, biosenso s, and beyond.
24,25
Howe e , he p oduc ion o sc een-p in ed elec odes also
mee s challenges, la gely due o he use o so-called binde s.
Binde s a e (elec ochemically) ine polyme ic o elas ome ic
ma e ials ha a e essen ial o ensu ing he op imal dispe sion
o ca bon pa icles, managing he iscosi y o he solu ion,
gua an eeing good adhe ence o he su ace, and achie ing he
desi ed p in hickness o he inal s uc u es. Howe e , hey
o en ha e addi ional e ec s on he inal unc ionali y o
p in ed s uc u es. Fo ins ance, oo high binde concen-
a ions can esul in s uc u es o lowe conduc i i y, o
undesi able su ace p ope ies such as supe hyd ophobici y
and poo elec on ans e a es.
26,27
Typical binde s used in comme cial inks may include a ious
cellulosic ma e ials, such as e hyl cellulose and ni ocellulose,
28
ac yla es, such as me hyl-me hac yla e-based,
29
and e en
luo ina ed polyme s, such as Vi on.
30
The choice o a speci ic
binde depends on he applica ion, which implies adhesion o a
pa icula kind o subs a e, pa icula chemical and he mal
esis ance and compa ibili y, and esis ance o wea , among
o he conside a ions.
Some comme cial g aphi e inks a e designed o he
cons uc ion o elec odes sui able o elec ochemical
applica ions, such as he case o Gwen and Henkel pas es
used he e. O he inks, such as he GST ink also used in his
wo k, may be designed as mul ipu pose, a o dable conduc ing
inks o eplace mo e expensi e me al-based inks in a wide
ange o applica ions beyond elec ochemis y. All hese inks
a e he mally cu ed a a mode a e empe a u e, o e apo a e
he sol en wi hou damaging he binde composi ion, which
ensu es good p in adhesion and mechanical s abili y. Unde
hese ci cums ances, i is no possible o ob ain a g aphi e-only
su ace, and binde esidues a e always p esen . These binde
esidues a e de imen al o elec ochemical p ocesses, which
has mo i a ed he in es iga ion o su ace cleaning and
ac i a ion p o ocols.
31
Mos o hese me hods ely on we
me hods, which limi hei p ac ical use. Lase ea men , in
con as , can be applied as pa o he ab ica ion p ocess, so
ha he use does no need o apply any p econdi ioning s eps.
He e, we p opose he use o ca bon-based sc een-p in ed
elec odes ea ed wi h lase pos p ocessing o enhance hei
elec ochemical p ope ies, ensu ing imp o ed ECL pe o m-
ance. Employing a CO2lase a low ene gy densi ies (7−12
mJ·cm−2) unde a mosphe ic condi ions emo es su ace
impu i ies, including binde s, and induces he o ma ion o
mo e c ys alline g aphi e wi h la ge su ace a eas. This esul s
in imp o ed elec on ans e and al e ed we abili y, which
collec i ely impac ECL gene a ion.
32,33
2. EXPERIMENTAL SECTION
All he ma e ials we e pu chased om Sigma-Ald ich. 2.8 μm beads
coa ed wi h s ep a idin we e ob ained om The moFishe Scien i ic
(Dynabeads beads).
2.1. Elec ode Fab ica ion and Cha ac e iza ion
G aphi e elec odes we e sc een-p in ed ollowing p e iously epo ed
me hods.
33,34
B ie ly, Au os a WP20 subs a es (MacDe mid
Au o ype, UK) we e sc een-p in ed wi h ca bon pas es
C20305194P4 and GST 4500 (Sun Chemical-Se ilan, ES) and
Loc i e EDAG 440B (Te achim, FR). These elec odes will be
e e ed o as Gwen , GST, and Henkel, espec i ely. Sil e pas e
Loc i e EDAG 725A and UV-cu able dielec ic Loc i e EDAG PF
455B (Te achim, FR) we e also used. Elec ode designs we e c ea ed
Chemical & Biomedical Imaging pubs.acs.o g/ChemBioImaging A icle
h ps://doi.o g/10.1021/cbmi.4c00070
Chem. Biomed. Imaging 2024, 2, 835−841
836
using Vec o Wo ks 2024 S uden Edi ion (Techlimi s, ES). Film-
posi i es and sc eens wi h 77 h eads·cm−1and 40 μm ibe diame e
SEFAR 1500 ab ic we e ab ica ed on aluminum ames (Paymse ,
ES).
