Senso s and Ac ua o s B 229 (2016) 646–654
Con en s lis s a ailable a ScienceDi ec
Senso s and Ac ua o s B: Chemical
jou nal homepage: www.else ie .com/loca e/snb
Regene a i e biosenso chips based on swi chable mu an s o
a idin—A sys ema ic s udy
Dominik Zaune a, Ba ba a Taskinenb,c, Daniel Eichinge a, Clemens Fla inge a,
Bianca Ru manna, Claudia Knoglinge a, Lukas T axle a, And eas Ebne a,
He mann J. G ube a,∗, Vesa P. Hy önenb,c
aIns i u e o Biophysics, Johannes Keple Uni e si y, G ube s asse 40, 4020 Linz, Aus ia
bBioMediTech, Uni e si y o Tampe e, Bioka u 6, FI-33520 Tampe e, Finland
cFimlab Labo a o ies, Bioka u 4, FI-33520 Tampe e, Finland
a i c l e i n o
A icle his o y:
Recei ed 8 Oc obe 2015
Recei ed in e ised o m 8 Feb ua y 2016
Accep ed 10 Feb ua y 2016
A ailable online 11 Feb ua y 2016
Keywo ds:
Biosenso
Bio in su ace
A idin mu an
Re e sible immobiliza ion
Senso chip egene a ion
a b s a c
Bio inyla ed bai molecules can be immobilized on bio inyla ed senso chips by o ma ion o
bio in–a idin–bio in b idges which a e e y s able when using wild- ype (s ep )a idin. S able immo-
biliza ion o bio inyla ed bai s is impo an o moni o ing e e sible binding and dissocia ion o p ey
molecules. Fo measu emen s wi h ano he bai molecule, howe e , i is desi able o eplace all immo-
bilized p o eins by esh (s ep )a idin and new bio inyla ed bai . In his s udy, fi e a idin mu an s
ha e been cha ac e ized wi h espec o hei abili y o o m swi chable bio in–a idin–bio in b idges
on bio inyla ed chip su aces, as needed o comple e chip egene a ion. All fi e mu an s o med s able
bio in–a idin–bio in b idges a pH 7, we e mo e o less s able a pH 2–3, and equi ed he combina ion
o pH 2 wi h SDS o quan i a i e emo al om he chip su ace. Mu an #3 (“swi cha idin”) showed he
bes combina ion o p ope ies, i.e., low nonspecific adso p ion o p o ein and nucleic acids, high binding
capaci y, and good s abili y a pH 2–3, as ypically used o quan i a i e emo al o p ey molecules in
epea ed measu emen cycles.
© 2016 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY license
(h p://c ea i ecommons.o g/licenses/by/4.0/).
1. In oduc ion
Biosenso s moni o binding o soluble p ey molecules o immo-
bilized bai molecules. Subsequen ly, all bound p ey molecules
mus be emo ed be o e he nex sample can be measu ed [1].
Usually he bai molecules a e immobilized by co alen bonds o
by a idin–bio in in e ac ion [2–5]. Since nei he is e e sible in a
easonable ime window, a new measu emen se ies can only be
s a ed a e exchanging he senso chip and immobilizing ano he
bai molecule. A desi able al e na i e is eplacemen o he old
bai molecules o new ones. Exchange o he bai on he chip
con e s significan ad an ages: (i) i sa es he cos and ime o
chip exchange. (ii) No human in e en ion is equi ed, allowing o
swi ching o bai s in p og ammed o e nigh uns. (iii) I elimina es
chip- o-chip a ia ion, which is a p oblem wi h some p oduc lines.
(i ) The mos u gen need o in-si u exchange o bai molecules
is encoun e ed i he ha sh condi ions (e.g., 100 mM HCl o NaOH)
∗Co esponding au ho .
E-mail add ess: [email p o ec ed] (H.J. G ube ).
ypically used o emo al o bound p ey molecules [1] cause dena -
u a ion o he bai .
In o de o be use ul in p ac ical applica ion, any me hod o bai
exchange mus obey s ic c i e ia: (a) he bai mus emain s ably
bound o hou s o days, un il all planned measu emen s ha e been
comple ed wi h one kind o bai molecule. (b) I mus be possible o
quan i a i ely emo e all bound bai molecules wi hin minu es. (c)
The binding capaci y o new bai molecules mus be ully e ained
o a la ge numbe o egene a ion cycles. (d) The eagen s used
o chip egene a ion mus be compa ible wi h he flow cells o
common biosenso s. (e) The eagen s and he chip su ace mus be
s able unde ambien condi ions (i.e., no sensi i e o oxida ion o
hyd olysis). ( ) The senso su ace used o swi chable bai immobi-
liza ion mus no bind any p ey o side componen s o he sample.
(g) P e e ably, he me hod should be easy o implemen in many
ypes o biosenso s.
Two published me hods ulfill hese c i e ia o a high deg ee. In
bo h me hods, a idin o (s ep )a idin is e e sibly immobilized,
p o iding o immobiliza ion o bio inyla ed bai molecules be o e
a measu emen se ies, and o apid emo al o (s ep )a idin
plus bai a he end. In he fi s me hod, (s ep )a idin is
h p://dx.doi.o g/10.1016/j.snb.2016.02.039
0925-4005/© 2016 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/).
D.
Zaune
e
al.
/
Senso s
and
Ac ua o s
B
229
(2016)
646–654
647
Fig.
1.
Re e sible
unc ionaliza ion
o
bio inyla ed
senso
chips
wi h
bio inyla ed
p obe
molecules.
(a)
Illus a ion
o
chip
ecycling
wi h
swi chable
bio in–a idin–bio in
b idges.
(b)
Tes ing
o
di e en
a idin
mu an s
o
e e sible
immobiliza ion
o
bio inyla ed
an ibodies.
(c)
SPR
aces
showing
binding
o
mu an
#1
in
bo h
flow
cells
and
o
bio in–IgG
in
FC2
(blue
dashed
line),
as
well
as
emo al
by
SDS/ci ic
acid.
The
numbe s
1–4
in
(c)
co espond
o
he
s ages
1–4
in
(b).
(d)
Compa ison
wi h
espec
o
he
mu an
laye s
in
bo h
cells,
bio in–IgG
bound
in
FC2,
and
baseline
d i
om
s age
1
o
4.
(e)
Calcula ed
pI
o
he
a idin
mu an s
(Table
S1).
(Fo
in e p e a ion
o
he
e e ences
o
colo
in
his
figu e
legend,
he
eade
is
e e ed
o
he
web
e sion
o
his
a icle.)
e e sibly
immobilized
on
ca boxyme hyldex an
ia
DNA
double
s and
o ma ion
(BIAco e
applica ion
no e
“Bio in
CAP u e
Ki ”,
GE
da a
file
28-9577-47
AA,
h ps://www.geli esciences.com/
gehcls
images/GELS/Rela ed%20Con en /Files/1314787424814/
li doc28957747AA1
20110831132219.pd ).
This
me hod
ulfills
c i e ia
(a)–(e)
bu
i
is
no
applicable
o
DNA-binding
p o eins
and
only
a ailable
o
one
b and
o
biosenso s.
In
he
second
me hod,
he
a idin
mu an
M96H
is
used
as
a
swi chable
link
be ween
bio inaly ed
senso
su aces
and
bio inyla ed
bai
molecules
[6,7].
Mu a ion
M96H
is
loca ed
a
he
subuni
in e ace
(Supplemen a y
Fig.
S1)
and
i
con e s
sensi i i y
o
low
pH
[8].
