De elopmen o Simple and Rapid Bead-Based Cy ome ic
Immunoassays Using Supe pa amagne ic Hyb id Co e−Shell
Mic opa icles
Cha lie Tobias, Daniel López-Pue ollano, An onio Abad-Somo illa, Josep V. Me cade ,
An onio Abad-Fuen es, and Knu Ru ack*
Ci e This: ACS Meas. Sci. Au 2024, 4, 678−688
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ABSTRACT: Flow cy ome y-based immunoassays a e aluable in biomedical
esea ch and clinical applica ions due o hei high h oughpu and mul ianaly e
capabili y, bu hei adop ion in a eas such as ood sa e y and en i onmen al
moni o ing is limi ed by long assay imes and complex wo k lows. Rapid, simpli ied
bead-based cy ome ic immunoassays a e needed o make hese me hods iable o
poin -o -need applica ions, especially wi h he inc easing accessibili y o
minia u ized cy ome e s. This wo k in oduces supe pa amagne ic hyb id
polys y ene-silica co e−shell mic opa icles as p omising al e na i es o con en-
ional polyme beads in compe i i e cy ome ic immunoassays. These beads,
ea u ing high speci ici y, sensi i i y, and excellen handling capabili ies ia magne ic sepa a ion, we e e alua ed wi h h ee di e en
an ibodies and binding me hods, showing a ia ions in signal in ensi y based on he an ibody and i s a achmen me hod. The
op imal pe o mance was achie ed h ough a seconda y an ibody binding app oach, p o iding s ong and consis en signals wi h
minimal unce ain y. The op imized p o ocol made i possible o achie e a de ec ion limi o 0.025 nM in a o al assay ime o only
15 min and was success ully used o de ec och a oxin A (OTA) in aw lou samples. This wo k highligh s he po en ial o hese
beads as e sa ile ools o low cy ome y-based immunoassays, wi h signi ican implica ions o ood sa e y, animal heal h,
en i onmen al moni o ing, and clinical diagnos ics.
KEYWORDS: an ibody-based, bead-based assay, co e−shell pa icles, cy ome y, myco oxins
■INTRODUCTION
Flow cy ome y-based compe i i e immunoassays a e widely
used in biomedical esea ch and clinical applica ions o
quan i y and de ec di e en analy es in complex biological
samples, since hey combine he high- h oughpu po en ial o
immunoassays wi h he mul ianaly e capabili y o liquid
ch oma og aphy (LC) echniques.
1−3
Al hough such ap-
p oaches also hold p omise in ields like ood sa e y, animal
heal h, and en i onmen al moni o ing, hey a e only slowly
inding hei way in o hese a eas.
4−12
On he one hand, his is
due o he he e ogenei y o he low molecula weigh a ge
analy es and he limi ed a ailabili y o an ibodies o hem, bu
on he o he hand also o he usually a he long assay imes
and mul is ep wo k lows, which o en ake 1 h o longe due o
(se e al) incuba ion s eps and hus canno e en compe e wi h
LC me hods in e ms o ime o esul . Howe e , simplici y
and speed would gene ally be desi able i immunoanaly ical
me hods we e o make he s ep o poin -o -need applica ions,
o which hey a e gene ally well sui ed due o he ad an age
ha obus low cy ome e s become inc easingly a ailable; o
ins ance, o esou ce-limi ed se ings,
13−15
hei measu emen
pe iphe y can be minia u ized
7,16
and as e mix-and- ead
immunoassay o ma s a e being de eloped.
9
Cen al as a model analy e o he p esen wo k is och a oxin
A (OTA), a myco oxin commonly ound in g ain p oduc s,
co ee, cacao, g apes, and po k, posing se e e heal h isks.
17−19
To add ess hese isks, he Eu opean Union (EU) has
ins i u ed maximum le els (ML) o OTA ac oss a ious
oods u s.
20
While se e al me hods exis o OTA analysis,
immunochemical and ch oma og aphic echniques domina e.
Among he i s g oup, enzyme-linked immunoso ben assays
(ELISA) and la e al low immunoassays (LFIA) a e widely
used,
21,22
wi h se e al ki s being al eady a ailable on he
ma ke . Rega ding ch oma og aphic echniques, high-pe o m-
ance liquid ch oma og aphy (HPLC) combined wi h luo-
escence de ec ion, a e immunoa ini y column cleanup, is he
Recei ed: July 9, 2024
Re ised: Augus 9, 2024
Accep ed: Sep embe 4, 2024
Published: Sep embe 17, 2024
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S anda diza ion.
23
Ne e heless, o en mass spec ome y
de ec ion (MS/MS) is p e e ed o OTA de e mina ion a
ace le els.
24,25
While ELISA and LFIA a e known o hei
obus ness, hey ha e d awbacks wi h ega d o speed and
handling s eps (ELISA) as well as he capabili y o
mul iplexing o o p ocess a high numbe o samples (LFIA),
making hem sui able o ou ine analysis o a limi ed se o
myco oxins and/o samples. Ch oma og aphy-based echni-
ques a e p e e ed o egula o y asks ha equi e highe
accu acy, sensi i i y, o a mul ianaly e app oach, ye such
analyses a e slow and cos ly. As a po en ial al e na i e, as
men ioned abo e, bead-based immunoassays u ilizing low
cy ome y o e a lo o ad an ages. Howe e , only ew
examples ha e been desc ibed o OTA, using ela i ely hea y
pa icles in all cases and equi ing long assay imes.
26−29
As we a e in e es ed in de eloping cy ome ic and
mic o luidic immunoassays o on-si e use, we s a ed some
ime ago o de elop a modula pa icle pla o m ha should
e en ually lead o a sui able app oach. The pla o m uses
polys y ene-silica co e−shell beads
30,31
which ha e also been
success ully used by o he s
32
and a e cu en ly being
comme cialized.
33
In addi ion, we ha e ecen ly ex ended
his pla o m by inco po a ing magne ic ea u es.
34
These
hyb id beads consis o a polys y ene co e and a silica shell in
which magne ic nanopa icles a e embedded, acili a ing
handling du ing washing and e en ion in analy ical assays.
The ou e silica su ace can be easily modi ied by silane
chemis y so ha an ibodies o o he molecules o in e es can
be a ached. In addi ion, he pa icle co e can be doped wi h
luo escen dyes, enabling use in mul iplexed assays o
simul aneous de ec ion o mul iple a ge s.