Sil e con ac s and acks, and pseudo e e ence elec odes we e
i s p in ed on WP20 Polye hylene e eph hala e (PET) subs a es
and cu ed in a con ec ion o en a 115 °C o 15 min. Nex , g aphi e
wo king and auxilia y elec odes we e p in ed using he inks desc ibed
abo e, and cu ed as pe hei espec i e echnical da ashee s. To
p o ec he conduc i e acks and delinea e he wo king, auxilia y, and
pseudo e e ence elec ode egions, a dielec ic coa ing was applied.
This dielec ic coa ing was cu ed by lood exposu e in a UVAcube 400
UV lamp (Honle UV Technology, DE) o 45 s. Lase pos p ocessing
was ca ied ou using a 30W Epilog Mini 18 eng a e (Epilog, US)
unde as e mode a 600 dpi. Ras e speed was ixed a 50%,
equi alen o app oxima ely 830−860 mm·s−1. Lase powe was ixed
be ween 9 and 12%, wi h co esponding applied ene gies be ween 7.7
and 10.2 mJ·cm−2. Subsequen o ea men , he elec odes we e
acuum-sealed in RP-1 Agen (Mi subishi Gas Company, JP) gas-
ba ie bags o p ese e hei in eg i y. The mo phology o he
wo king elec odes was analyzed using scanning elec on mic oscopy
(FEG-SEM Hi achi S-4800) a 15 kV. Raman spec oscopy was
pe o med using an InVia Raman spec ome e (Renishaw) equipped
wi h a Leica DMLM mic oscope and an a gon ion lase (Modu-Lase )
wi h a 514 nm wa eleng h. X- ay pho oelec on spec oscopy (XPS)
measu emen s we e conduc ed using a SPECS sys em (Be lin,
Ge many) equipped wi h a Phoibos 150 1D-DLD de ec o and a
monoch oma ic Al KαX- ay sou ce (1486.7 eV). Con ac angle
analysis was ca ied ou using an Ossila con ac angle goniome e
(so wa e e sion 3.1.2.2 o Windows 10). A 2 μL wa e d ople was
used o de e mine he con ac angle in ai .
2.2. Func ionaliza ion o 2.8 μm Beads wi h
Ru(bpy)2-bpy-NHS
The 2.8 μm diame e magne ic beads we e unc ionalized wi h Ru-
NHS ( u henium N-hyd oxysuccinimide) dilu ed in 0.01 M PBS
bu e (1×, pH = 7.4) o a concen a ion o 2.7 ×10−2mg·mL−1.
Fo he unc ionaliza ion, 217 μL o 2.8 μm beads we e aken and
washed wice in PBS using magne ic suppo (Pu eP o eome
Magne ic S and, Sigma-Ald ich). A e washing, he supe na an was
emo ed, and he Ru henium solu ion was added. The solu ion was
con inuously s i ed un il he nex day o op imize bead
unc ionaliza ion.
The nex day, he beads we e washed h ee imes in 1x PBS,
emo ing he supe na an each ime. Finally, hey we e dispe sed in
217 μL o PBS o main ain he ini ial beads’ concen a ion (0.72 mg·
mL−1).
2.3. Func ionaliza ion o An ibody wi h Bio in and
[Ru(bpy)3]2+
The p ocedu e has been desc ibed in ou p e ious wo k.
35
The
an ibody (IgG om Vec o Labo a o ies) was unc ionalized by
incuba ing a 1 mg·mL−1solu ion in PBS wi h a high mola excess o
bio in, EDC, NHS, and [Ru(bpy)2(mcbpy-O-Su-es e )] (85 equi ).
A e a 90 min incuba ion, he solu ion was pu i ied using Millipo e
Amicon Ul a 0.5 mL cen i ugal il e de ices wi h a 5 kDa cu o
memb ane o emo e excess u henium complex and bio in.
2.4. Func ionaliza ion o 2.8 μm Beads wi h Ru-Labeled
Bio inyla ed An ibody
A solu ion o 1 nM o an ibody labeled wi h bio in and [Ru(bpy)3]2+
was used o he beads’ unc ionaliza ion.
800 μL o he bead solu ion was pipe ed in o a 20 mL ube,
magne ically ga he ed o 2 min, and he supe na an was emo ed.