In
he
bio in-bound
s a e,
howe e ,
his
mu an
e ains
ull
unc ion
down
o
pH
2.7,
unless
a idin
M96H
is
in en ionally
dissocia ed
in o
ou
non unc ional
subuni s
by
combina ion
o
ci ic
acid
wi h
sodium
dodecyl
sul a e
(SDS)
as
ou lined
in
Fig.
1a
[6].
An
ob ious
d awback
o
a idin
M96H
is
i s
posi i e
cha ge
a
neu al
pH
(pI
∼
9.5,
see
Fig.
1e).
Mos
p o eins
and
all
nucleic
acids
a e
nega i ely
cha ged
a
neu al
pH,
esul ing
in
nonspecific
adso p ion
o
immobilized
a idin
M96H,
especially
in
case
o
DNA
[6,7].
Nonspecific
p o ein
adso p ion
was
la gely
supp essed
by
blocking
wi h
bio in–BSA
[6],
whe eas
supp ession
o
DNA
adso p-
ion
equi ed
addi ional
mu a ions,
which
lowe ed
he
pI
owa ds
7
(mu an
#3
in
Fig.
1e
and
Table
S1)
[7].
In
he
p esen
s udy,
fi e
a idin
mu an s
(Table
S1
and
Fig.
S1)
we e
cha ac e ized
along
c i e ia
(a)–(g)
and
he
limi s
o
he
me hod
we e
iden ified.
Mu an
#3
(“swi cha idin”
[7])
was
ound
o
be
he
op imal
choice,
combining
high
s abili y
o
he
bio in–a idin–bio in
b idge
wi h
low
nonspecific
adso p ion
o
p o ein
and
DNA.
2.
Ma e ials
and
me hods
2.1.
Ma e ials
The
a idin
mu an s
we e
cons uc ed
o
bac e ial
exp ession
in
Esche ichia
coli
by
in oducing
mu a ions
o
cDNA
encod-
ing
chicken
a idin
con aining
ompA
signal
pep ide
in
pET101/D
[9,10]
by
QuikChange
mu agenesis
acco ding
o
manu ac u e ’s
648
D.
Zaune
e
al.
/
Senso s
and
Ac ua o s
B
229
(2016)
646–654
ins uc ions
(S a agene,
La
Jolla,
CA,
USA)
o
by
using
s anda d
PCR
echniques,
whe e
mul iple
mu a ions
we e
in oduced
by
o e lapping
mu a ed
DNA
agmen s
amplified
using
DNA
oligonu-
cleo ides
con aining
he
desi ed
mu a ions,
ollowed
by
subcloning
o
pET101/D
wi h
he
help
o
TOPO
cloning
( o
de ails
see
Re .
[7]).
All
DNA
cons uc s
we e
confi med
by
DNA
sequenc-
ing.
The
p o eins
we e
p oduced
and
pu ified
as
desc ibed
[7].
The
componen s
o
he
mixed
sel -assembled
monolaye
(SAM)
we e
syn hesized
and
mixed
as
desc ibed
in
Re .
[6].
Bio in-cap-
NHS,
bio inyla ed
p o ein
G
(bio in–p o ein
G),
immunoglobulin
G
(IgG)
om
goa ,
human
IgG2␬,
and
lysozyme
we e
ob ained
om
Sigma–Ald ich.
Bo ine
se um
albumin
(BSA,
a y
acid- ee)
was
pu chased
om
Roche
Applied
Science.
Bio in–IgG
(wi h
6–7
bio ins/IgG)
was
p epa ed
as
published
[11]
and
bio in–BSA
was
p epa ed
using
he
same
mass
concen a ions
o
p o ein
and
bio in-cap-NHS.
All
single-s anded
DNA
molecules
we e
cus om-syn hesized
by
VBC
Genomics
(Vienna)
wi h
99
±
0.5%
coupling
e ficiency
and
he
uncapped
final
p oduc
was
pu ified
by
HPLC
(posi i e
selec ion
o
he
5- e minal
dime hoxy i yl
g oup
by
e e sed
phase
ch oma og aphy).
The
bio in-p obe
had
he
s uc u e
5-bio in-GCACCTGACTCCTGTGGAGAAGTCTGCCGT-
3[5],
he
“unlabeled
p obe”
had
he
same
sequence
bu
lacked
bio in.
The
digoxigenin-labeled
analy e
was
complemen a y
o
he
p obe
(digoxigenin-5-ACGGCAGACTTCTCCACAGGAGTCAGGTGC-
3)
and
he
“unlabeled
analy e”
had
he
same
sequence
bu
lacked
digoxigenin
[5].
Bio in-N19T
con ained
1:1:1:1
mix-
u es
o
A:C:G:T
in
posi ions
1–19
and
hymine
in
posi-
ion
20.
Phospha e-bu e ed
saline
(PBS
7.3)
con ained
140
mM
NaCl,
2.7
mM
KCl,
10
mM
Na2HPO4,
and
1.8
mM
KH2PO4,
yielding
pH
7.3.
I
was
degassed
by
s e ile
fil a ion
(0.2
␮m)
wi h
s ong
aspi a o
suc ion
e e y
day.
Bio in–BSA,
Bio in–IgG,
BSA,
IgG,
and
lysozyme
we e
pu ified
by
gel
fil a ion
in
PBS
7.3
on
Supe dex
200
(1
×
30
cm,
GE
Heal hca e)
a
0.5
ml/min
o
emo e
agg ega es.
The
p o ein
concen a ions
we e
adjus ed
o
1
mg/ml
and
small
aliquo s
we e
ozen
in
liq-
uid
ni ogen
and
s o ed
a
−25 ◦C.
Bio in–p o ein
G,
IgG2␬
and
he
oligonucleo ides
we e
dissol ed
in
PBS
(20
␮M,
7
␮M,
and
10
␮M,
espec i ely)
and
s o ed
a
−25 ◦C.
The
aliquo s
we e
hawed
by
sho
imme sion
in
wa e
(∼20 ◦C),
dilu ed
o
he
desi ed
concen-
a ion
by
addi ion
o
PBS
7.3,
s o ed
a
4◦C,
and
used
wi hin
ou
days.
2.2.
Su ace
plasmon
esonance
expe imen s
Cleaning
o
ba e
glass
chips,
e apo a ion
o
ch omium
(3
nm)
and
gold
(41
nm),
as
well
as
cleaning
o
he
gold
su ace
and
coa -
ing
wi h
a
mixed
bio in
SAM
(Fig.
S2)
was
pe o med
as
desc ibed
[6].
The
chips
we e
moun ed
on
he
chip
suppo s
wi h
double-
sided
adhesi e
ape
(non-pe manen )
and
inse ed
in
a
BIAco e
X
de ice
o
measu emen
o
binding
by
su ace
plasmon
esonance
(SPR).
Degassed
bu e
(PBS
7.3)
was
un
o e
he
chip
su ace
a
10–20
␮l/min,
as
s a ed
in
he
figu e
legends.
The
esonance
angle
was
eco ded
a
1
s
in e als
in
bo h
flow
cells
and
exp essed
in
esonance
uni s
(1
RU
=
0.0001◦).
3.
Resul s
and
discussion
Fig.
1a
shows
he
wo
kinds
o
chip
egene a ion
used
in
his
s udy.
“No mal
egene a ion”
means
quan i a i e
emo al
o
p ey
molecules
only,
as
con en ionally
pe o med
on
all
kinds
o
biosen-
so s
[1].
“Rigo ous
egene a ion”
means
quan i a i e
dissocia ion
o
he
bio in–a idin–bio in
b idges
on
he
chip
su ace,
he eby
eco e ing
he
ba e
bio inyla ed
chip
on
which
esh
a idin
and
a
new
kind
bio inyla ed
bai
is
immobilized
o
a
new
se ies
o
expe imen s.