35
Compa ed o
pa icles doped wi h me allic nanopa icles o gi e hem
magne ic p ope ies, hese hyb id beads a e ligh e , esul ing in
slowe sedimen a ion and making hem mo e sui able o low
cy ome y.
36−38
These p ope ies enable e icien analysis o
la ge sample olumes, as hea y pa icles can lead o
sedimen a ion, clogging, signal sa u a ion, and op ical in e e -
ences, which a ec s da a quali y and ins umen pe o mance
in low cy ome y. When ligh e pa icles a e a ailable as an
op ion, hey a e gene ally p e e ed o a oid hese p oblems
and ensu e mo e accu a e low cy ome y measu emen s.
39
In he p esen s udy, we used a compe i i e immunoassay
app oach wi h a luo escen compe i o o de ec OTA in o de
o e alua e he applicabili y o hese new ma e ials in
cy ome y assays. By quan i ying he amoun o compe i o
bound o he beads, we we e able o de e mine he OTA
concen a ion, which allowed o p ecise and accu a e
de ec ion wi hou a washing o isola ion s ep. Ou second
objec i e was o e alua e he unc ionaliza ion p o ocol o he
supe pa amagne ic hyb id co e−shell beads. Fo his pu pose,
we in es iga ed h ee di e en an i-OTA an ibodies ha had
also been p e iously p oduced by ou g oup.
40
In addi ion,
h ee me hods o immobilizing he p ima y an ibodies on he
bead su ace we e es ed, namely, di ec binding o an ibodies
o he bead su ace, binding ia p o ein G, and binding ia a
seconda y goa -an imouse (GAM) an ibody. Finally, by
employing he op imal assay con igu a ion, we analyzed eal
lou samples ob ained om a mill o OTA, ealizing a low
limi o de ec ion in a a o able o e all assay ime.
■MATERIALS AND METHODS
Poly( inylpy olidone) (PVP10, 10 kDa, Sigma), s y ene (Sigma),
basic alumina (Al2O3, B ockmann I, Ac os), and azo-biscyano ale ic
acid (ACVA, MP Biomedicals) we e used o he PVP-coa ed
polys y ene co e syn hesis. FeCl3·6H2O (AppliChem) and FeCl2·
4H2O (Bake ) we e used o he p epa a ion o magne ic nano-
pa icles. Te ae hoxyo hosilica e (TEOS, Me ck) and ammonia
solu ion (NH3, 32%, Supelco) we e used o he silica coa ing. (3-
Aminop opyl) ie hoxysilane (APTES, Ald ich) was used o amino
unc ionaliza ion and succinic anhyd ide (Me ck) o la e ca boxylic
acid unc ionaliza ion. 1-E hyl-3-[3-(dime hylamino)p opyl]-
ca bodiimide hyd ochlo ide (EDC, Me ck) and N-hyd oxysul osucci-
nimide sodium sal (sNHS, Sigma) we e used o pa icle ac i a ion.
A iniPu e Goa An i-Mouse (GAM) IgG, Fcγ agmen speci ic,
unconjuga ed seconda y an ibody om Jackson ImmunoResea ch,
and Pie ce Recombinan P o ein G we e used o u he pa icle
unc ionaliza ion. Phospha e-bu e ed saline (PBS, pH 7.4, 10 mM,
130 mM NaCl), 2-(N-mo pholino)e hanesul onic acid bu e (MES,
pH 6.0, 10 mM), and bica bona e bu e (pH 9.6, 50 mM) we e
p epa ed in Milli-Q g ade wa e . T is bu e was p epa ed using 10
mM T is−HCl (pH 7.5), 120 mM NaCl, 20 mM CaCl2, and 40 mM
MgCl2in Milli-Q g ade wa e . The OTA luo escein compe i o
conjuga e (OTA-F) and he OTA s anda d we e pu chased om
Aokin. Monoclonal an i-OTA an ibodies we e p e iously p oduced
and cha ac e ized by ou g oup.
40
Measu emen s we e conduc ed ia low cy ome y using a BD
Accu i C6 ins umen equipped wi h 488 and 640 nm lase s o
exci a ion and included he eco ding o he o wa d sca e (FSC)
and sidewa d sca e (SSC) signals o he pa icles a angles o 180
and 90°, espec i ely. Addi ionally, he luo escence signal in he FL1
channel (488 nm, 533/30.H il e ) was cap u ed. To de e mine he
IC50 alues, he da a ob ained om he compe i i e assay we e
analyzed and plo ed in he O igin so wa e (O iginLab) using a ou -
pa ame e sigmoidal i ing. Fo SEM imaging, he pa icles we e
dispe sed in e hanol and subjec ed o ul asonica ion o 5 min. To
p epa e he samples o analysis, hey we e d op-cas ed on o
con en ional ca bon TEM g ids. Imaging o indi idual pa icles was
pe o med using a Zeiss Sup a 40 scanning elec on mic oscope
(Zeiss), equipped wi h a high- esolu ion ca hode (Scho ky ield
emi e ), an E e ha −Tho nley seconda y elec on (SE) de ec o ,
and an SE InLens de ec o . Fo ansmission elec on mic oscopy
mode (TSEM o STEM-in-SEM), a dedica ed “ ansmission” sample
holde was u ilized.
Syn hesis o Hyb id Pa icles
The supe pa amagne ic mic opa icles we e p epa ed by a ou e
adap ed om e 34, elabo a ing ou ea lie a chi ec u e desc ibed in
e s 30,31,35 o he p esen one wi h polys y ene (PS) as he co e,
i on oxide (Fe3O4) nanopa icles as a magne izable in e laye , and an
ou e silica shell o p o ec ion and u he unc ionaliza ion. The
syn hesis o he PS pa icles was ca ied ou ia dispe sion
polyme iza ion by eac ing a solu ion o 170 mg o PVP10 in 10
mL o E OH wi h 1 mL o s y ene, il e ed h ough basic aluminum
oxide, in a glass ial a e lushing he mix u e wi h a gon o 30 min
and subsequen ly ini ia ing i by he addi ion o 0.5 mL o a solu ion
o 105 mg o ACVA in 10 mL o MeOH, lushed wi h a gon, unde
s i ing a 70 °C in an a gon a mosphe e o e nigh . The esul ing
pa icles we e cen i uged, washed wi h wa e and E OH mul iple
imes, and hen d ied a oom empe a u e.