The beads we e hen washed wice wi h 10 mL o 0.01 M PBS o 5
min each. The beads we e hen incuba ed wi h 18 mL o Ru-labeled
an ibody solu ion o 3 h a 37 °C on a ube o a o . The beads we e
magne ically collec ed o 2 min, and he supe na an was emo ed.
Finally, beads we e s o ed in PBS wi h a o al olume o 800 μL, o
main ain he ini ial concen a ion o he beads’ solu ion. The same
p ocedu e was used wi h a p og essi e dilu ion o he s a ing solu ion
o bio inyla ed Ru-labeled an ibody o ob ain beads unc ionalized
wi h di e en an ibody loadings.
2.5. Single-Bead ECL Mic oscopy Analysis
The ECL/op ical imaging was pe o med using a Raman ec low cell
a achmen o SPE holde wi h homemade modi ica ions, comp ising
SPCE as wo king elec ode (0.096 cm2). P wi e and Ag/AgCl (3 M
KCl) we e used as coun e and e e ence elec odes, espec i ely. The
di ec mic oscope is om Nikon (Chiyoda, Tokyo, Japan) and can
wo k ei he in ansmission o in e lec ion mode. The mic oscope
was shielded om ex e nal ligh by a cus om-made da k box and was
i ed wi h a mo o ized s age (Co us, Ma zhause , We zla ,
Ge many) o elec ochemical cell posi ioning and igh ocal plane
obse ing.
Fo image acquisi ion, an ul asensi i e Elec on-Mul iplying CCD
came a (EM-CCD 9100−13 om Hamama su, Hamama su Japan)
wi h a esolu ion o 512 ×512 pixel and a size o 16 ×16 μm was
connec ed o he mic oscope. Finally, a long-dis ance wa e dipping
objec i e was used (100x, 1.1 nume ical ape u e, 2.5 mm wo king
dis ance).
The in eg a ed sys em also includes a po en ios a om BioLogic
(SP-300) o gene a e he ECL emission. The EMCCD came a
in eg a ion ime was se o 200 ms, allowing he acquisi ion o 5
images pe second. The sys em was igge ed by he po en ios a ,
ensu ing ha image acquisi ion by he EMCCD commenced
ins an aneously wi h he s a o he po en ial scan. A pos p ocessing
s ep allows he co ela ion o he ECL in ensi y in each image o he
exac po en ial scanned a he ime he image was cap u ed.
2.6. Quan i a i e ECL Analysis
A pho omul iplie ube (Hamama su R928) was posi ioned a a ixed
dis ance abo e he 3D-p in ed elec ochemical cell o de ec ECL
signals. To minimize ex e nal ligh in e e ence, bo h he cell and he
PMT we e housed wi hin a da k box. A high- ol age powe supply
wi h a ans-impedance ampli ie (Hamama su C6271) p o ided a
750 V bias o he PMT, igge ed ex e nally by he po en ios a ’s DAC
module. The ampli ied PMT ou pu signal was acqui ed by he
po en ios a ’s ADC module (BioLogic SP-300) o gene a e ligh /
cu en / ol age g aphs.
3. RESULTS AND DISCUSSION
Ou ini ial s udy ocuses on he cha ac e iza ion o SPCE made
wi h h ee di e en ca bon pas es, which will be named Gwen ,
Henkel and GST in his wo k. XPS cha ac e iza ion (Figu e
S1) shows he emo al o sp3ca bon, p esen mainly in he
binde polyme chains, and he en ichmen o p edominance
o sp2ca bon ollowing lase ea men . Raman spec a also
con i m lase ea men inc eases he G (1600 cm−1) band
ela i e o he D (1360 cm−1) band, which means mo e
c ys alline g aphi e has eme ged (Figu e S2). S ep a idin-
coa ed mic obeads we e unc ionalized wi h Ru(bpy)2-bpy-
NHS ( u henium N-hyd oxysuccinimide) (Figu e S3), exploi -
ing he NH2si es p esen on he s ep a idin. These beads
o e a subs an ial bene i by p o iding a g ea e numbe o
binding si es, which allows o he immobiliza ion o a
maximum Ru loading capaci y. To ho oughly assess he
ECL emission ea u es, we employed ECL mic oscopy using
an EM-CCD came a (Figu e S4 and Supplemen a y Mo ie 1).