In
his
s udy,
we
examined
he
ep oducibili y
o
chip
egene a ion
(Sec ion
3.1),
nonspecific
binding
o
p o ein
(Sec ion
3.2)
and
nucleic
acids
(Sec ion
3.6),
mul i alen
bai –p ey
in e -
ac ions
(Sec ion
3.3),
as
well
as
pe ec
di e en ia ion
be ween
sample
cell
and
e e ence
cell
(Sec ion
3.5).
O
pa icula
in e -
es
was
he
sui abili y
o
me hods
o
quan i a i e
emo al
o
p ey
(“no mal
egene a ion”)
wi hou
losing
bio inyla ed
bai
molecules
om
he
chip
su ace
(Sec ion
3.4).
3.1.
Binding
o
bio inyla ed
p o ein
o
bio inyla ed
su aces
by
using
di e en
a idin
mu an s
In
p eceding
s udies
[6,7],
h ee
di e en
a idin
mu an s
we e
shown
o
p o ide
o
e e sible
o ma ion
o
bio in–a idin–bio in
b idges,
as
ou lined
in
Fig.
1a.
Re e sibili y
is
made
possible
by
mu a ion
M96H
which
is
loca ed
a
he
subuni
in e ace
(Fig.
S1),
causing
subuni
dissocia ion
when
ea ed
wi h
SDS/ci ic
acid
[6].
Mu an
#2
con ains
he
addi ional
mu a ion
R114L
which
low-
e s
he
pI
alue,
educes
nonspecific
binding,
and
enhances
he
a fini y
o
bio inyla ed
molecules
[7],
due
o
i s
loca ion
nex
o
he
bio in-binding
si e
(Fig.
S1).
The
h ee
addi ional
mu a ions
in
mu an
#3
a e
loca ed
on
he
ou e
su ace
o
a idin
(Fig.
S1,
Table
S1)
and
shi ed
he
pI
alue
owa ds
7
(Fig.
1e),
esul -
ing
in
e y
low
nonspecific
adso p ion
o
p o eins
and
nucleic
acids
[7].
In
he
p esen
s udy,
he
newly
p epa ed
a idin
mu an
#4
was
analogous
o
mu an
#3,
excep
ha
mu a ion
R114L
was
eplaced
by
mu a ion
R26N
which
had
li le
e ec
on
he
pI
alue
(Fig.
1e).
The
in en ion
was
o
demons a e
he
beneficial
e ec
o
mu a-
ion
R114L
o
he
s abili y
o
he
bio in-bound
s a e,
and
his
was
confi med
by
he
da a
(see
below).
Finally,
mu an
#5
con ained
all
mu a ions
a
he
same
ime,
causing
u he
lowe ing
o
he
pI
alue
(Fig.
1e,
Table
S1).
Mu an s
#1–5
we e
sys ema ically
es ed
o
hei
pe o mance
in
e e sible
biosenso
unc ionaliza ion.
The
fi s
es
conce ned
binding
o
he
a idin
mu an s
o
he
bio inyla ed
chip
and
o
bio in–IgG
on
op
o
a idin
(s ages
2
and
3
in
Fig.
1b),
as
well
as
emo al
o
he
bound
p o eins
wi h
a
mix u e
o
SDS
and
ci -
ic
acid
(s age
4
in
Fig.
1b).
As
exemplified
in
Fig.
1c,
he
a idin
mu an
was
injec ed
in
bo h
flow
cells
and
bio in–IgG
in
FC2
only.
The
expe imen s
we e
pe o med
in
iplica es
wi h
all
fi e
a idin
mu an s
and
he
signal
ampli udes
we e
highly
ep oducible
(Fig.
1d).
The
ex en
o
mu an
binding
(s age
2
minus
s age
1)
was
simila
o
mu an s
#1–4
(∼2000
RU,
ci cles
in
Fig.
1d),
only
mu an
#5
was
less
e ec i e.
Binding
o
bio in–IgG
(s age
3
minus
s age
2)
showed
a
significan
dec ease
wi h
inc eased
mu an
numbe
( iangles
in
Fig.
1d).
All
mu an s
allowed
o
good
e e sibili y
o
binding
(s age
4
minus
s age
1,
squa es
in
Fig.
1d).
Mu an
#3
pe o med
bes
in
his
espec ,
wi h
a
d i
o
13
±
18
RU
in
FC1
and
13
±
14
RU
in
FC2.
I
is
impo an
o
no e
ha
all
es ed
chips
could
be
egene a ed
o
an
unlimi ed
numbe
o
cycles,
excep
ha
he
binding
capac-
i y
o
he
chips
s a ed
o
dec ease
a e
h ee
weeks
o
con inued
use.
3.2.
Nonspecific
adso p ion
o
p o ein
on
monolaye s
o
di e en
a idin
mu an s
Sensi i e
and
selec i e
biosensing
implies
ha
no
bind-
ing/adso p ion
o
any
componen
o
he
sample
occu s
on
he
chip
su ace,
excep
o
specific
cap u e
o
p ey
o
bai .
Fig.
2a
exempli-
fies
he
s anda d
es
[6]
which
consis s
o
consecu i e
injec ions
o
lysozyme,
BSA,
and
goa
IgG.
The
esul s
o
BSA
and
IgG
a e
summa ized
in
Fig.
2b.
Mu an
#3
showed
he
lowes
adso p ion
o
BSA
(ci cles)
and
IgG
( ian-
D.
Zaune
e
al.
/
Senso s
and
Ac ua o s
B
229
(2016)
646–654
649
Fig.
2.
Non-specific
adso p ion
o
p o eins
on
monolaye s
o
he
a idin
mu an s
#1–5.
(a)
Tes
o
p o ein
adso p ion
o
mu an
#2.
(b)
The
expe imen
in
(a)
was
pe o med
wi h
mu an s
#1–5.
The
amoun s
o
p o ein
which
emained
bound
a
he
end
o
he
injec ions
o
BSA
and
goa
IgG
a e
shown.
“BSA
injec ion
2”
and
“IgG
a e
BSA”
o
mu an s
#1–3
we e
epo ed
be o e
[7].
Fig.
3.
Kine ics
o
human
IgG2␬
binding
o
bio in–p o ein
G
on
op
o
mu an
#3.
(a)
Schema ic
o
binding
o
immobilized
p o ein
G
in
FC1
( ed
ace
in
(c)).
(b)
Schema ic
o
he
con ol
injec ion
in
FC2
(blue
ace
in
(c)).
(c)
Repea ed
associa ion
and
dissocia ion
o
IgG2␬,
using
100
mM
glycine
(pH
2.7)
o
emo al
o
IgG2␬.
The
di e en
IgG2␬
samples
we e
p epa ed
by
se ial
dilu ion
o
7
␮M
IgG2␬
(in
PBS
7.3)
wi h
sample
bu e
(1
␮M
BSA
in
PBS
7.3).
Double
e e encing
[6,14]
was
used
o
ob ain
he
expe imen al
binding
cu es
(solid
aces
in
(d)
and
(e)).
(d)
The
do ed
lines
show
he
bes
global
fi
o
he
Langmui
model
o
he
expe imen al
binding
cu es
(solid
lines).
(e)
Analogous
fi
as
in
(d),
using
he
“bi alen
analy e
model”.
(Fo
in e p e a ion
o
he
e e ences
o
colo
in
his
figu e
legend,
he
eade
is
e e ed
o
he
web
e sion
o
his
a icle.)
gles).
