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Scien i ic RepoR s | 7: 4752 | DOI:10.1038/s41598-017-04970-5
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Di ec p o ein quan i ica ion in
complex sample solu ions by
su ace-enginee ed nano od p obes
S e an Sch i wiese 1, Bea iz Pelaz2,6, Wol gang J. Pa ak 2,3, Se gio Len ijo-Mozo4,7,
Ka e ina Soulan ica 4, Jan Dieckho 5,8, F ank Ludwig5 & Joe g Scho e 1
De ec ing bioma ke s om complex sample solu ions is he key objec i e o molecula diagnos ics.
Being able o do so in a simple app oach ha does no equi e labo ious sample p epa a ion,
sophis ica ed equipmen and ained s a is i al o poin -o -ca e applica ions. He e, we epo on he
speci ic de ec ion o he b eas cance bioma ke sHER2 di ec ly om se um and sali a samples by a
nano od-based homogeneous biosensing app oach, which is easy o ope a e as i only equi es mixing
o he samples wi h he nano od p obes. By ca e ul nano od su ace enginee ing and homogeneous
assay design, we demons a e ha he o ma ion o a p o ein co ona a ound he nanopa icles does
no limi he applicabili y o ou de ec ion me hod, bu on he con a y enables us o conduc in-si u
e e ence measu emen s, hus u he s eng hening he poin -o -ca e applicabili y o ou me hod.
Making use o sandwich assays on op o he nano ods, we ob ain a limi o de ec ion o 110 pM and 470
pM in 10- old dilu ed spiked sali a and se um samples, espec i ely. In conclusion, ou esul s open up
nume ous applica ions in di ec p o ein bioma ke quan i ica ion, speci ically in poin -o -ca e se ings
whe e esou ces a e limi ed and ease-o -use is o essence.
Molecula diagnos ics employed o he diagnosis and p ognos ics o a wide ange o diseases is based on he
de ec ion o bioma ke s in complex sample solu ions and is o eno mous scien i ic and clinical in e es 1, 2. Wi hin
he ange o me hods employed o bioma ke de ec ion, homogenous measu emen echniques a e o special
ele ance o poin -o -ca e (PoC) es ing se ings, as hey allow o omi ing complex sample p epa a ion s eps.
Thus, he ime o sample analysis may be educed, whils ensu ing a he same ime maximal ease-o -use3. He e,
magne ic nanopa icles play a majo ole due o hei added capabili y o magne ic manipula ion, which can
be exploi ed, o example, o accele a e binding p ocesses o o enhance he signal o noise a io4. Al e na i e
non-magne ic nanopa icle-based bio sensing echniques include su ace-enhanced Raman spec oscopy5, 6 o
me hods elying on luo escen nanopa icle p ope ies7. I has been shown ha he combina ion o nanopa -
icle labels and su ace-enhanced Raman spec oscopy allows o de ec a ious bioma ke s in complex sample
solu ions8, 9.
In he p esen a icle, we show he applicabili y o ou p e iously in oduced magne ic nanopa icle-based
homogeneous measu emen p inciple o molecula diagnos ics in complex samples, i.e. se um and sali a sam-
ples. The me hod elies on changes o he hyd odynamic nanopa icle olume upon analy e molecule binding. To
ha end, an ibody- unc ionalized magne ic nano ods (‘nanop obes’) a e exci ed in solu ion by a o a ing mag-
ne ic ield (RMF), which esul s in a o a ional nanop obe mo ion. The hyd odynamic olume o he nanop obes
induces a o a ional d ag o que wi h he esul ha he nanop obes lag behind he RMF by a cha ac e is ic phase
lag. Binding o he an igen causes an inc ease o he hyd odynamic nanop obe olume, which can be obse ed
1Molecula Diagnos ics, AIT Aus ian Ins i u e o Technology, Vienna, Aus ia. 2Fachbe eich Physik, Philipps-Uni e si ä
Ma bu g, Ma bu g, Ge many. 3CIC Biomagune, San Sebas ian, Spain. 4Labo a oi e de Physique e Chimie des Nano-
obje s (LPCNO), Uni e si é de Toulouse; INSA, UPS, CNRS, Toulouse, F ance. 5Ins i u e o Elec ical Measu emen and
Fundamen al Elec ical Enginee ing, TU B aunschweig, B aunschweig, Ge many. 6P esen add ess: Cen o Singula
de In es igación en Química Biológica y Ma e iales Molecula es (CiQUS) y Depa amen o de Física de Pa ículas,