Syn hesis o Supe pa amagne ic I on Oxide Nanopa icles
(SPIONs)
To syn hesize SPIONs, 0.465 g o FeCl3·6H2O and 0.172 g o FeCl2·
4H2O we e dissol ed in 100 mL o Milli-Q wa e in a ound-bo om
lask. The solu ion was pu ged wi h a gon o 20 min be o e slowly
adding a solu ion o 4 g o PVP10 in 58 mL o NH3solu ion (16%).
The eac ion mix u e was s i ed a 150 pm o 1.5 h using a
mechanical s i e . The pa icles we e hen washed se e al imes wi h
wa e using magne ic sepa a ion and s o ed in a e ige a o a a
concen a ion o app oxima ely 3% (w/ ) in Milli-Q wa e .
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Coa ing o PS Co es wi h SPIONs (SPION@PS)
The PS co es we e coa ed wi h a laye o SPIONs by suspending 60
mg o PS co es and 2 mL o Fe3O4pa icles (3% in wa e ) in 30 mL
o Milli-Q wa e in Falcon ubes. The coa ing p ocess was ca ied ou
by placing he ubes on a o a o pla e a 40 pm o 1.5 h. A e wa d,
he pa icles we e washed wice wi h wa e and once wi h e hanol,
using magne ic sepa a ion, be o e d ying.
Coa ing o SPION@PS Pa icles wi h Silica Shell
(SiO2@SPION@PS)
555 μL o NH3solu ion (32%) was added o a dispe sion o 60 mg o
SPION@PS pa icles in 30 mL o E OH and 1 mL o Milli-Q wa e
while s i ing a 150 pm using a mechanical s i e . Then, 555 μL o
TEOS was added d opwise. The mix u e was s i ed o e nigh a 38
°C, ollowed by mul iple washes wi h wa e and E OH, using
magne ic sepa a ion. Finally, he pa icles we e d ied a oom
empe a u e.
Func ionaliza ion o SiO2@SPION@PS wi h Amino G oups
Acco ding o a p e iously epo ed p o ocol,
35
he SiO2@SPION@PS
pa icles we e unc ionalized wi h amino g oups by i s ac i a ing he
pa icles’ su ace a e suspending 20 mg o he pa icles in 800 μL o
E OH, adding 400 μL o 1 M HCl in E OH, and sonica ing he
mix u e in a sonica ion ba h o 5 min. A e wa d, he pa icles we e
washed wice wi h 400 μL o E OH and edispe sed in 400 μL o
E OH. Fo amino modi ica ion, 8 μL o APTES was added o he
pa icle dispe sion and he mix u e was allowed o eac in a
he momixe (800 pm) a 40 °C o e nigh . Subsequen ly, he
pa icles we e washed h ee imes wi h a mix u e o E OH:H2O in a
1:1 a io, be o e d ying in acuum a oom empe a u e.
Re unc ionaliza ion o SiO2@SPION@PS wi h Ca boxylic
Acid G oups
To modi y he su ace o he ma e ials wi h ca boxylic acid g oups o
acile biomolecule a achmen , 5 mg o he co esponding amino-
modi ied pa icles was dispe sed in 1.5 mL o absolu e E OH in a 2
mL Eppendo ube. A solu ion o 30 μL 10% w/ o succinic
anhyd ide in dime hyl o mamide was added o he pa icle dispe sion,
and he mix u e was hen allowed o eac in a he momixe (800
pm) a 40 °C o e nigh . A e wa d, he pa icles we e washed h ee
imes wi h a mix u e o E OH:H2O in a 1:1 a io. In he inal s ep, 500
μL o e hanol was added o he pa icles o ob ain a s ock solu ion
wi h a inal concen a ion o 1% (w/ ).
Coupling o P o ein G and GAM o Re unc ionalized
SiO2@SPION@PS
F om he 1% (w/ ) s ock solu ions, 10 μL o SiO2@SPION@PS
pa icles was dispe sed in 100 μL o MES bu e . To his pa icle
dispe sion, 80 μL o a eshly p epa ed solu ion o EDC and 160 μL o
s-NHS solu ion, bo h in MES bu e and wi h a concen a ion o 1%
(w/ ), we e added. The mix u e was incuba ed a oom empe a u e
o 15 min, and hen 50 μL o ei he p o ein G (2.4 mg mL−1) o
GAM (2.4 mg mL−1) was added and incuba ed o e nigh on a o a o
pla e a 40 pm and oom empe a u e. A e wa d, he pa icles we e
washed wice wi h 500 μL o PBS and edispe sed in 200 μL o esh
PBS o each a s ock solu ion concen a ion o 0.05% (w/ ).
Di ec Binding o he P ima y An ibody
Fo he di ec binding o he an i-OTA mAbs, he p e iously
desc ibed p o ocol o p o ein G o GAM was ollowed. In each case,
20 μL a 1 mg mL−1in PBS o each o he mAbs was used. The
washing was pe o med as p e iously desc ibed o he cap u e
p o ein-modi ied pa icles.
Coupling o he P ima y An ibodies o he P o ein G- and
GAM-Modi ied Pa icles
A suspension con aining 0.035% p o ein G o GAM-modi ied
pa icles was p epa ed in 300 μL o PBS, and hen 20 μL o solu ions
con aining he mAbs a 1 mg mL−1in PBS was added. The mix u e
was incuba ed on a o a o pla e o 1 h a oom empe a u e.
Subsequen ly, he pa icles we e washed once wi h PBS and he
olume was es o ed o he o iginal 200 μL, yielding a inal pa icle
concen a ion o 0.05% (w/ ).
Inhibi ion Tes s
The inhibi ion es s we e pe o med in la -bo omed PS 96-well
pla es. To do so, 10 μL (0.05%) o unc ionalized pa icles was placed
in o indi idual wells con aining 100 μL o PBS. Nex , 5 μL o OTA-F
a di e en concen a ions (0.1, 0.01, 0.001, and 0.0001 μM plus a
blank con ol) was added. The same p ocedu e was epea ed o a
second se o wells, bu wi h he addi ion o 5 μL o a 1 μM OTA
solu ion. The pla e was incuba ed unde gen le shaking o 15 min.
The inhibi ion a e (in pe cen ) was calcula ed as he quo ien o he
signals (mean coun s in FL1) measu ed o he wells con aining OTA
(subsc ip xOTA) and he wells wi hou OTA (subsc ip noOTA),
ep esen ing he maximum signal, using he same OTA-F concen-
a ion in all wells:
Inhibi ion a e FL FL1 / 1
xOTA noOTA
=
Compe i i e Assays
In he compe i i e assay, 10 μL (0.05%) o pa icles was dispensed
in o indi idual wells o a well pla e along wi h 100 μL o PBS bu e .
