Creation of glycoprotein imprinted self-assembled monolayers … · 2020. 3. 4. · S-1 Supporting...
Transcript of Creation of glycoprotein imprinted self-assembled monolayers … · 2020. 3. 4. · S-1 Supporting...
S-1
Supporting Information
for
Creation of glycoprotein imprinted self-assembled
monolayers with dynamic boronate recognition sites and
imprinted cavities for selective glycoprotein recognition
Xianfeng Zhanga,b
and Xuezhong Dua,*
a Key Laboratory of Mesoscopic Chemistry (Ministry of Education), State Key
Laboratory of Coordination Chemistry, Collaborative Innovation Center of Chemistry
for Life Sciences, and School of Chemistry and Chemical Engineering, Nanjing
University, Nanjing 210023, People’s Republic of China
b School of Material and Chemical Engineering, Bengbu University, Bengbu 233030,
People’s Republic of China
E-mail: [email protected]
Electronic Supplementary Material (ESI) for Soft Matter.This journal is © The Royal Society of Chemistry 2020
S-2
Synthesis of DHAP
DHAP was synthesized according to our previous work.S1
The synthetic route to
DHAP was described in the following:
S S
OH
O
(1) HOBt, DMAP, DCC, CH2Cl2
(2) NH2(CH2)6NH2, DIPEA
S S
NH
O
NH2
1a
HO(CH2CH2O)3Hbenzyl chloride
NaOHHO(CH2CH2O)3CH2Ph
NaH, THF
BrCH2COOC(CH3)3
OO
3O
O
TFA, CH2Cl2O
O3
OH
O
1b
1c 1d
(1) HOBt, DMAP, DCC, CH2Cl2
(2) 1a, DIPEA
OO3
NH
O
NH6
O
HOO
3NH
O
NH6
O
1e
DHAP
S S
AlCl3, N,N-dimethylaniline
CH2Cl2, r.t.
4 S S
S-3
Synthesis of MDTG
MDTG was synthesized according to our previous work.S1
The synthetic route to
MDTG was described in the following:
BrBr
HO(CH2CH2O)3H, t-BuOK
THF
BrO
OH3
H2NCSNH2
CH3CH2OH
H2NCH2CH2NH2
CH3CH2OH
HSO
OH3
MDTG
S-4
200 220 240 260 280-6.0M
-4.0M
-2.0M
0.0
2.0M
4.0M
6.0M
CD
/ d
me
g
Wavelength / nm
0 L ethanol
10 L
20 L
Fig. S1 CD spectra of HRP (46 μg/mL) in 0.5 mL PBS solution (pH = 7.4) upon
addition of ethanol with different amounts.
In the UV region of CD spectra, the primary chromophores of proteins are
peptides related to the information on conformations of proteins. Two negative peaks
at 222 and 208 nm and one positive peak around 190 nm are indicative of -helix
conformation. The CD spectra of HRP in the PBS solution remained almost
unchanged in the presence of 1020 μL ethanol, which indicates that the
conformations of HRP were unaffected upon addition of a very small amount of
ethanol.
S-5
0 2 4 6 8 10 12 140
1
2
3
4
5
6
7
8
9
Curr
ent / A
Time / h
Fig. S2 DPV cathodic peak current of HRP-imprinted SAM coated electrode from
DHAP and PMBA/PATP as a function of elution time with aqueous solution of acetic
acid (10 mM, pH = 3.1) and subsequently with double-distilled water. The DPV
curves of the HRP-imprinted electrodes were measured in 10 mM PBS solution (pH =
7.4) of 2.5 mM Fe(CN)64−/3−
and 0.1 M KCl.
S-6
Fig. S3 Change in DPV cathodic peak current (Δi) after and before HRP binding and
the ratio of peak current change of the HRP-imprinted SAM coated electrodes to
non-imprinted one against the molar ratio of DHAP:PMBA:PATP: (A) the molar ratio
of PMBA:PATP was fixed at 1:1; (B) the amount of DHAP was fixed. The DPV
curves of the HRP-imprinted and non-imprinted electrodes were measured in 10 mM
PBS solution (pH = 7.4) of 2.5 mM Fe(CN)64−/3−
and 0.1 M KCl.