Uni e sidade de San iago de Compos ela, San iago de Compos ela, Spain. 7P esen add ess: NABLA Lab, Biological
and En i onmen al Sciences and Enginee ing (BESE) Di ision, King Abdullah Uni e si y o Science and Technology
(KAUST), Thuwal, 23955-6900, Saudi A abia. 8P esen add ess: Diagnos ic and In e en ional Radiology Depa men
and Clinic, Uni e si y Medical Cen e Hambu g-Eppendo , Hambu g, Ge many. Co espondence and eques s o
ma e ials should be add essed o S.S. (email: [email p o ec ed])
Recei ed: 6 Ap il 2017
Accep ed: 5 June 2017
Published: xx xx xxxx
OPEN
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Scien i ic RepoR s | 7: 4752 | DOI:10.1038/s41598-017-04970-5
di ec ly ia a change o he phase lag. This e ec can be u he enhanced by also adding seconda y an ibodies o
o m a sandwich- ype immunoassay on op o he nanop obe su ace. The phase lag is de e mined op ically by
measu emen s o he ac ual nanop obe alignmen . This is made possible by he elonga ed nanopa icle geome y
ha causes aniso opic abso p ion and sca e ing. When applying linea ly pola ized inciden ligh , his e ec
allows o deducing he ac ual nanop obe o ien a ion in he sample solu ion ia ansmission measu emen s. By
co ela ing he measu ed ac ual nanop obe o ien a ion wi h he momen a y ec o o he applied RMF, he phase
lag angle can be de e mined, and ou signal is de ined by he change in phase lag angle (Δα) be ween he sample
and a sui able e e ence 10–12. Nex o he inhe en ad an ages o homogenous magne ic nanopa icle-based
measu emen me hods, we show ha ou me hod is capable o de e mining quan i a i e bioma ke concen a ion
le els in complex samples by in-si u e e encing.
As model p o ein we ha e chosen he soluble domain o he human epide mal g ow h ac o ecep o 2 -
sHER2, which is he ex acellula domain o HER2, a ecep o -like y osine kinase ha is epo ed o be in ol ed
in se e al ypes o human ca cinomas13. The ex acellula sHER2 p o ein is shed in o he blood s eam so ha i
can be ound in se um as well as in sali a samples, cu en ly being mainly o in e es o he diagnosis as well as
o he p ognosis o b eas cance 14, 15. Cu en ly, he clinical cu -o alue o sHER2 in se um is 170 pM14, while
he clinical cu -o alue o sali a is one o de o magni ude below he se um alue15.
In he ollowing sec ions, we desc ibe he measu es ha a e aken o de ec he sHER2 analy e p o ein in com-
plex samples o se um and sali a, which we e spiked wi h sHER2. These measu es include he igh choice o ype
and concen a ion o seconda y an ibodies as well as he dilu ion ac o o he complex sample solu ions. Finally,
we conclude by summa izing he majo esul s and by gi ing an ou look on how o u he apply and imp o e he
measu emen me hod.
Resul s and Discussion
Simples mix-and-measu e de ec ion o sHER2 analy e in complex solu ion spiked wi h sHER2 could be execu ed
by adding he nanop obes o he sample solu ion, ollowed by de e mining he phase lag di e ence Δα wi h
espec o a e e ence sample. To ha end, ini ial measu emen s ha e been execu ed in 10- old dilu ed se um
samples. Wha a a i s glance seemed o be a p omising measu emen app oach did no esul in an analy e
molecule concen a ion-dependen signal (see Supplemen a y Fig.S1). This we a ibu e o he o ma ion o a
p o ein co ona in complex samples su ounding he nanop obe su ace16, which sc eens he measu emen e ec
o bound analy e molecules. Hence, addi ional measu es had o be aken o gain speci ici y. This can be achie ed
by employing seconda y an ibodies (2nd Abs) in a sandwich- ype immunoassay o ma (see ske ch in Fig.1a). The
2nd Abs a ach o he nanop obe-bound analy e molecules, and, when ex ending he p o ein co ona hickness,
e ie e he concen a ion dependen measu emen signal ha has p e iously been obse ed o spiking analy e
molecules alone in o bu e solu ions10. The exac composi ion and s uc u e o he p o ein co ona o med on
op o he nanop obes canno be answe ed he e, while in p inciple we assume a single laye o adso bed p o eins.