The
da a
se
“IgG
a e
BSA”
is
mo e
ele an
han
“only
IgG
in
FC1”
because
BSA
is
o en
used
o
passi a e
he
chip
su ace
be o e/du ing
injec ion
o
samples
(see
Fig.
3).
On
mu an
#3,
bind-
ing
o
IgG
amoun ed
o
11
±
5
RU
a e
BSA
and
27
±
6
RU
wi hou
BSA
ea men ,
co esponding
o
0.4%
o
1%
o
an
IgG
monolaye ,
espec i ely
[12].
Only
a
small
amoun
o
lysozyme
was
adso bed
on
any
a idin
mu an
(Fig.
S3),
due
o
he
high
pI
alue
o
lysozyme
(11.2
[13]).
3.3.
Biological
in e ac ion
analysis
on
a idin
mu an - unc ionalized
chip
su aces
In
a
p eceding
s udy
[6]
we
showed
ha
bio inyla ed
p o ein
G
and
human
IgG2␬
p o ide
o
a
c i ical
unc ional
es
o
chip
pe -
o mance,
o
wo
easons:
(i)
he
in e ac ion
is
mul i alen
in
he
sense
ha
one
soluble
IgG
molecule
is
cap u ed
by
wo
bio inyla ed
p o ein
G
molecules
on
he
chip
su ace
(Fig.
3a).
(ii)
In
his
case,
he
epea ed
emo al
o
IgG
a e
each
IgG
injec ion
(Fig.
3c)
is
ypically
pe o med
wi h
100
mM
glycine
bu e
(pH
2.7,
GE
Heal hca e
Da a
File
18-1012-91
AC,
en i led
“A fini y
Ch oma og aphy:
P o ein
G
Sepha ose
4
Fas
Flow”,
a ailable
a
he
in e ne
om
h ps://www.
geli esciences.com/gehcls
images/GELS/Rela ed%20Con en /Files/
1314774443672/li doc18101291AC
20110831095008.pd )
which
seems
dange ously
close
o
“ igo ous
egene a ion”
(Fig.
1a)
wi h
SDS/ci ic
acid.
In
Re .
[6]
i
was
shown
ha
he
bio in–a idin–bio in
b idges
o med
by
mu an
#1
we e
only
a ec ed
by
SDS/ci ic
acid
(pH
2.0)
bu
no
by
glycine
(pH
2.7).
We
now
show
ha
he
same
is
also
ue
o
mu an s
#2
(Fig.
S4c)
and
#3
(Fig.
3c).
The
a idin
mu an
unde
inspec ion
was
immobilized
in
bo h
flow
cells,
FC2
650
D.
Zaune
e
al.
/
Senso s
and
Ac ua o s
B
229
(2016)
646–654
Fig.
4.
Tes
o
specific
hyb idiza ion
and
non-specific
adso p ion
o
single-s anded
DNA
on
mu an
#5.
(a)
Schema ic
o
hyb idiza ion
o
unlabeled
analy e
DNA,
as
used
in
(c).
(b)
Analogous
schema ic
wi h
digoxigenin-labeled
analy e
DNA.
(c)
Hyb idiza ion
o
unlabeled
analy e
DNA
wi h
complemen a y
bio in-DNA
which
had
been
immobilized
on
mu an
#5
in
FC2
only
(blue
ace).
(d)
Segmen
om
(c).
(e)
Analogous
expe imen
as
in
(d),
using
digoxigenin-labeled
analy e
(compa e
(a)
and
(b)).
(Fo
in e p e a ion
o
he
e e ences
o
colo
in
his
figu e
legend,
he
eade
is
e e ed
o
he
web
e sion
o
his
a icle.)
was
blocked
wi h
bio in–BSA
(Fig.
3b)
and
FC1
was
unc ionalized
wi h
bio in–p o ein
G
(Fig.
3a).
A e
u he
passi a ion
o
bo h
flow
cells
wi h
bio in–BSA
and
BSA,
di e en
concen a ions
o
IgG2␬
we e
injec ed
and
epea edly
emo ed
wi h
glycine
(pH
2.7),
as
shown
in
Fig.
3c.
FC2
was
sub ac ed
om
FC1
and
he
esul ing
ace
o
sample
bu e
injec ion
(Fig.
3c)
was
sub ac ed
om
all
o he
injec ion
aces
(“double
e e encing
me hod”
[14]),
esul -
ing
in
he
expe imen al
binding
cu es
(solid
aces
in
panels
(d)
and
(e),
Figs.
3
and
S4).
The
kine ic
da a
could
well
be
fi ed
by
he
“bi alen
analy e
model”
(Figs.
3
e
and
S4e,
do ed
lines),
which
assumes
binding
o
each
IgG
molecule
by
wo
adjacen
p o ein
G
molecules
on
he
chip
su ace
(Fig.
3a),
in
con as
o
he
simple
Langmui
model
(Figs.
3
d
and
S4d,
do ed
lines)
which
assumes
1:1
binding.
The
use ulness
o
all
h ee
a idin
mu an s
(#1–3)
o
biological
in e ac ion
analysis
is
p o en
by
he
good
ag eemen
o
he
cal-
cula ed
kine ic
cons an s
(Table
1),
yielding
a e ages
wi h
small
s anda d
de ia ions
(las
column).
In
spi e
o
he
less
pe ec
fi ,
he
Langmui
model
has
he
ad an age
ha
i
yields
an
e ec i e
Kd
alue
wi h
he
usual
dimension
“nM”
(no
“RU”,
as
in
he
bi alen
analy e
model,
see
Table
1),
allowing
o
compa ison
wi h
li e -
a u e
da a.
Kd=
710
nM
was
epo ed
om
a fini y
adso p ion
o
mixed
human
IgG
[15].
The
Fc
agmen
o
human
IgG1
ga e
Kd
alues
o
47
nM
in
BIAco e
expe imen s
on
a
CM5
chip
[16]
and
310
nM
in
a
homogeneous
fluo escence
assay
[17].
The
disc epancy
wi h
Kd=
2
nM
in
Table
1
is
in
pa
explained
by
he
ac
ha
bi a-
len
in e ac ion
was
only
possible
on
ou
dense
a idin
monolaye s
which
allow
o
close
p oximi y
o
immobilized
bio in–p o ein
G
(Fig.
3a).
Ob iously
ou
chip
is
able
o
mimic
he
na u al
unc ion
o
p o ein
G,
which
is
also
p esen
a
high
la e al
densi y
on
he
su ace
o
S ep ococcus
sp.
[18].
Table
1
Fi
pa ame e s
o
he
Langmui
model
(do ed
cu es
in
Fig.
3d)
and
o
he
bi a-
len
analy e
model
(do ed
cu es
in
Fig.
3e)
by
which
binding
o
human
IgG2␬
owa ds
immobilized
bio in–p o ein
G
(Fig.
3a)
was
analyzed
on
op
o
mu an
#3.
The
co esponding
da a
o
mu an
#2
a e
om
Fig.
S4,
hose
o
mu an
#1
om
Re .
[6].
Mu an
#1
#2
#3
#1–3
Langmui
model
10−5kaM
s
4.1
4.1
4.2
4.1
±
0.1
104kds
11
7.4
8.4
9.0
±
1.9
KD/nM
2.7
1.8
2.0
2.2
±
0.5
Rmax/RU
1370
1360
1290
1340
±
44
Bi al.
analy e
model
10−5ka1 M
s
1.8
1.6
1.8
1.7
±
0.1
104kd1 s
17
14
14
15
±
0.3
KD1/nM
9.4
8.8
7.5
8.6
±
0.9
104ka2 RU
s
1.7
2.7
3.1
2.5
±
0.7
kd2 s
0.013
0.013
0.017
0.014
±
0.003
KD2/RU
77
47
56
60
±
15
Rmax/RU
1800
1930
1740
1820
±
100
3.4.