This echnique enabled he examina ion o ECL signals
gene a ed by he mic obeads, e ec i ely supp essing back-
g ound noise. This app oach allowed o op imal spa ial
cha ac e iza ion o he elec ode su ace, including i s
homogenei y and elec ical ac i i y. To mimic a comme cial
ECL immunoassay sys em, he ECL om mic obeads a ge ed
wi h a Ru-labeled bio inyla ed an ibody (Figu e S5) was
quan i a i ely measu ed using a pho omul iplie ube de ec-
o .
34
Chemical & Biomedical Imaging pubs.acs.o g/ChemBioImaging A icle
h ps://doi.o g/10.1021/cbmi.4c00070
Chem. Biomed. Imaging 2024, 2, 835−841
837
Single-bead ECLM e ealed an inc ease in cu en o
Henkel and GST elec odes (Figu e 1c,e), accompanied by an
enhanced single-bead ECL in ensi y (Figu e 1d, ) a e lase
ea men . In he case o he Gwen elec ode, he e was no
no able al e a ion in he cu en (Figu e 1a) and a educ ion in
he ECL signal (Figu e 1b). No ably, o Gwen and Henkel
SPEs, he p onounced inc ease in hyd ophobici y
33
has
esul ed in a lack o ep oducibili y o measu emen s and
be ween di e en beads deposi ed on he same SPCE.
Al hough lase ea men o g aphi e elec odes enhances
elec on ans e a es (Figu e 2a), su ace we abili y may also
be impai ed depending on he pas e composi ion and
mo phology. As he ma e ial becomes mo e g aphi ic, elec on
ans e is enhanced, bu hyd ophobici y also inc eases. This
g ea e hyd ophobici y limi s solu ion access o he en i e
elec ode su ace, signi ican ly educing ECL gene a ion o
Gwen and Henkel SPEs (Figu e 2b and Table 1).
Table 1 summa izes he con ac angle analysis o he
di e en elec odes be o e and a e lase ea men . Gwen
and Henkel elec odes exhibi ed a subs an ial ise in con ac
angle, om 102° o 155°and 102° o 140°, espec i ely. This
inc ease in con ac angle indica es an g ow h in su ace
Figu e 1. Single-bead ECL in ensi y collec ed du ing he cyclic ol amme y o Gwen , Henkel and GST elec odes be o e and a e he lase
ea men . In g aphs a,cand e, he cu en end o he h ee elec odes be o e and a e lase ea men , ed and blue lines espec i ely, a e shown.
Do ed lines, g aphs b,dand , ep esen he single-bead ECL in ensi y a each 200 ms. G een do ed lines co espond o he ECL in ensi y o he
bes lase ed elec ode. The inse images show he ECL emission o he indi idual beads aken a he maximum o he cyclic ol amme y ECL
emission (Scale ba 5 μm). Po en ial scan om 0 V s OCP o 2 V s e , e ( e e ence elec ode) is Ag/AgCl (KCl sa .), he coun e elec ode is
P wi e, scan a e 50 mV·s−1, EM-CCD in eg a ion ime 200 ms, sensi i i y gain 800, gain 5, magni ica ion 100x. Image p ocessing in ol es
in eg a ing a 50 ×50 pixel squa e a ea cen e ed on he magne ic bead ac oss all analysis ames (one image cap u ed e e y 200 ms). The
backg ound is calcula ed om he same a ea on a bead ha is no unc ionalized wi h u henium and sub ac ed om he signal o he
unc ionalized bead. Finally, each image is co ela ed wi h he applied po en ial a ha momen o ob ain a end o he ECL in ensi y as a unc ion
o he scanned po en ial. Each measu emen is an a e age o a leas N= 5 magne ic beads.
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hyd ophobici y. While Gwen elec odes expe ienced simila
educ ions in ECL gene a ion a e lase ea men , he ECL
yielded a lase - ea ed GST elec odes became he b igh es in
he se ies, highligh ing he c i ical ole o elec ode
we abili y.
33
In he case o GST, al hough he emo al o amo phous
ca bon also exposes mo e c ys alline g aphi e, which is mainly
esponsible o he hyd ophobic beha io obse ed, he binde
is much mo e e ec i ely emo ed han in he case o Henkel
and Gwen elec odes, lea ing a mo e po ous su ace (SEM
images in Figu es S6−S9). This highe po osi y compensa es
o he hyd ophobici y o he g aphi e and allows he solu ion
o pe mea e he p in ed laye , hus imp o ing we ing.