De ailed s udies applying exac ly he same condi ions will be equi ed o gain a deep unde s anding o he unda-
men al p inciples o p o ein co ona o ma ion o ou nanop obes.
To cha ac e ize he e ec o adding 2nd Abs o ou measu emen echnique, we i s de e mined hei op imal
concen a ion. Figu e1b shows he dependence o he measu emen signal in bu e solu ion o nanop obes
unc ionalized by he p ima y an ibody Ab-a on adding a ying concen a ion o he seconda y an ibody Ab-b
o wo dis inc concen a ions o sHER2 analy e molecules (2 nM and 8 nM). Clea ly, he signal inc eased o
ising 2nd Ab concen a ions up o a sa u a ion le el, which co ela es wi h he concen a ion o analy e molecules.
Figu e 1. Signal dependence on seconda y an ibody concen a ion. (a) Ske ch o he nano od (NR) based
sandwich- ype immunoassay employing p ima y (1s ) and seconda y (2nd) an ibodies (Ab); (b) Phase lag
di e ence Δα in measu emen bu e solu ion o wo ixed concen a ions o sHER2 analy e molecules (2 and
8 nM) in dependence o he added concen a ion o 2nd an ibodies.
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Speci ically, he signal sa u a ed o bo h analy e concen a ions when adding a leas i e imes highe 2nd Ab
concen a ions. The addi ion o 2nd Abs wi hou sHER2 did no esul in a measu able signal.
In an ac ual assay ecipe, a ixed concen a ion o added 2nd Abs needs o be selec ed. He e, ou chosen 2nd Ab
concen a ion o 25 nM allows maximal and oughly linea signal ampli ica ion wi hin a measu emen ange up
o abou 5 nM sHER2 ( he limi was ob ained om he obse ed signal sa u a ion a abou i e imes he analy e
molecule concen a ion).
Fu he mo e, he assay can be op imized by ca e ully choosing he bes sui ed an ibody pai s. To ha end, we
es ed all combina ions o Ab-a and Ab-b as p ima y and seconda y an ibodies. Co esponding measu emen
esul s a e shown in he Supplemen a y TableS1 and indica e ha he applica ion o Ab-b as bo h p ima y and
seconda y an ibody esul ed in maximal measu emen signal (case A). This is due o he polyclonal na u e o
Ab-b and he associa ed highe binding a ini y compa ed o monoclonal an ibodies. Consequen ly, when Ab-a
is applied as p ima y an ibody ins ead o Ab-b (case B), less an igen binds o he nanop obes, esul ing in lowe
signal on adding Ab-b as seconda y an ibody (case B s. case A). As expec ed, as only a single epi ope is a ge ed,
he applica ion o he monoclonal an ibody ype Ab-a as bo h p ima y and seconda y an ibody esul ed in he
lowes measu emen signal (case D). When combining Ab-a and Ab-b, he signal was subs an ially la ge o
applying Ab-a as p ima y and Ab-b as seconda y an ibody (case B) as ice e sa (case C). This we a ibu e o
s e ic hind ance o he epi ope a ge ed by he monoclonal Ab-a when he an igen is bound o he nanop obes ia
p ima y an ibody Ab-b. Based on hese esul s, all subsequen measu emen s we e ca ied ou wi h Ab-b bo h as
p ima y and seconda y an ibody (case A).
The nex aspec o be add essed in o de o op imize he assay o complex sample solu ions like sali a and
se um is he de e mina ion o he op imum dilu ion ac o . Dilu ion is an impo an aspec o ou measu emen
app oach as he s uc u e and he composi ion o he p o ein co ona depends on he ypes and he concen a ions
o he p o eins p esen in he sample solu ion and, he e o e, on he sample dilu ion17. In addi ion, he nanop obe
o a ion is also in luenced by he concen a ion o non-bound p o eins in solu ion, he eby a ec ing he d ag
o que and measu ed phase lag18. Consequen ly, by adjus ing he dilu ion ac o , he measu emen signal can be
maximized. To ha end, di e en sali a dilu ions we e es ed. He e, we ha e chosen a high ini ial concen a ion
o sHER2 o 10 nM spiked in o pu e sali a o ensu e ha he concen a ion a e dilu ion emains high enough
o be easily de ec ed. On he o he hand, he ini ial concen a ion was chosen low enough o gua an ee less han
5 nM e en in low sample dilu ions o op imal signal enhancemen by he 2nd Abs as shown abo e. The ob ained
measu emen signals a di e en dilu ions o sali a a e shown in Supplemen a y TableS2. A dilu ion ac o o 10
esul ed in he highes measu emen signal and was, hus, chosen in he ollowing. Fo bes possible compa abili y
o se um and sali a samples, he same dilu ion ac o was chosen also o se um samples.