Sensi i i y
o
a idin
mu an - unc ionalized
chips
o
acid
o
SDS
As
men ioned
abo e,
he
selec i e
emo al
o
IgG
om
p o ein
G
by
100
mM
glycine
(pH
2.7)
in
Fig.
3c
seems
a he
close
o
he
SDS/ci ic
acid
mix u e
(pH
2.0,
Fig.
S5)
used
o
emo al
o
all
p o eins
a
he
end
o
Fig.
3c.
A
close
look
a
he
fi s
IgG2␬
injec-
ion
indeed
shows
ha
he
subsequen
injec ion
o
glycine
(pH
2.7)
leads
o
a
lowe
baseline
(−188
RU)
han
be o e
injec ion
o
200
nM
IgG2␬.
P obably
a
small
ac ion
o
mu an
#3
is
mo e
sensi i e
o
pH
2.7
han
he
es ,
being
bound
o
only
one
bio in
esidue
on

D.
Zaune
e
al.
/
Senso s
and
Ac ua o s
B
229
(2016)
646–654
651
he
bio in–SAM
[19].
Howe e ,
such
baseline
d i
was
no
seen
in
he
subsequen
cycles
wi h
lowe
concen a ions
o
IgG2␬.
Thus,
such
a
baseline
shi
du ing
he
bai –p ey
in e ac ion
s udy
can
easily
be
a oided
when
applying
a
dummy
injec ion
o
glycine
(pH
2.7)
be o e
he
fi s
injec ion
o
IgG2␬.
The
same
obse a ions
we e
made
wi h
mu an s
#1
[6]
and
#2
(Fig.
S4c).
Un o una ely,
pH
2.7
is
insu ficien
o
emo al
o
an igens
om
immobilized
an ibodies
(o
ice
e sa).
Repo ed
egene a ion
con-
di ions
ange
om
pH
2.5
o
pH
1.75
[1].
We,
he e o e,
examined
he
esis ance
o
he
chip-bound
a idin
mu an s
#1–5
o
low
pH,
using
2.5%
ci ic
acid
wi hou
sal
(pH
2.0)
o
wi h
150
mM
NaCl
(pH
1.9,
Fig.
S5).
The
esul s
a e
shown
in
Figs.
S6–S9.
A
pH
2,
only
mu an
#1
showed
s able
binding
o
he
bio inyla ed
chip
( ed
squa es
in
Fig.
S7b).
Fo una ely,
he
sensi i i y
o
pH
2
was
only
seen
i
he
a idin
mu an s
we e
he
only
p o ein
on
he
chip
su -
ace
(s a e
2
in
Fig.
1b).
I
bio inyla ed
an ibody
was
bound
on
op
o
a idin
(s a e
3
in
Fig.
1b)
hen
all
fi e
a idin
mu an s
we e
esis an
o
pH
2
(blue
ci cles
in
Fig.
S7b).
In
spi e
o
i s
lowe
pH
(1.9,
Fig.
S5),
he
combina ion
o
ci ic
acid
wi h
150
mM
NaCl
caused
much
less
emo al
o
he
a idin
mu an s
om
he
bio inyla ed
chip
(Figs.
S6–S9).
A
simila
beneficial
e ec
o
ele a ed
ionic
s eng h
is
also
expec ed
o
he
100
mM
glycine
bu e s
ypically
used
o
an ibody–an igen
sepa a ion.
De e gen s
a e
also
used
o
emo al
o
p ey
om
bai
[1].
The e-
o e,
we
es ed
he
esis ance
o
mu an s
#1–5
o
0.5%
SDS
a
neu al
pH
(Figs.
S10
and
S11).
Mu an
#1
was
e y
s able,
while
mu an
#3
showed
losses
o
∼5%
(i espec i e
o
whe he
bio in–IgG
was
bound
on
op
o
he
a idin
laye
o
no ).
Inclusion
o
NaCl
a o ded
inc eased
esis ance
o
SDS.
The
epo ed
expe imen s
wi h
ci ic
acid,
NaCl,
and
SDS
esul ed
in
he
ollowing
ules
o
“no mal
egene a ion”
(Fig.
1a)
o
chips
unc ionalized
wi h
he
es ed
a idin
mu an s:
i.
The
egene a ion
bu e
(pH
1.9
o
SDS)
should
ha e
physiolog-
ical
ionic
s eng h
(e.g.,
150
mM
NaCl)
because
hen
much
less
a idin
mu an
(and
bio inyla ed
bai )
is
dissocia ed
om
he
chip.
ii.
Mu an
#3
is
mos
a ac i e
because
o
low
nonspecific
adso p-
ion,
bu
his
mu an
will
esis
pH
1.9
only
i
i
is
c osslinked
by
a
mul iply
bio inyla ed
p o ein
(ske ch
(d)
in
Fig.
S7).
P o-
nounced
s abiliza ion
o
a idin
by
such
c osslinking
has
been
demons a ed
be o e
[6].
This
s abiliza ion
is
high
in
case
o
IgG
ca ying
6–7
bio in
esidues
on
a e age
(blue
ci cles
in
Fig.
S7b).
iii.
The
ex en
o
c osslinking,
and
o
s abiliza ion
a
pH
1.9,
will
be
much
weake
in
case
o
small
p o eins
wi h
ew
bio in
esidues
and
no
s abiliza ion
is
expec ed
o
bai s
wi h
only
one
bio in
esidue.
The
la e
si ua ion
is
equi alen
o
a
simple
a idin
monolaye
(ske ch
(c)
in
Fig.
S7).
He e,
only
mu an
#1
is
su fi-
cien ly
esis an
o
pH
1.9
( ed
squa es
in
Fig.
S7b).
Fo una ely,
i
is
possible
o
elimina e
nonspecific
adso p ion
o
p o ein
on
mu an
#1
[6].
In
case
o
DNA,
howe e ,
i
is
necessa y
o
use
mu an
#3
and
o he
me hods
han
pH
1.9
o
“no mal
egen-
e a ion”
(see
Sec ion
3.6).
i .
A
small
loss
o
a idin
plus
bio inyla ed
bai
will
equen ly
be
expec ed
du ing
he
fi s
egene a ion
ound,
e en
wi h
milde
egene a ion
a
pH
2.7
(Fig.
3).
Fo una ely,
no
such
loss
is
seen
in
subsequen
injec ions.
The e o e,
a
dummy
injec ion
wi h
egene a ion
bu e
should
be
pe o med
be o e
epea ed
binding
and
dissocia ion
o
p ey
molecules.
Ini ial
emo al
o
he
weekly
bound
a idin
molecules
has
no
ad e se
e ec s
on
subsequen
measu emen
cycles
o
bai –p ey
in e ac-
ion.
In
conclusion,
he
choice
o
egene a ion
condi ions
and
he
choice
o
mu an
#1
e sus
#3
mus
be
made
wi h
ca e,
in
o de
o
exploi
he
ull
po en ial
o
hese
mu an s
o
egene a i e
biosens-
ing.
3.5.
How
o
p e en
bai
molecule
immobiliza ion
in
he
e e ence
cell
Label- ee
biosensing
equi es
injec ion
o
he
sample
in
he
ac i e
cell
wi h
immobilized
bai
molecule
and
in
a
e e ence
cell
lacking
he
bai
molecules
(see
Fig.
3a,b).
Fig.
3c
(and
Fig.