To ini ia e he assay, 5 μL o OTA (a di e en concen a ions,
including a blank) was added o he wells. Subsequen ly, 5 μL o
OTA-F was in oduced in o each well, and he assay was gen ly
shaken o 10 min in he da k. Finally, he well pla e was subjec ed o
measu emen using a low cy ome e wi h a measu emen ime o 1
min o a single well.
Sample Ex ac ion
2 g o sample was placed in a 15 mL ube along wi h 8 mL o CH2Cl2
and 200 μL o H3PO46M. The ube was hen placed on a o a o
pla e o 15 min a 40 pm and hen cen i uged a 8000 pm o 10
min. F om he esul ing mix u e, 1 mL o he CH2Cl2phase was
ans e ed o a 2 mL Eppendo ube and 500 μL o bica bona e
bu e was added. The Eppendo ube was mixed on a o a o pla e
o 10 min, la e cen i uged a 6000 pm o 3 min, and he aqueous
phase was collec ed. Finally, he ex ac was dilu ed in assay bu e
(PNa75, phospha e bu e pH 7.4, 75 mM) using a dilu ion ac o o
1/25.
Unce ain y Budge Calcula ions
The ba ch- o-ba ch ep oducibili y o he syn hesized pa icles is gi en
by he coe icien o a ia ion CV, as de e mined ia he o wa d
sca e ing pa ame e measu ed wi h he low cy ome e , esul ing in
CV
sc= 5% o he PS co e, CV
sc= 7% o he pa icles a e he i on oxide
coa ing s ep and CV
sc= 13% a e silica coa ing.
The coupling o an ibodies o SiO2@SPION@PS accoun s o an
addi ional combined unce ain y o u el
T1=3%, including
a) Weighing o 5 mg o he co esponding amino- unc ionalized
pa icles (balance Me le Toledo lab 205 ±0.01 mg) u el
a=
0.2%
b) Dispe sing he pa icles in 500 μL o E OH a e eac ion wi h
succinic anhyd ide (Eppendo e e ence pipe 500 μL±3μL)
u el
b= 0.6%
c) Dilu ing 10 μL o he suspension in 100 μL o MES bu e
(Eppendo e e ence pipe es ±0.1 μL o 10 μL and ±0.8 μL
o 100 μL pipe ) u el
c= 1.8%
d) Adding 80 μL o a solu ion o EDC (1%) and 160 μL o a
solu ion o NHS (1%) u el
d=1.3%
e) Adding 50 μL o ei he GAM o p o ein G o he solu ion; u el
e
= 1%
) Cen i uging and washing (2×) and esuspending in 200 μL
PBS; u el
= 0.6%
g) Adding 20 μL o he di e en mAbs u el
= 1.5%
The unce ain ies o he p epa a ion and execu ion o he assay
include dilu ions o he s ock solu ions o he analy e, i.e., dilu ing 5
μL o s ock solu ion in 100 μL o bu e (u el
V= 2%), u he dilu ing
s ock solu ions o OTA in wa e o ob aining s anda d OTA o OTA-
F, in PBS successi e dilu ion o he mo he solu ion: n×u el
V,nmax = 9,
mixing o 10 μL o pa icle suspension (0.05%) and 100 μL o bu e
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+ 5 μL o OTA and 5 μL o OTA-F (u el
a= 4.8%) and he
con ibu ions om he cy ome e measu emen s:
a) Rela i e unce ain y o coun s o pa icles egis e ed: u el
cy ≤
1.5%
b) Expe imen al s anda d de ia ion o eplica e measu emen s:
u el
≤2% amoun ing o a o al ela i e unce ain y o u el
T= 5.8%
o he assay.
■RESULTS AND DISCUSSION
Ou s udy add esses he applica ion o a nex gene a ion o
magne ic hyb id mic opa icles o he de e mina ion o small-
molecule analy es in apid cy ome y assays using monoclonal
an ibodies as he de ec ion en i y. While he p esen wo k
demons a es he pe o mance o he app oach wi h he use
case o de ec ing OTA in whea lou ex ac s, he pa icle
a chi ec u e al eady includes a u u e use o he beads in
mul iplexed au oma ized assays. In addi ion, he aim was o
u he exploi a s aigh o wa d mix-and- ead app oach as ou
beads a e ideally sui ed o use wi h many di e en an ibodies
wi hou he need o change he p o ocol o he beads. Exis ing
magne ic pa icles ace challenges ela ed o s abili y,
unc ionaliza ion, and weigh (sedimen a ion). To o e come
hese limi a ions, we ecen ly imp o ed ou p omising hyb id
pa icle pla o m, being ad an ageous in e ms o s abili y,
p ac ical weigh , acile unc ionaliza ion, and cus omizable
su ace a ea,
30,31,35
wi h ano he ea u e, a magne ic unc ion-
ali y.
34
The p esen wo k epo s on he unc ionaliza ion,
deploymen , and pe o mance o hese beads in analy ical
assays, o e ing ad an ages in e ms o e iciency and eliabili y
compa ed o p e ious app oaches.
The design a ionale o he supe pa amagne ic co e−shell
mic opa icles and hei s epwise syn hesis is as ollows.
Poly( inylpy olidone)-s abilized (PVP10, a e age molecula
weigh 10 kDa) PS mic opa icles cons i u e he co e (Figu e
1a, (i)), because hey can be acilely p epa ed in sizes well
sui able o single-pa icle assays (1−3μm) wi h high
monodispe si y and he possibili y o dope hem subsequen ly
wi h o ganic dyes ia a simple swelling p ocedu e.
31
A hin
laye o SPIONs ha does no co e he su ace comple ely is
hen coa ed on o he PS co es o endow magne ic p ope ies
while a oiding sedimen a ion issues and lea ing space o any
op ical moie ies doped in o he co e o be exci ed in an
applica ion (Figu e 1a, (ii)). Finally, a closed seconda y silica
shell is coa ed on o he i s shell o p o ec ion and u he
acile unc ionaliza ion ia silane chemis y (Figu e 1a, (iii)).
The esul ing pa icles as obse ed in a scanning elec on
mic oscope (SEM) a e shown in Figu e 2. They ha e an
o e all diame e o 1.8 ±0.1 μm. The silica shell, measu ing ca.