Based on he in o ma ion gained by he esul s p esen ed abo e, we conduc ed phase lag di e ence meas-
u emen s in dependence o he analy e concen a ion in solu ions o pu e bu e , se um, and sali a. Fo all hese
measu emen se ies, we de e mined he ele an limi s o de ec ion.
Figu e2 shows he measu emen esul s o he sHER2 assay conduc ed in pu e bu e solu ion. The phase
lag di e ence signal Δα clea ly inc eased wi h inc easing analy e concen a ion up o a sa u a ion le el, and was
subs an ially la ge when 2nd Abs we e also added. In his case, he signal sa u a ed a abou 5 nM sHER2 concen-
a ion, and dec eased o highe concen a ions. This dec ease is due o he now non-op imal a io o 2nd Abs o
analy e molecules (see abo e). The achie ed limi s o de ec ion we e abou 400 pM o he assay wi hou 2nd Ab
addi ion and abou 170 pM a e addi ion o 2nd Abs.
Finally, we conduc ed assay measu emen s in 10- old dilu ed solu ions o spiked se um and sali a wi h
addi ion o 2nd Abs. Gene ally, he esul s compa ed well wi h he ones ob ained wi h 2nd Ab addi ion in bu e
Figu e 2. Phase lag di e ence signal Δα in pu e bu e solu ion and i s dependence on he an igen
concen a ion. Measu emen signal wi h (black cu e) and wi hou (g ey cu e) he addi ion o 2nd Abs.
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solu ion. Speci ically, he signals again ose up o a sa u a ion le el o abou 5 nM sHER2 concen a ion. While
in dilu ed se um, we deduced a limi o de ec ion o abou 470 pM, in dilu ed sali a samples sHER2 could be
de ec ed down o abou 110 pM. The eason o he be e measu emen sensi i i y in sali a compa ed o se um
we a ibu e o di e en p o ein composi ion o se um and sali a samples and he di e ence in o al p o ein con-
en 15, 19. Speci ically, he lowe e e ence phase lag in sali a allowed o a la ge ela i e phase lag inc ease due o
analy e and 2nd Ab binding12. We asc ibe he sligh ly be e de ec ion limi in dilu ed sali a samples compa ed o
bu e solu ion o adso p ion o e ia y p o eins o he bound 2nd Abs, which u he inc eases he signal.
Conclusions
In his wo k, we demons a ed ha ou p e iously in oduced nanop obe-based homogeneous biosensing
app oach is also easible o speci ically de ec analy e p o eins in complex sample solu ions (i.e. se um and sali a).
While in bu e solu ion, di ec analy e quan i ica ion is possible, he o ma ion o a p o ein co ona in com-
plex samples sc eens he di ec measu emen signal. Consequen , he inc ease in hyd odynamic diame e due
o an ibody-media ed binding o a ge p o ein canno be dis inguished om non-speci ic adso p ion (i.e. he
o ma ion o a p o ein co ona). Howe e , upon addi ion o seconda y an ibodies o he sample solu ion, he e
again is a speci ic u he inc ease in hyd odynamic nanop obe size, which esul s in a de ec ion signal. Thus,
wha a i s glance seems o be a disad an age, can in ac be exploi ed o conduc in-si u e e ence measu emen s
by simply no adding 2nd Abs, which is an impo an ea u e o PoC es ing en i onmen s.