S12c)
show
ha
he
bai
(bio in–p o ein
G)
was
only
p esen
in
he
sample
cell
i
he
e e ence
cell
was
unc ionalized
wi h
bio in–BSA
p io
o
injec ion
o
bio in–p o ein
G
o
he
ac i e
cell.
In
con as ,
p o-
nounced
con amina ion
o
he
e e ence
cell
wi h
bio in–p o ein
G
was
obse ed
i
p o ein
G
was
injec ed
be o e
bio in–BSA
(Fig.
S12b).
The
eason
lies
in
he
design
o
mic ofluidic
flow
cells
which
allows
o
di usion
be ween
he
cells
e en
i
he
flow
is
blocked
in
one
cell
(see
Fig.
S12a).
Fig.
4c
shows
ano he
success ul
s a egy
by
which
bai
immobi-
liza ion
is
es ic ed
o
he
ac i e
cell.
Mu an
#5
was
fi s
injec ed
in o
he
ac i e
cell
only
(FC2,
blue
ace
in
Fig.
4c),
ollowed
by
injec-
ion
o
bio inyla ed
bai
(bio in-p obe).
In
his
si ua ion,
mu an
#5
is
s ill
absen
in
he
e e ence
cell
(FC1,
ed
ace)
and
no
bio in-
DNA
can
be
immobilized
in
FC1,
e en
i
a
ace
o
i
di uses
in o
FC1.
Subsequen ly,
mu an
#5
was
injec ed
in o
he
e e ence
cell
(FC1,
ed
ace),
gene a ing
an
ine
su ace
whe e
no
DNA
was
bound
in
he
nex
s eps.
The
p o ocol
in
Fig.
4c
always
ensu es
es ic ion
o
bai
o
he
ac i e
flow
cell.
The
simple
p o ocol
in
Fig.
3c
is
only
applicable
o
la ge
bai s
(such
as
p o eins)
which
canno
bind
on
a idin
a e
injec ion
o
bio in–BSA.
3.6.
Specific
and
nonspecific
binding
o
DNA
on
a idin-mu an - unc ionalized
chip
su aces
In
a
p eceding
s udy
[7],
mu an s
#1
and
#2
exhibi ed
high
nonspecific
adso p ion
o
DNA,
whe eas
only
specific
binding
(hyb idiza ion,
Fig.
4a)
was
seen
on
mu an
#3.
The
same
expe i-
men
was
now
pe o med
on
mu an s
#4
(Fig.
S15)
and
#5
(Fig.
4c).
The
sample
cell
was
unc ionalized
wi h
a idin
and
bio inyla ed
p obe
DNA,
ollowed
by
a idin
binding
in
he
e e ence
cell.
Subse-
quen ly,
absence
o
nonspecific
p o ein
adso p ion
was
seen
wi h
BSA
and
absence
o
nonspecific
DNA
adso p ion
when
injec ing
unlabeled
p obe
DNA
which
had
same
nucleo ide
sequence
as
he
bio inyla ed
DNA
on
he
chip
su ace
(Fig.
4c).
Subsequen
injec-
ion
o
unlabeled
analy e
DNA
ga e
p onounced
hyb idiza ion
in
he
sample
cell
(blue
aces
in
Fig.
4c
and
d)
and
no
nonspe-
cific
esponse
in
he
e e ence
cell
( ed
aces
in
Fig.
4c
and
d).
Howe e ,
when
he
expe imen
was
epea ed
using
digoxigenin-
labeled
DNA
(Fig.
4b)
in
place
o
unlabeled
DNA
(Fig.
4a),
p onounced
binding
was
obse ed
in
he
e e ence
cell
( ed
ace
in
Fig.
4e).
No
such
e ec
was
seen
wi h
mu an
#4
(Fig.
S15)
and
mu an s
#1–3
(see
below)
e en
when
using
digoxigenin-labeled
DNA.
Fig.
5
compa es
mu an s
#1–4
wi h
espec
o
specific
bind-
ing
(hyb idiza ion)
and
nonspecific
adso p ion.
The
op
ow
(a)–(c)
conce ns
expe imen s
wi h
mu an
#1.
Panel
(a)
shows
p onounced
binding
o
analy e
DNA,
bo h
in
he
ac i e
cell
which
con ained
he
complemen a y
bio inyla ed
p obe
DNA
(blue
ace),
and
in
he
e e ence
cell
wi h
he
ba e
monolaye
o
mu an
#1
( ed
ace).
We
suspec ed
ha
he
con ibu ion
o
nonspecific
DNA
adso p ion
in
he
ac i e
cell
(blue
ace)
was
much
lowe
han
he
pu ely
nonspecific
signal
in
he
e e ence
cell
( ed
ace),
because
he
posi i ely
cha ged
mu an
#1
was
co e ed
wi h
nega i ely
cha ged
bio in-DNA
in
he
ac i e
cell
bu
no
in
he
e e ence
cell.
This
hypo hesis
was
e ified
in
panel
(b)
whe e
he
e e -
ence
cell
con ained
a
degene a ed
oligonucleo ide
(bio in-N19T),
652
D.
Zaune
e
al.
/
Senso s
and
Ac ua o s
B
229
(2016)
646–654
Fig.
5.
DNA
hyb idiza ion
expe imen s
pe o med
on
monolaye s
o
mu an
#1
(panels
a–c),
and
o
mu an s
#2,
#3,
and
#4
(panels
d– ),
using
he
p o ocol
o
Fig.
4c.
Red
aces
show
adso p ion
o
analy e
DNA
o
he
a idin
mu an s
in
FC1,
blue
aces
he
sum
o
specific
and
nonspecific
binding
in
FC2
o
a idin
ca ying
bio in-labeled
p obe
DNA.
The
g een
aces
in
(g),
(h),
and
(i)
we e
calcula ed
by
sub ac ion
o
he
ed
aces
om
he
blue
aces
in
(d),
(e),
and
( ),
espec i ely.
The
analy e
DNA
always
ca ied
a
digoxigenin
label,
excep
in
(c).
In
(b)
and
(c)
bo h
flow
cells
we e
ea ed
wi h
bio in-N19T
be o e
injec ion
o
analy e
DNA.
(Fo
in e p e a ion
o
he
e e ences
o
colo
in
his
figu e
legend,
he
eade
is
e e ed
o
he
web
e sion
o
his
a icle.)
while
he
ac i e
cell
con ained
bio inyla ed
p obe
DNA
plus
sub-
sequen ly
injec ed
bio in-N19T.
This
esul ed
in
low
nonspecific
binding
no
only
in
he
e e ence
cell
( ed
ace)
bu
also
in
he
ac i e
cell
(blue
ace).
Panel
(c)
confi ms
he
beneficial
e ec
o
bio in-N19T
and
a
e y
mino
con ibu ion
o
he
digoxigenin
label
o
nonspecific
adso p ion
o
digoxigenin-labeled
DNA
o
mu an
#1.
The
cen e
ow
(d)–( )
shows
he
pe o mance
o
mu an s
#2–4
unde
he
same
condi ions
as
used
o
mu an
#1
in
panel
(a)
(i.e.,
wi h
digoxigenin-labeled
DNA,
in
absence
o
bio in-N19T).
Nonspe-
cific
binding
o
DNA
on
mu an
#2
( ed
ace
in
(d))
was
only
sligh ly
lowe
han
on
mu an
#1.
Nonspecific
DNA
adso p ion
was
absen
on
mu an s
#3
and
#4
( ed
aces
in
(e)
and
( )).
The
small
an-
sien
displacemen
o
he
ed
ace
in
panel
( )
is
due
o
a
bulk
e ec
(di e en
e ac o y
index
o
sample
and
unning
bu e ).