30 nm in hickness, inco po a es 5 nm SPIONs. The image in
Figu e 2a shows he whole pa icle, while he image in Figu e
2b highligh s he su ace a ea and i s oughness. To isualize
he SPION laye , TSEM images we e aken as seen in he
image in Figu e 2c. Fi s , hese pa icles ha e a simila su ace
a ea o comme cial mic opa icles o analy ical applica ions
(e.g., Dynabeads, The mo Fishe ) while p esen ing no able
ad an ages: Poly inylpy olidone (PVP) se es as a s abilize
Figu e 1. (a) Wo k low o bead p epa a ion: coa ing o PS beads (i) wi h SPIONs (ii) and an insula ing SiO2shell (iii); he amino
unc ionaliza ion and ca boxy e unc ionaliza ion s eps a e omi ed o simplici y. Binding o an ibodies o beads h ough one-s ep di ec
a achmen (i , ou e DA) o in wo s eps ia p o ein G ( , ou e PG) o goa an imouse Ab ( , ou e GAM). (b) Wo k low o mix-and- ead assay:
pipe ing o h ee solu ions in o wells ( i), shaking ( ii), and cy ome ic measu emen and analysis ( iii).
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in he pa icles, no only o e ing a long- e m s abili y o he
SPIONs by hemsel es
41
bu also acili a ing a s aigh o wa d
deco a ion wi h SPIONs be o e o e g ow h o an insula ing
silica coa ing. They a e ligh e in weigh han con en ional
silica o PS pa icles con aining he magne ic i on oxide
nanopa icles in he mic obead’s co e,
10,12
making hem mo e
sui able o longe use in a ious applica ions. The pa icles’
dis inc i ely ex u ed, high-su ace a ea s uc u e can be
a ibu ed o he p esence o SPIONs pa ially deco a ing he
pu e PS co e. This laye , combined wi h a dense silica coa ing,
esul s in a oughe su ace, as p e iously desc ibed.
34
An impo an new aspec o hese pa icles is his ype o
laye o med by he SPIONs. Al hough hey a e no magne ic
ou side a magne ic ield due o he size o he nanopa icles,
he en i e co e−shell pa icle can easily be mo ed by a magne .
The p esence o a a he loose laye ins ead o a closed shell o
nanopa icles also enables op ical encoding o he co es, since a
dense shell o nanopa icles would esul in op ically opaque
co e pa icles.
35
Las ly, he silica coa ing no only p o ides mul iple a enues
o unc ionaliza ion bu also ac s as a p o ec i e ba ie ,
enabling he pa icles o be used in acidic en i onmen s whe e
i on would ypically oxidize and lose i s magne ic p ope ies. In
ou case, his silica ou e shell was employed o include amino
g oups h ough classical silane chemis y using APTES. These
amino g oups we e la e con e ed in o ca boxylic acid g oups,
ia succinic anhyd ide, o he u he immobiliza ion o he
p o eins.
To de elop an easy- o-handle assay p o ocol using he
supe pa amagne ic hyb id PS-co e magne ic silica-shell beads,
se e al es s we e conduc ed. These es s included he binding
o di e en p o eins, such as p ima y monoclonal an ibody,
seconda y polyclonal an ibody, o p o ein G o he pa icle
su ace (Figu e 1a, (i ) and/o ( )), as well as he in es iga ion
o an ibody in e ac ions wi h he selec ed luo escen
compe i o and he a ge analy e, OTA. Conside ing ou
p e ious expe ience wo king wi h his myco oxin, h ee
di e en monoclonal an i-OTA an ibodies we e selec ed o
e alua ion (e#115, #223, and b#311; labeling ollows he
labeling in e 40). These h ee an ibodies we e selec ed
because hey we e able o in e ac wi h a compe i o o OTA
p epa ed h ough he same posi ion as he luo escen
luo escein-con aining compe i o (OTA-F) selec ed o his
s udy. Figu e 3 shows he s uc u e o OTA, OTA-F, and he
hap ens ha we e p e iously used o an ibody gene a ion.
Each o he hap ens was used o he gene a ion o one o he
an ibodies es ed he e, as indica ed by he hap ens and he
le e in he an ibody code.
To bind he di e en p o eins o he pa icle su ace, we
used EDC/sNHS chemis y o o m co alen amide bonds
be ween he amino g oups o he lysine esidues o he p o eins
wi h he ca boxylic acid g oups on he su ace o he pa icles.
Addi ionally, he pu pose o using p o ein G (Figu e 1a, ou e
PG) o GAM (Figu e 1a, ou e GAM) as assis ing cap u e
p o eins was o e alua e he impo ance o he o ien a ion o
he p ima y an i-OTA an ibody. This was done wi h he aim o
minimize s a is ical e o s, ensu ing ha he p ima y an ibody
would consis en ly ha e he co ec o ien a ion (pa a ope
acing he analy e) on he pa icles. As a con ol, he p ima y
an i-OTA an ibodies we e also di ec ly a ached (Figu e 1a,
ou e DA) o he su ace using he same EDC/sNHS
app oach. Conside ing he inal pa icle assay applica ion,
low cy ome y was chosen as he p e e ed me hod o all
es ing due o i s use - iendly ea u es, including he
a ailabili y o an au osample and apid assay eadou (Figu e
1b).
The luo escence emi ed by he OTA-F compe i o on he
su ace o he pa icle is cen e ed a 518 nm and was de ec ed
h ough a 533/30.H bandpass il e be o e co ela ion wi h he
FSC signal o dis inguish i om he excess o compe i o s ill
in solu ion.
In a p elimina y s udy, o es he capabili y o hese
an ibodies o ecognize he OTA-F compe i o , h ee
immobiliza ion app oaches ia ou es DA, PG, and GAM
we e e alua ed o he h ee an i-OTA an ibodies. The
maximum signal om he su ace o a single pa icle was
measu ed in he absence o OTA (mos OTA-F bound), while
also a p onounced inhibi ion a e was assessed o a ela i ely
Figu e 2. SEM images ((a) ull iew and (b) close-up o su ace) and
TSEM images ((c) close-up o su ace) o he polys y ene-co e
magne ic silica-shell beads.
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high concen a ion o OTA (40 nM), ca ying ou hese
expe imen s o all combina ions. The inhibi ion a e is de ined
he e as he quo ien o he signals in he wells wi h OTA and
he signals in he wells wi hou OTA, wi h all wells ha ing he
same OTA-F concen a ion; see Ma e ials and Me hods o
de ails.