We sys ema ically elabo a ed measu emen condi ions o de e mine sHER2 analy e p o ein di ec ly in spiked
complex sample solu ions by es ablishing a homogeneous sandwich assay di ec ly a he su ace o ou dispe sed
nanop obes. Recalcula ed o undilu ed samples, ou sHER2 limi o de ec ion cu en ly is abou 4.7 nM in se um
and 1.1 nM in sali a, which is abou one o de o magni ude abo e he clinical cu -o alue o se um14, and,
due o he subs an ially lowe concen a ion, abou wo o de s o magni ude abo e he clinical cu -o alue o
sali a15. Howe e , ou esul s demons a e ha by ad ancing he nanop obe assay condi ions, he de ec ion limi
can s ill be imp o ed subs an ially. Fo example, in bu e solu ion we obse ed a signi ican enhancemen o he
signal and de ec ion limi by in oducing 2nd Abs, which can be expec ed o u he imp o e by applying labelled
2nd Abs o bind la ge molecula weigh biopolyme s. In addi ion, while we es ed some an ibody combina ions
and obse ed a subs an ial e ec on ou measu emen signal, he e is a wide a ie y o di e en an ibodies a ail-
able o de e mine he bes -sui ed pai .
In summa y, o ou simple mix-and-measu e ype homogeneous biosenso we succeeded o o e come a
majo hu dle ha nanopa icle biosenso s o en su e om, which conce ns signal sc eening by p o ein co ona
o ma ion. This opens up nume ous applica ions in p o ein bioma ke de ec ion, speci ically in PoC se ings
whe e esou ces a e limi ed and ease-o -use is o essence.
Me hods
Nanop obe p epa a ion. The nanop obe p epa a ion comp ises he syn hesis o ba e Co nano ods (NRs),
which we e co e ed by a noble me al shell o Au and P acco ding o al eady published p ocedu es20. A e wa ds,
he NRs we e ans e ed o aqueous solu ions by o e -coa ing hem wi h an amphiphilic polyme as desc ibed
ea lie 10, 21. The unc ionaliza ion o he NRs wi h an ibodies (Abs) was achie ed by ca boxy-amine linke chem-
is y. To ha end, we employed EDC (N-(3-(dime hylamino)p opyl)-N′-e hylca bodiimide hyd ochlo ide) and
S-NHS (N-hyd oxysul osuccinimide sodium sal ) a a ios o 1 × 104 EDC molecules and 3 × 104 S-NHS mole-
cules pe NR. The NR concen a ion was de e mined by induc i ely coupled plasma mass spec ome y and by
Figu e 3. Phase lag di e ence signal Δα in 10- old dilu ed solu ions o spiked se um and sali a and i s
dependence on he an igen concen a ion. Measu emen signal in sali a (blue cu e) and in se um ( ed cu e)
unde addi ion o 2nd Abs.
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he geome ic NR dimensions de e mined by ansmission elec on mic oscopy as desc ibed elsewhe e10. We
employed a NR concen a ion o 730 pM and 2-(N-mo pholino)-e hanesul onic acid (MES) bu e solu ion a
a concen a ion o 50 mM and a a pH o 5.5 h oughou he whole unc ionaliza ion p ocedu e. In a i s unc-
ionaliza ion s ep, he NRs we e incuba ed wi h EDC and S-NHS o 15 min a oom empe a u e (RT) and hen
dialyzed agains MES o 30 min a RT o educe he concen a ion o EDC and S-NHS. The dialysis was pe -
o med wi h a Floa -A-Lyze G2 dialysis de ice wi h an app oxima e molecula weigh cu -o alue o 1000 kDa.
A e wa ds, he Abs dilu ed in MES we e added a a olume compa able o he NR/EDC/S-NHS solu ion a a
a io o 200 Abs pe NR, and he whole solu ion was incuba ed o 100 min a RT. Abs applied he e we e ei he
as uzumab in he o m o he He cep in he apeu ic d ug (Ab-a) o comme cial HER2 an ibodies (Ab-b) pu -
chased om R&D Sys ems unde ca alogue numbe AF1129. He e, Ab-a is a monoclonal an ibody, while Ab-b
is o polyclonal na u e. In a nex s ep, 100 bo ine se um albumin molecules pe NR we e added, ollowed by
ano he incuba ion s ep o e nigh a 4 °C o block any emaining binding si es. Finally, o emo e all unbound
eagen s, he unc ionalized NRs we e dialyzed agains MES a a pH o 5.5 o 48 h a 4 °C wi h a change o he
dialysis solu ion a e 20 h.