The
find-
ings
a e
well
explained
by
he
high
pI
alues
o
mu an s
#1
and
#2
(Fig.
1e),
which
implies
a
posi i e
ne
cha ge,
and
by
he
neu al
pI
o
mu an s
#3
and
4,
which
elimina es
elec os a ic
a ac ion
o
DNA.
The
bo om
ow
(g)–(i)
shows
he
ex en
o
specific
binding
(hyb idiza ion),
as
calcula ed
by
sub ac ion
o
nonspecific
binding
in
he
e e ence
cell
( ed
aces
in
(d)–( ))
om
o al
DNA
binding
in
he
ac i e
cell
(blue
aces
in
(d)–( )).
The
biphasic
cu e
o
mu an
#2
(panel
(g))
is
caused
by
much
highe
nonspecific
adso p ion
in
he
e e ence
cell
han
in
he
ac i e
cell
(as
explained
abo e
o
mu an
#1).
In
con as ,
panels
(h)
and
(i)
eflec
he
ue
hyb idiza-
ion
signals
on
mu an s
#3
and
#4,
due
o
absence
o
nonspecific
adso p ion
on
bo h
mu an s
( ed
aces
in
(e)
and
( )).
Mu an
#3
exhibi s
a
highe
binding
capaci y
han
mu an
#4,
he e-
o e
i
appea s
bes
sui ed
o
measu emen
o
DNA-con aining
samples.
In
his
s udy
we
p o ide
no
me hod
o
he
“no mal
egen-
e a ion”
(Fig.
1a)
o
DNA- unc ionalized
chips,
i.e.,
o
comple e
dissocia ion
o
analy e
DNA
wi hou
loss
o
bio inyla ed
DNA.
Mu an
#3
is
no
su ficien ly
s able
o
use
100
mM
HCl
(as
used
in
he
“Bio in
CAP u e
Ki ”)
and
he
mo e
acid- esis an
mu an
#1
s ongly
adso bs
DNA.
The
mos
p omising
eagen
seems
con-
cen a ed
u ea;
i
ensu es
comple e
dissocia ion
o
DNA
duplexes
[20],
while
a idin
is
known
o
esis
9
M
u ea
wi hou
losing
i s
bio in-binding
capaci y
[21].
Howe e ,
es ablishing
he
exac
con-
di ions
will
equi e
a
subs an ial
amoun
o
expe imen s
in
a
u u e
s udy.
4.
Conclusions
All
fi e
es ed
a idin
mu an s
a e
sui able
o
e e sible
immo-
biliza ion
o
bio inyla ed
bai s
on
bio inyla ed
senso
chips.
The
s ably
o med
bio in–a idin–bio in
b idges
can
be
quan i a i ely
dissol ed
when
desi ed.
Mu an
#1
showed
he
highes
s abili y
a
pH
2
and
he
highes
binding
capaci y
o
bio inyla ed
bai
molecules.
Nonspecific
bind-
ing
o
p o eins
was
mode a e,
especially
when
bio in–BSA
was
used
o
passi a e
he
e e ence
cell
and
he
unoccupied
bio in-binding
si es
in
he
ac i e
cell.
The
majo
weakness
o
mu an
#1
was
high
D.
Zaune
e
al.
/
Senso s
and
Ac ua o s
B
229
(2016)
646–654
653
nonspecific
binding
o
nucleic
acids.
Passi a ion
wi h
degene a e
bio in-N19T
caused
a
s ong
educ ion
o
DNA
adso p ion
bu
did
no
elimina e
i
comple ely.
In
conclusion,
mu an
#1
may
be
ideal
o
biological
in e ac ion
analysis
be ween
pu ified
p o eins,
bu
i
appea s
unsui able
o
biosensing
whe e
nucleic
acids
a e
likely
o
be
p esen .
Mu an
#2
esembles
mu an
#1
in
all
espec s,
excep
ha
all
posi i e
and
nega i e
aspec s
a e
somewha
mode-
a ed.
Mu an
#3
showed
sligh ly
lowe
binding
capaci y
and
s a-
bili y
a
pH
2,
ne e heless
i
can
well
be
used
o
in e ac ion
s udies
be ween
an ibodies
and
an igens
whe e
pH
2.3
is
ypi-
cally
used
o
epea ed
emo al
o
he
analy e.
The
eason
is
ha
s a is ically
bio inyla ed
an ibodies
o
an igens
cause
c osslinking
o
mu an
#3,
which
con e s
high
s abili y
down
o
pH
2.
This
ac
is
impo an
because
mu an
#3
appea ed
ideal
in
all
o he
aspec s:
i
showed
he
lowes
nonspecific
binding
o
p o ein
and
DNA
and
he
highes
pe o mance
in
chip
ecycling
wi h
SDS/ci ic
acid.
Mu an s
#4
and
#5
exhibi ed
also
low
nonspecific
binding
o
p o ein
and
DNA.
Howe e ,
hey
we e
no
s able
a
pH
2
and
hei
binding
capaci y
o
bio inyla ed
bai
molecules
was
conside ably
lowe
han
ha
o
mu an
#3.
In
conclusion,
mu an
#3
showed
he
bes
o e all
pe o mance
in
chip
ecycling
and
applica ion
o
ecycled
chips
in
biosensing.
Acknowledgemen s
This
wo k
was
suppo ed
by
he
go e nmen
o
Uppe
Aus ia
(p ojec
DK
Nanocell)
and
by
g an s
om
he
Academy
o
Finland
(p ojec
numbe s
136288
and
140978
o
V.P.H.).
We
acknowl-
edge
he
in as uc u e
suppo
by
Biocen e
Finland.
We
hank
Sand a
Posch
and
Felix
Faschinge
o
help ul
commen s
on
he
manusc ip .
Appendix
A.
Supplemen a y
da a
Supplemen a y
da a
associa ed
wi h
his
a icle
can
be
ound,
in
he
online
e sion,
a
h p://dx.doi.o g/10.1016/j.snb.2016.02.039
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S ay on,
C.T.
Campbell,
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(2000)
9421–9432.
[20]
Sou he n,
E.M.
2002.
Dena u ing
gel
elec opho esis
o
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u ea-polyac ylamide
gel.
In:
eLS.
John
Wiley
&
Sons
L d.,
Chiches e .
h p://
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[doi:
10.1038/npg.
els.0003778].
[21]
N.M.
G een,
Biochem.
J.
89
(1963)
609–620.
Biog aphies
Dominik
Zaune
ecei ed
his
M.Sc.
deg ee
om
he
Ins i u e
o
O ganic
Chemis y
a
Johannes
Keple
Uni e si y
Linz,
Linz,
Aus ia,
in
2015.
He
did
his
mas e
hesis
as
gues
o
P o .
He mann
J.
G ube
(see
below)
whe e
he
syn hesized
new
componen s
o
sel -assembled
monolaye s
and
cha ac e ized
hei
use
o
enewable
biosenso
su aces.
Ba ba a
Taskinen
ecei ed
he
B.Sc.
deg ee
(2006)
and
he
M.Sc.
deg ee
(2008)
in
Biochemis y
a
he
Uni e si y
o
Be ne,
Be ne,
Swi ze land.
She
did
he
Ph.D.
hesis
in
he
g oup
o
P o .
Vesa
P.
Hy önen
a
BioMediTech
a
he
Uni e si y
o
Tampe e,
Tampe e,
Finland
and
ecei ed
he
Ph.D.
deg ee
in
Medical
Technology
and
Bio echnology
(2014).