Ha ing es ablished ha he GST sc een-p in ed elec ode
demons a ed supe io pe o mance in bo h ECL emission and
ep oducibili y, subsequen expe imen s in ol ed consis en ly
deposi ing a uni o m olume o beads on o he elec ode and
using a pho omul iplie ube as a de ec o (Figu e S10). The
easibili y o u ilizing he SPCE o quan i a i e analysis o
de e mine di e en an ibody concen a ions was e alua ed,
aiming o es ablish he elec ode as a pla o m o bioma ke
de ec ion.
Fo quan i a i e bead measu emen s, a cus om cell was
ab ica ed using a Fused Deposi ion Modeling (FDM) p in e
(Ul imake S33D) as shown in Figu e S11. In comme cial
sys ems, he Ru con en on he magne ic mic obead su ace is
p opo ional o he immobilized analy e. In ou se up,
bio inyla ed an ibodies unc ionalized wi h he Ru dye we e
immobilized on he beads. Knowing he an ibody concen-
a ion, he bound concen a ion o [Ru(bpy)3]2+ was
de e mined using UV− is spec oscopy by calcula ing he
[Ru(bpy)3]2+ concen a ion om he MLCT abso p ion band
a 450 nm, wi hin an abso bance ange below 0.1, based on he
Lambe −Bee law. Gi en ha up o 6 Ru complexes can be
di ec ly immobilized on an an ibody, he minimum de ec able
amoun o an ibody can be de e mined.
Va ious bead samples we e p epa ed o assess i he selec ed
elec ode could p o ide an ECL esponse p opo ional o he
an ibody amoun on he beads. Di e en an ibody quan i ies
we e inco po a ed in o he beads using p og essi ely mo e
Figu e 2. Compa ison be ween single-bead cu en a) and ECL signal b) du ing cyclic ol amme y o lase - ea ed SPEs p oduced wi h di e en
pas es. Scan a e 50 mV·s−1, EM-CCD in eg a ion ime 200 ms, sensi i i y gain 800, gain 5, magni ica ion 100×. Each measu emen is an a e age o
a leas N= 5 magne ic beads.
Table 1. Al e a ion o Con ac Angles in Th ee-Elec ode
Ma e ials Induced by Lase T ea men
elec odes con ac angle be o e lase con ac angle a e lase
Gwen 102°155°
Henkel 102°140°
GST 84°48°
Figu e 3. a) Quan i a i e beads ECL in ensi y du ing cyclic ol amme y o di e en [Ru(bpy)3]2+ concen a ions: 4392 (black line); 2196 ( ed
line); 1098 (blue line); 549 (pink line); 275 (da k g een line); 137 (ligh g een line); 69 (pu ple line) Ru dyes·μm−2quan i ied by p e ious ICP-
MS analysis. b) Calib a ion cu e ob ained a e he in eg a ion o he a ea below he ECL cu e plo ed as a unc ion o he Ru amoun o each
bead. Scan a e 50 mV·s−1, PMT cu en ampli ica ion 000.0 nA. The equa ion o he linea calib a ion cu e is Y= (1.7 ×10−4±1.0 ×10−5)X+
(3.4 ×10−2±2.1 ×10−2), R2= 0.98, whe e Yis he in eg a ed ECL signal and Xis he numbe o Ru dye pe μm2o bead’s su ace. Each da um is
he mean ±SD o h ee independen expe imen s.
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Chem. Biomed. Imaging 2024, 2, 835−841
839
dilu ed solu ions o bead unc ionaliza ion, wi h he mos
concen a ed solu ion co esponding o a inal concen a ion o
4392 Ru dyes·μm−2.
ECL signal dec eases wi h dec easing u henium concen-
a ion on he beads (Figu e 3a). Plo ing he in eg al o he
ECL cu es agains he concen a ion o u henium on he
beads yields a calib a ion cu e ha in e pola es he poin s
qui e well wi hou ou lie s (Figu e 3b). E alua ing he blank
signal wi h he same expe imen al se up wi h a no - unc ion-
alized magne ic mic obead, we calcula ed he limi o de ec ion
as he minimum concen a ion we can de ec in ou
expe imen al condi ions which esul s in a LOD o 65 Ru
dyes·μm−2, which means a ound 11 an ibodies·μm−2.