Sample p epa a ion and measu emen condi ions. The employed se um (pool o heal hy male indi-
iduals) was ob ained comme cially om Sigma-Ald ich (p oduc numbe H4522), while uns imula ed whole
sali a was collec ed in he mo ning om a single heal hy male indi idual who e ained om ea ing be o e sam-
ple aking. The non-in asi e sali a sample has been dona ed by a s a scien is o AIT ollowing in o med consen
and app o al by he Local E hics Commi ee o he Ci y o Vienna22. All me hods we e pe o med in acco dance
wi h he ele an guidelines and egula ions. Sali a was il e ed by a Wha man glass ib e sy inge il e wi h a
nominal po e size o 0.45 μm and s o ed in he idge un il usage. Fil e ing o he sali a was necessa y o educe
he iscosi y by emo ing glycop o eins and la ge molecules wi hou losing analy e molecules23. This p ocedu e
allows o p epa ing samples o a iscosi y le el compa able o se um by a e y simple way sui able o PoC
analysis.
The measu emen bu e (MB) a a pH alue o 7.4 was composed o 10 mM 4-(2-hyd oxye hyl)
pipe azine-1-e hanesul onic acid (HEPES) sodium sal , 150 mM NaCl and 0.05% / Tween 20. Samples spiked
wi h sHER2 we e p epa ed by mixing he MB o dilu ions o se um/sali a (dilu ed by he MB) wi h he sHER2
an igen a a chosen concen a ion. Nex , he nanop obes we e added o he sample solu ion. A e an incuba ion
ime o 30 min a RT, he seconda y an ibodies dilu ed in MB (o olume-equi alen MB solu ions o samples
wi hou added 2nd Abs) we e added, and he whole sample was incuba ed o ano he 60 min a RT. The o e all
sample olume amoun ed o 240 µl, and he nanop obe concen a ion was 10 pM. Measu emen s we e conduc ed
a a RMF ampli ude o 10 mT and a o a ional equency o 1 kHz. These pa ame e s a e chosen o op imal
measu emen signals o de ec a ge p o eins by ou speci ic nanop obes10, 24. Phase lag di e ences we e eco ded
wi h espec o a sui able e e ence alue. Fo he measu emen s execu ed in bu e solu ion only, we employed
e e ence samples comp ising all eagen s excep o he analy e sHER2 molecules. In complex sample solu ions o
se um and sali a, he e e ence phase lag alue was de e mined wi h samples con aining no seconda y an ibod-
ies. Addi ion o he seconda y an ibodies only wi hou analy e molecules did no esul in a measu able signal.
The e o s o each measu emen we e de e mined by he s anda d de ia ions o he e e ence and he espec i e
analy e-spiked samples and he e o p opaga ion law.
Fo de e mining he limi o de ec ion, we de ined a phase lag di e ence h eshold. To ha end, we added
3- imes he ob ained e o o he lowes spiked sHER2 analy e concen a ion (i.e. 0.125 nM) o he e e ence
phase lag. Nex , he assay esul s o he measu ed phase lag di e ences we e i ed by a 4-pa ame e logis ic i
model, and he limi o de ec ion was ob ained by using he h eshold alue in he i ing cu e equa ion.
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Acknowledgemen s
We hank F auke Al es and Julia Bode om he Max-Planck-Ins i u e o Expe imen al Medicine in Gö ingen
o he supply wi h he He cep in he apeu ic d ug. We also wan o hank Da id Fe nandez and Amagoia
Ame zazu a om P ogenika Biopha ma o p o iding he se um. This esea ch was suppo ed by he Eu opean
Commission FP7 NAMDIATREAM p ojec (EU NMP4-LA-2010–246479), and he Ge man Resea ch
Founda ion (DFG g an PA 794/25-1).
Au ho Con ibu ions
The co e-shell nano ods we e syn hesized by S.L.-M. and K.S., while B.P. and W.J.P. s abilized he pa icles ia
polyme -coa ing in aqueous solu ion. S.S. immobilized he an ibodies on o he nano ods. The expe imen al se up
was designed and cons uc ed by S.S., J.S., J.D. and F.L. The expe imen s we e concei ed and pe o med by S.S.
and J.S. The manusc ip was w i en by S.S. and J.S. wi h inpu om all co-au ho s. All au ho s e iewed he
manusc ip .
Addi ional In o ma ion
Supplemen a y in o ma ion accompanies his pape a doi:10.1038/s41598-017-04970-5
Compe ing In e es s: The au ho s decla e ha hey ha e no compe ing in e es s.
Publishe 's no e: Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in published maps and
ins i u ional a ilia ions.
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