Since
2014
she
is
a
pos doc o al
ellow
a
he
Uni e si y
o
Washing on,
Depa men
o
Genome
Sciences,
Sea le,
USA
whe e
she
s udies
he
e olu ion
o
y osine
kinases.
Daniel
Eichinge
ecei ed
his
B.Sc.
deg ee
in
Medical
Enginee ing
a
he
Uni e si y
o
Applied
Sciences
o
Uppe
Aus ia,
Linz,
Aus ia,
in
2013.
He
did
his
bachelo
hesis
as
gues
o
P o .
He mann
J.
G ube
(see
below).
Clemens
Fla inge
ecei ed
his
B.Sc.
deg ee
in
Medical
Enginee ing
a
he
Uni e -
si y
o
Applied
Sciences
o
Uppe
Aus ia,
Linz,
Aus ia,
in
2013.
He
did
his
bachelo
hesis
as
gues
o
P o .
He mann
J.
G ube
(see
below).
Bianca
Ru mann
did
he
bachelo
s udies
in
“Biological
Chemis y”,
a
join
s udy
p og am
o
he
Uni e si y
o
Sou h
Bohemia,
Budejo ice,
Czech
Republic,
and
o
Johannes
Keple
Uni e si y
Linz,
Linz,
Aus ia.
She
ecei ed
he
Bachelo
deg ee
om
he
Ins i u e
o
Ino ganic
Chemis y
a
Johannes
Keple
Uni e si y
Linz,
Linz,
Aus ia
(2012).
He
mas e
s udies
we e
in
“Molecula
Biosciences”,
a
join
s udy
p og am
o
he
Pa is
Lod on
Uni e si y,
Salzbu g,
Aus ia,
and
o
Johannes
Keple
Uni e si y
Linz,
Linz,
Aus ia
whe e
she
ecei ed
he
M.Sc.
deg ee
in
2014.
He
mas-
e
hesis
in
he
g oup
o
P o .
He mann
J.
G ube
(see
below)
was
conce ned
wi h
new
fluo escen
bio in
de i a i es
and
wi h
enewable
senso
su aces.
P esen ly
she
is
p oduc
enginee
a
GE
Heal hca e,
Linz,
Aus ia.
Claudia
Knoglinge
ecei ed
he
B.Sc.
deg ee
in
Mic obiology
and
Gene ics
om
he
Uni e si y
o
Vienna
in
2013
and
he
M.Sc.
deg ee
in
Molecula
Biology
(wi h
emphasis
on
Biochemis y)
in
2015
om
he
same
uni e si y.
F om
2014–2015
she
did
he
mas e
hesis
as
gues
o
P o .
He mann
J.
G ube ,
Ins i u e
o
Biophysics,
Johannes
Keple
Uni e si y
Linz
(Aus ia),
cha ac e izing
di e en
s a egies
o
sen-
so
su ace
egene a ion.
P esen ly
she
is
p oduc
enginee
a
GE
Heal hca e,
Linz,
Aus ia.
Lukas
T axle
ecei ed
his
B.Sc.
deg ee
(2011)
and
his
M.Sc.
deg ee
(2013)
in
Med-
ical
Enginee ing
om
he
Uni e si y
o
Applied
Sciences
o
Uppe
Aus ia,
Linz,
Aus ia.
He
did
his
mas e
hesis
as
gues
o
D .
And eas
Ebne
(Ins i u e
o
Bio-
physics,
Johannes
Keple
Uni e si y
Linz,
Linz,
Aus ia)
in
he
field
o
biosensing
(QCM
and
SPR).
In
2014
he
ecei ed
a
M.Sc.
deg ee
in
Law
and
Economics
om
Johannes
Keple
Uni e si y
Linz,
Linz,
Aus ia.
P esen ly
he
is
a
Ph.D.
s uden
o
P o .
He mann
J.
G ube
in
he
Ph.D.
P og am
“Nanocell”,
a
join
p og am
o
Johannes
Keple
Uni e si y
Linz,
Linz,
Aus ia,
o
he
Ins i u e
o
Science
and
Technology,
Klos e neubu g,
Aus ia,
and
o
Vienna
Uni e si y
o
Technology,
Vienna,
Aus ia.
And eas
Ebne
ecei ed
his
Ph.D.
in
Technical
Sciences
a
he
Ins i u e
o
Biophysics
a
he
Johannes
Keple
Uni e si y
(JKU)
Linz,
Aus ia
in
2007.
He
wo ked
as
so wa e
de elope
o
Ebne
Enginee ing
(1991–1999),
as
esea ch
associa e
a
he
Ins i u e
o
Biophysics
JKU
(2000)
and
a
he
Uppe
Aus ian
Resea ch
(2000–2001).
Fu -
he mo e
he
was
scien ific
consul an
o
Ame sham
Biosciences
(2002–2003)
and
was
employed
as
scien ific
assis an
by
Molecula
Imaging,
USA.
A
he
ins i u e
o
biophysics
he
is
g oup
leade
(since
2007)
and
assis an
p o esso
(since
2011).
He
published
>70
pee
e iewed
scien ific
a icles,
and
>10
scien ific
book
chap e s.
His
key
compe ences
a e
biosensing
a omic
o ce
mic oscopy
echniques
like
single
molecule
o ce
spec oscopy
and
ecogni ion
imaging.
He mann
J.
G ube
ecei ed
his
Ph.D.
in
chemis y
om
he
Ins i u e
o
O ganic
Chemis y
a
Ka l–F anzens–Uni e si y
in
G az,
Aus ia
in
1983.
He
was
pos doc o al
ellow
in
he
g oup
o
P o .
P.
S.
Low
in
he
Biochemis y
Di ision
o
he
Chemis y
Depa men
a
Pu due
Uni e si y,
Wes
La aye e
(1983–1985)
and
in
he
g oup
o
P o .
H.
Schindle
a
he
Ins i u e
o
Biophysics
o
Johannes
Keple
Uni e si y
Linz,
Linz,
Aus ia
(1985–1995),
whe eupon
he
became
assis an
p o esso
(1995–2001)
and
associa e
p o esso
(since
2001)
a
he
same
ins i u e.
He
has
published
>130
scien ific
a icles.
In
pa icula ,
he
es ablished
a
oolbox
o
linke s
and
p ocedu es
o
flexible
e he ing
o
single
biomolecules
o
he
measu ing
ip
o
o ce
mic o-
654
D.
Zaune
e
al.
/
Senso s
and
Ac ua o s
B
229
(2016)
646–654
scopes
and
published
many
s udies
on
new
fluo escen
bio in
de i a i es
and
hei
applica ion
in
Bioanaly ics.
P esen ly
he
de elops
enewable
senso
su aces
ha
acili a e
biosensing
and
biological
in e ac ion
analysis.
Vesa
P.
Hy önen
is
head
o
he
P o ein
Dynamics
esea ch
g oup
and
ac s
as
a
lec u e
in
BioMediTech
a
he
Uni e si y
o
Tampe e,
Tampe e,
Finland.
A e
g ad-
ua ing
as
a
Ph.D.
om
he
Uni e si y
o
Jy äskylä,
Jy äskylä,
Finland
a
2005,
he
conduc ed
pos doc o al
aining
a
ETH
Zu ich,
Zü ich,
Swi ze land
(2005–2007).
He
hen
con inued
as
a
pos doc o al
esea che
a
he
Uni e si y
o
Tampe e
and
es ablished
an
independen
esea ch
g oup
a
2010.
His
esea ch
in e es s
a e
mechanobiology,
p o ein
enginee ing
and
accine
esea ch,
and
he
has
published
>90
scien ific
a icles.