A his poin , he concen a ion o an ibody was kep
cons an a 300 ng o an ibody pe μg o pa icle. Figu e 4
shows he g aphs ep esen ing he inhibi ion a es as do ed
lines and he maximum signals as ba s o he nine ypes o
pa icles used in he s udy. The le y-axis deno es he
maximum signal achie able wi h he compe i o o each ype
o binding, while he igh y-axis ep esen s he inhibi ion o
he compe i o in he p esence o 40 nM OTA. To ensu e he
lowes a e o alse-nega i e esul s, i was c ucial o employ he
lowes possible concen a ion o compe i o . Excess luo es-
cence om he le o e compe i o could po en ially lead o
inaccu a e ou comes ia unspeci ic binding. The highes
inhibi ions we e obse ed when an ibody e#115 was di ec ly
bound o he pa icles ( ou e DA, 80%) o when an ibody
#223 was used in conjunc ion wi h GAM ( ou e GAM, 85%).
Con e sely, hese es ing s eps e ealed ha an ibody b#311
showed no signi ican in e ac ion wi h he compe i o ,
p obably due o he lack o a spa ial linke be ween he
OTA and luo escein subuni s, as epo e s wi h longe linke s
we e able o in e ac wi h his an ibody in ELISA.
21
The e o e,
an ibody b#311 was excluded om u he in es iga ions he e.
To de e mine he op imal signal- o-noise a io, we e alua ed
di e en concen a ions o compe i o wi h he o he wo
an ibodies. The bes a io was achie ed wi h a concen a ion o
0.01 μM o OTA-F. While an ibody e#115 displayed he
highes o e all signal, i s inhibi ion a e eached 80% in he
case o an ibody binding along ou e DA. On he o he hand,
an ibody #223 exhibi ed success ul pe o mance wi h he
seconda y an ibody a achmen ia ou e GAM and an
imp o ed inhibi ion a e o 84%, aligning wi h he objec i e
Figu e 3. S uc u es o (a) OTA, (b) luo escen compe i o OTA-F,
and (c) hap ens used o an ibody gene a ion.
Figu e 4. Maximum signal o he bound compe i o in he absence o
analy e in he assay (le y-axis) and inhibi ion a e o a concen a ion
o 40 nM o OTA ( igh y-axis) o (a) an ibody e#115, (b) an ibody
#233, and (c) an ibody b#311 a ached ia ou es DA, PG, and GAM
o he i le beads.
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o his p ojec . Consequen ly, u he es ing in ol ed a
compa ison o e#115/DA and #223/GAM.
To gain mo e insigh in o he an ibody immobiliza ion s ep,
we decided o de e mine he equi ed amoun o an ibody o
maximum co e age o he pa icles. In ha ega d, he assay
wi h an ibody e#115 was chosen o his e alua ion since i was
pe o ming be e in he DA app oach.
To accoun o any unce ain ies ega ding he beha io o
he an ibody on he su ace, ideally ull unc ionaliza ion o he
su ace is desi able. Because o ha , we conduc ed u he es s
using wo di e en concen a ions o his an ibody: 300 ng o
an ibody μg−1o pa icle, a simila concen a ion o he one
employed in he p elimina y es s, and 450 ng o an ibody μg−1
o pa icle.
The calib a ion cu es we e gene a ed by es ing di e en
concen a ions o OTA in he p esence o a 0.01 μM
compe i o concen a ion. Figu e 5 (bo om) shows ha
bo h concen a ions ul ima ely yielded simila signal in en-
si ies, indica ing ha he maximum binding capaci y o he
pa icles’ su ace had been eached wi h he lowe concen-
a ion es ed. The hal inhibi o y concen a ion (IC50)
commonly iden i ied as he concen a ions a he in lec ion
poin when he minimal asymp o e ends o ze o o hese wo
assays we e de e mined o be 1.4 and 1.7 nM o he
concen a ions o 300 and 450 ng o an ibody μg−1o pa icle,
espec i ely.
Conside ing he simila beha io o bo h concen a ions
shown, he lowes one was selec ed o u he op imiza ion.
Since an ibody #223 was showing a be e inhibi ion in he
p elimina y es when immobilized ia ou e GAM, his assay
was chosen o u he de elopmen . In he same way as be o e,
wo di e en an ibody concen a ions we e es ed, 300 and 450
ng o an ibody μg−1o pa icle.
A his poin , i mus be no ed ha he pa icles we e
p e iously unc ionalized wi h a la ge excess o cap u e
an ibody GAM (960 ng o seconda y an ibody μg−1o
pa icle) o sa u a e he pa icle su ace and elimina e he
a ailabili y o ac i e places as a limi ing ac o . Figu e 5 ( op)
p esen s a signal inc ease wi h he amoun o p ima y an ibody
#223 used, sugges ing ha he amoun o cap u e an ibody is
adequa e o he es ed concen a ions o monoclonal an i-
body.
The e o e, he decisi e ac o in selec ing he op imal assay
was a comp omise be ween an adequa e maximal signal and
minimal IC50. The IC50 alues o he wo an ibody
concen a ions es ed, 300 and 450 ng o an ibody μg−1o
pa icle, we e 0.7 and 1.0 nM, espec i ely. The ac ha he
maximum speci ic binding (Bmax) and IC50 a e di e en ly
a ec ed by a change in an ibody concen a ion o ou e GAM
compa ed o ou e DA, i.e., a educ ion in Bmax wi h simila
IC50 o ou e GAM compa ed o simila Bmax and IC50 o
ou e DA, is p esumably due o he di e en ways o an ibody
a achmen . As men ioned abo e, an ibodies e#115 a e
co alen ly ancho ed o he su ace o he beads in ou e DA
and bo h concen a ions esul ed in a i ually iden ical
unc ionaliza ion densi y, leading o a e age FL1 signals o
3310 ±60 coun s and a 20% highe IC50 o he highe
an ibody concen a ion. Howe e , wi h ou e GAM, wo
biomac omolecules a e sequen ially bound o he su ace o
he beads, so ha no only he co alen unc ionaliza ion
densi y wi h GAM is decisi e, bu also he nonco alen binding
o #223 o GAM plays a ole. Ob iously, he occupa ion o he
GAM uni s by #223 is no ye quan i a i e a he lowe
concen a ion, which leads o a 25% highe Bmax a he 450 ng
o an ibody μg−1o pa icle. In addi ion, IC50 is also 45%
highe o he highe an ibody concen a ion.
As lowe IC50 alues co espond o imp o ed de ec ion
limi s and he absolu e signal in ensi ies measu ed a e su icien
o all assay combina ions, he op imized assay u ilized he
lowe an ibody amoun o 300 ng o an ibody μg−1o pa icle.