4. CONCLUSIONS
This s udy in es iga ed he e ec s o pos ab ica ion lase
ea men on sc een-p in ed ca bon elec odes made om
h ee di e en ca bon pas es. We ound ha lase ea men
signi ican ly imp o ed he elec ochemical and elec ochemi-
luminescence p ope ies o Henkel and GST elec odes, while
i did no enhance, and e en educe, he ECL signal o Gwen
elec odes. GST elec odes, in pa icula , showed supe io ECL
pe o mance and ep oducibili y.
Using ECL mic oscopy, we isualized and analyzed single-
bead ECL signals, p o iding de ailed insigh s in o elec ode
su ace p ope ies. Quan i a i e analysis demons a ed he high
sensi i i y o GST elec odes o bioma ke de ec ion, wi h a
limi o de ec ion co esponding o abou 11 an ibodies·μm−2.
These esul s sugges ha lase - ea ed GST elec odes a e
well-sui ed o comme cial ECL immunoassays and o he
biosensing applica ions, o e ing enhanced sensi i i y and
eliabili y. Fu u e wo k should u he op imize lase ea men
pa ame e s o enhance elec ode pe o mance ac oss di e en
composi ions.
■ASSOCIATED CONTENT
Da a A ailabili y S a emen
Expe imen al da a a e a ailable in AMS Ac a a h ps://
amsac a.unibo.i /id/ep in /7883.
*
sı Suppo ing In o ma ion
The Suppo ing In o ma ion is a ailable ee o cha ge a
h ps://pubs.acs.o g/doi/10.1021/cbmi.4c00070.
Addi ional expe imen al de ails, he expe imen al se up
o single-bead ECL mic oscopy and collec i e beads
analysis. cha ac e iza ion o SPCEs su ace (XPS, Raman
and SEM) (PDF)
Supplemen a y Mo ie 1: ECL emission om single
beads du ing cyclic ol amme y on a lase - ea ed GST
SPCE, illus a ing he dynamic change in ECL in ensi y
as he applied po en ial a ies (MP4)
■AUTHOR INFORMATION
Co esponding Au ho s
Gio anni Valen i −Depa men o Chemis y “G.Ciamician”,
Uni e si y o Bologna, 40129 Bologna, I aly; o cid.o g/
0000-0002-6223-2072; Email: [email p o ec ed]
F ancisco Ja ie Del Campo −BCMa e ials, Basque Cen e
o Ma e ials, Applica ions and Nanos uc u es, UPV/EHU
Science Pa k, 48940 Leioa, Vizcaya, Spain; IKERBASQUE,
Basque Founda ion o Science, 48009 Bilbao, Spain;
o cid.o g/0000-0002-3637-5782;
Email: [email p o ec ed]
Au ho s
Claudio Ignazio San o −Depa men o Chemis y
“G.Ciamician”, Uni e si y o Bologna, 40129 Bologna, I aly;
o cid.o g/0000-0003-4802-1671
Guille mo Conejo-Cue as −BCMa e ials, Basque Cen e o
Ma e ials, Applica ions and Nanos uc u es, UPV/EHU
Science Pa k, 48940 Leioa, Vizcaya, Spain
F ancesco Paolucci −Depa men o Chemis y
“G.Ciamician”, Uni e si y o Bologna, 40129 Bologna, I aly;
o cid.o g/0000-0003-4614-8740
Comple e con ac in o ma ion is a ailable a :
h ps://pubs.acs.o g/10.1021/cbmi.4c00070
Au ho Con ibu ions
C.I.S.: Me hodology, In es iga ion, W i ing-O iginal D a ;
G.C.C: Me hodology, In es iga ion; G.V., F.J.D.C., F.P.:
Concep ualiza ion, Supe ision, W i ing-Re iew and Edi ing.
No es
The au ho s decla e no compe ing inancial in e es .
■ACKNOWLEDGMENTS
This esea ch was unded by he Eu opean Union −Nex
Gene a ion EU, p ojec MEET codice g an numbe
20225P4EJC, CUP J53D23014570001. F.J.D.C. acknowledges
suppo om he Spanish Minis y o Science and Inno a ion
h ough p og ams RED2022-134120-T and PID2020-
113154RB-C22. This wo k was suppo ed by he Nano-
ImmunoE a p ojec ha has ecei ed unding om he
Eu opean Union’s MSCA S a exchange Ho izon Eu ope
p og amme G an Ag eemen Numbe 101086341.
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