We made addi ional e o s o enhance he assays by
expe imen ing wi h pos an ibody a achmen washing s eps
and modi ying he bu e composi ion. Howe e , hese
a emp s yielded no disce nible imp o emen .
The in oduc ion o an addi ional washing s ep wi h bu e ,
in addi ion o cen i uga ion and sol en exchange, did no lead
o any no iceable changes in assay signals. Simila ly,
ansi ioning om a PBS o a TRIS-based bu e did no
esul in an inc ease o dec ease in signal in ensi y beyond
measu emen unce ain y.
Thus, ad an ageously, he mix-and- ead app oach as shown
in Figu e 1b p o ed o yield op imum pe o mance while being
simple and as .
To assess he ep oducibili y o he de eloped assay, i s day-
o-day pe o mance was s udied. The assay was pe o med on
ou sepa a e days, wi h ou di e en ba ches o beads, wi h
h ee eplica es each, yielding he calib a ion cu es p esen ed
in Figu e 6.
Figu e 6 p o ides a di ec compa ison o he wo pa icle-
based immunoassays, i.e., in he op panel, OTA was again
de ec ed wi h an ibody e#115 a ached o he su ace o he
pa icles along ou e DA, while in he bo om panel, he
combina ion #223/GAM was used. The igu e shows ha he
de ec ion limi s exhibi ed minimal a iabili y h oughou he 4-
Figu e 5. Calib a ion cu es using e#115/DA ( op) and #223/GAM
(bo om) in di e en amoun s o de e mine he maximum co e age
o he pa icles.
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day es ing pe iod, and wi h an IC50 o 0.1 nM, he assay
in ol ing he use o GAM and monoclonal an ibody #223
appea s o be he mos e ec i e.
The la ge e o obse ed in pa icles using an ibody e#115
is likely due o he di ec binding me hod used in hei
p epa a ion. Wi hou con ol o e he o ien a ion o he
pa a ope egion, he unce ain y in measu ing lowe concen-
a ions o OTA is mainly in luenced by andom e o s due o
he lack in di ec ionali y a he han he p epa a ion o he
ca ie pa icles o he assay p o ocol.
The limi o de ec ion (LoD, acco ding o DIN 32645:2008-
11) achie ed by ou assay using he seconda y an ibody is
0.025 nM, which is in he lowe ange o cy ome y assays o
OTA epo ed in he li e a u e.
26−29
An essen ial imp o emen
in compa ison o comme cial assays is he o e all assay ime.
The en i e p ocess akes less han 15 min, 10 min o
incuba ion plus ans e ime o he low cy ome e , and ca. 1
min measu emen ime o a cy ome y un, in con as o 1 h
o mo e o mos epo ed examples,
4−7,10,12
he main
imp o emen being he simplici y and g ea ly educed
incuba ion ime.
The abili y o de ec low concen a ions o he a ge analy e
u he demons a es he obus ness and eliabili y o ou
de eloped assay. No ably, he achie ed de ec ion limi is
compa able o ha o many immunochemical me hods, such as
ELISA, and e en ou pe o ms ce ain echniques like elec o-
chemical analysis.
21,42,43
The ela i ely low and consis en e o obse ed in ou
measu emen s ensu es compliance wi h he myco oxin es ing
limi s se by he Eu opean Union (EU).
20
The e sa ili y o he analy ical me hod desc ibed in his
s udy allows o i s applicabili y in a ious pa icle-based assays,
only limi ed by he a ailabili y o a compe i o and i s
pe o mance wi h he a ailable an ibodies.
Fo u he p oo , we conduc ed a eal-li e example by
es ing whea lou ob ained om a local mill. The lou sample
was ob ained di ec ly om he mill wi hou any u he
p ocessing, e lec ing he ypical handling a he acili y. To
ensu e eliable measu emen s, a po ion o he sample was
spiked wi h OTA. The ini ial concen a ion o OTA was
known h ough a egula analysis by Eu o ins, commissioned
by he mill.
We spiked he samples wi h an OTA s anda d, esul ing in a
concen a ion o 6.1 μg kg−1, while he pu e lou sample
con ained 0.2 μg kg−1, a alue below he maximum limi
es ablished by he EU.
20
Following an ex ac ion me hod
p e iously desc ibed based on he acidic cha ac e is ics o
OTA,
21
we pe o med he assay and ob ained alues o 0.4 ±
0.1 μg kg−1 o he blank lou sample and 6.4 ±0.1 μg kg−1 o
he spiked sample. This esul ed in a eco e y a e o 104%,
which alls wi hin he commonly accep ed ange o 90−110%.
We a emp ed al e na i e ex ac ion me hods, including he
use o e hanol as he ma ix, bu un o una ely, hese e o s did
no yield any meaning ul esul s. Using e hanol only in he
ex ac ion s ep, no signal was de ec ed, and when measu e-
men s we e conduc ed in e hanol, a ma ix e ec was obse ed,
ul ima ely esul ing in inconclusi e ou comes.
The success ul eco e y demons a es he accu acy and
e iciency o his assay and u he highligh s i s sui abili y o
p ac ical applica ions. Addi ionally, he speed o he assay
shows by equi ing only a 10 min incuba ion ollowed by
measu emen s comple ed in less han 30 s.
The assay p esen ed in ou s udy demons a es a
compe i i e pe o mance o OTA de ec ion compa ed o
exis ing assays, in e ms o bo h de ec ion limi and analysis
ime. The o al ime equi ed o ou assay is less han 11 min
pe sample, which is on pa wi h he pe o mance o
LFIAs.
21,22,44,45
Unlike LFIAs, which ypically canno pe o m
pa allel analyses, ou assay u ilizes a well pla e au osample
wi h a cy ome e , allowing o apid sequen ial analysis. This
se up p o ides a signi ican ad an age in e ms o h oughpu
and speed and has he ad an age o ha ing much be e
mul iplexing po en ial han LFIAs due o he PS co e, which
can be easily encoded wi h dyes.
While ELISAs gene ally o e he capabili y o p ocess
mul iple samples simul aneously, making hem sui able o
high- h oughpu scena ios, hey a e commonly cha ac e ized
by signi ican ly longe analysis imes while showing com-
pa able de ec ion limi s.
21,46,47
Also, he use o o he binde s
such as nanobodies
48,49
o ap ame s
50
o he employmen o
no el ampli ica ion s a egies
51−53
has no changed he
si ua ion wi h espec o assay ime and has also no led o
d ama ic imp o emen s in de ec ion limi s. Compa ed wi h
ELISAs, ou assay o e s a much as e al e na i e wi hou
sac i icing sensi i i y. The assay de eloped he e is especially
ad an ageous in si ua ions equi ing immedia e decision-
making, like he p ocessing o a shipmen in a mill. I would
also be bene icial in labo a o ies whe e he h oughpu o LFIA
is needed wi h highe sensi i i y and speci ici y.
Figu e 6. Calib a ion cu es o he inal op imized assays; assay based
on combina ion an ibody e#115/DA ( op) and assay based on
combina ion an ibody #223/GAM (bo om).
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Wi h he ease and speed o his assay, u he wo k in ou
labo a o ies add esses mul iplexed analysis, allowing o he
de ec ion o mul iple oxins simul aneously.
■CONCLUSIONS
Ou p esen wo k ocused on he de elopmen and
op imiza ion o a pa icle-based immunoassay o he de ec ion
o och a oxin A. By employing no el pa icles wi h imp o ed
s abili y, easy unc ionaliza ion, and low p ac ical weigh , we
we e able o o e come he limi a ions o exis ing magne ic
pa icles and de elop a as mix-and- ead immunoassay o ma
wi h an o e all assay ime o <15 min.
Flow cy ome y p o ed o be a sui able me hod o he
measu emen o he assay, o e ing use - iendly handling and
quick eadou . The calib a ion cu es ob ained h ough he
assay demons a ed he maximum binding capaci y o he
pa icles and he impac o di e en an ibody a achmen and
concen a ions. Day- o-day measu emen s showed consis en
calib a ion cu es wi h minimal a ia ion especially o he
ou e employing a seconda y goa -an imouse an ibody o he
deco a ion o he pa icles wi h he p ima y an ibody.
No ably, he limi o de ec ion o he inal op imized assay,
using he seconda y an ibody, was de e mined o be 0.025 nM,
su passing he de ec ion limi s o many exis ing me hods,
including elec ochemical ones.
Fu he mo e, he de eloped assay demons a ed i s
p ac icali y and applicabili y in a eal-li e scena io by
success ully analyzing a lou sample ob ained om a mill
and de ec ing OTA wi h an excellen eco e y a e. This
unde sco es he ease and e iciency o he p oposed assay,
making i a aluable ool o apid myco oxin es ing in a ious
ood indus ies.
Wi h i s e sa ili y, po en ial o mul iplexing, and sui abili y
o simpli ied luidic sys ems due o he magne ic p ope ies o
he pa icles employed, his assay holds p omise o he
de ec ion o o he oxins o low molecula weigh pollu an s as
well. The esul s ob ained in his s udy con ibu e o he
ad ancemen o myco oxin de ec ion me hods and o e
p ac ical solu ions o ood sa e y analysis.
■AUTHOR INFORMATION
Co esponding Au ho
Knu Ru ack −Chemical and Op ical Sensing Di ision,
Bundesans al u Ma e ial o schung und -p u ung (BAM),
Be lin D-12489, Ge many; o cid.o g/0000-0002-5589-
5548; Email: [email p o ec ed]
Au ho s
Cha lie Tobias −Chemical and Op ical Sensing Di ision,
Bundesans al u Ma e ial o schung und -p u ung (BAM),
Be lin D-12489, Ge many; o cid.o g/0000-0003-2440-
3181
Daniel López-Pue ollano −Chemical and Op ical Sensing
Di ision, Bundesans al u Ma e ial o schung und -p u ung
(BAM), Be lin D-12489, Ge many; Depa men o O ganic
Chemis y, Uni e si y o Valencia, Bu jasso , Valencia 46100,
Spain; o cid.o g/0000-0001-7259-4918
An onio Abad-Somo illa −Depa men o O ganic
Chemis y, Uni e si y o Valencia, Bu jasso , Valencia 46100,
Spain; o cid.o g/0000-0002-5599-3682
Josep V. Me cade −Ins i u e o Ag icul u al Chemis y and
Food Technology (IATA), Spanish Council o Scien i ic
Resea ch (CSIC), Pa e na, Valencia 46980, Spain;
o cid.o g/0000-0002-1838-2647
An onio Abad-Fuen es −Ins i u e o Ag icul u al Chemis y
and Food Technology (IATA), Spanish Council o Scien i ic
Resea ch (CSIC), Pa e na, Valencia 46980, Spain;
o cid.o g/0000-0001-5672-1438
Comple e con ac in o ma ion is a ailable a :
h ps://pubs.acs.o g/10.1021/acsmeasu esciau.4c00038
Au ho Con ibu ions
CRediT: Cha lie Tobias concep ualiza ion, o mal analysis,
in es iga ion, me hodology, isualiza ion, w i ing - o iginal
d a ; Daniel Lopez-Pue ollano o mal analysis, unding
acquisi ion, in es iga ion, me hodology, p ojec adminis a ion,
isualiza ion, w i ing - e iew & edi ing; An onio Abad-
Somo illa me hodology, esou ces, w i ing - e iew & edi ing;
Josep Vicen Me cade me hodology, esou ces, w i ing -
e iew & edi ing; An onio Abad-Fuen es me hodology,
esou ces, w i ing - e iew & edi ing; Knu Ru ack
concep ualiza ion, unding acquisi ion, me hodology, p ojec
adminis a ion, supe ision, isualiza ion, w i ing - e iew &
edi ing.
No es
The au ho s decla e no compe ing inancial in e es .
■ACKNOWLEDGMENTS
This wo k has been pe o med as a pa o BAM’s Focus A ea
P ojec “MamaLoCA�Modula , mul iplexed, an ibody-based
lab-on-chip analyse o ood con ol” o which inancial
suppo by BAM is acknowledged (No. TF20). D.L.-P. was
suppo ed by a Ma ga i a Salas pos doc o al g an (MS21-068)
om he Minis e io de Uni e sidades, Spain, o he
equali ica ion o he Spanish uni e si y sys em, and inanced
by he Eu opean Union (Nex Gene a ionEU). We a e g a e ul
o Elbland Bio-Muhle GmbH, especially Sebas ian S ein, o
ui ul discussions and au hen ic lou samples and Sig id
Benemann (BAM’s Su ace Analysis and In e acial Chemis y
Di ision) o SEM measu emen s. The pa icles epo ed
he ein a e a ailable upon eques o esea ch pu poses.
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