0
1
2
3
4
5
6
7B
iimprinted
/i non-imprinted
4:0:14:0.5:14:1:14:1:0.54:1:0
0
2
4
6
8
10
12
i im
printe
d/
i non-im
printe
d
i / A
Molar Ratio of DHAP:PMBA:PATP
imprinted SAM
non-imprinted SAM
0
2
4
6
8
10
12
3:1:1 4:1:12:1:1 5:1:1
imprinted SAM
non-imprinted SAM
i / A
Molar Ratio of DHAP:PMBA:PATP
0
1
2
3
4
5
6
7A
iimprinted
/inon-imprinted
i im
printe
d/
i non-im
printe
d
S-7
-0.2 0.0 0.2 0.4 0.6
-30
-20
-10
0
10
20
30 (a)
Cu
rre
nt
/ A
Potential / V (vs. SCE)
HRP-Fe(CN)6
4/3
HRP
-0.2 0.0 0.2 0.4 0.6
-30
-20
-10
0
10
20
30 (b)
Cu
rre
nt
/ A
Potential / V (vs. SCE)
HRP-Fe(CN)6
4/3
HRP
-0.2 0.0 0.2 0.4 0.6
-30
-20
-10
0
10
20
30
Cu
rre
nt
/ A
Potential / V (vs. SCE)
Lac-Fe(CN)6
4/3
Lac
-0.2 0.0 0.2 0.4 0.6
-30
-20
-10
0
10
20
30 (d)(c)
Cu
rre
nt
/ A
Potential / V (vs. SCE)
Lac-Fe(CN)6
4/3
Lac
Fig. S4 CV and DPV curves of bare gold electrodes toward different proteins at 120
μg/mL in 10 mM PBS solution (pH = 7.4) in the absence and presence of Fe(CN)64−/3−
:
(a,b) HRP; (c,d) Lac; (e,f) Mb; (g,h) BHb.
The proteins themselves showed no or almost no redox peak. The decrease in peak
current in the presence of Fe(CN)64−/3−
was owing to the adsorption of protein onto the
gold electrode. The order of protein adsorption was BHb > Mb > HRP > Lac.
S-8
-0.2 0.0 0.2 0.4 0.6
-30
-20
-10
0
10
20
30 (e)
Cu
rre
nt
/ A
Potential / V (vs. SCE)
Mb-Fe(CN)6
4/3
Mb
-0.2 0.0 0.2 0.4 0.6
-30
-20
-10
0
10
20
30 (f)
Cu
rre
nt
/ A
Potential / V (vs. SCE)
Mb-Fe(CN)6
4/3
Mb
-0.2 0.0 0.2 0.4 0.6
-30
-20
-10
0
10
20
30 (g)
Cu
rre
nt
/ A
Potential / V (vs. SCE)
BHb-Fe(CN)6
4/3
BHb
-0.2 0.0 0.2 0.4 0.6
-30
-20
-10
0
10
20
30 (h)
Cu
rre
nt
/ A
Potential / V (vs. SCE)
BHb-Fe(CN)6
4/3
BHb
Fig. S4 (continued)
S-9
-0.2 0.0 0.2 0.4 0.6-30
-20
-10
0
10
20
30
Cu
rre
nt
/ A
Potential / V (vs.SCE)
PMBA-Fe(CN)6
4/3
PMBA
-0.2 0.0 0.2 0.4 0.6-30
-20
-10
0
10
20
30
(b)(a)
Cu
rre
nt
/ A
Potential / V (vs. SCE)
PATP-Fe(CN)6
4/3
PATP
Fig. S5 CV curves of (a) PMBA- and (b) PATP-modified gold electrodes in 10 mM
PBS solution (pH = 7.4) in the absence and presence of Fe(CN)64−/3−
.
The PMBA- and PATP-modified SAM coated electrodes themselves showed no
redox peak from respective CV curves, which indicates that PMBA and PATP
themselves could not affect the electrochemical measurements.
S-10
Rs Rct Wo
CPE
Fig. S6 Modified Randles’ equivalent circuit model was used to fit the EIS curves in
Fig. 1: Rs, solution resistance; Rct, charge transfer resistance; Wo, finite diffusion
impedance; CPE, constant phase angle element associated with double layer
capacitance.
Table S1 Electrochemical parameters extracted from the EIS curves of various
electrodes in Fig. 1.
electrode Rs (Ω) Rct (Ω) Wo-R (Ω) Wo-T (Ω) Wo-P (Ω) CPE-T (10–6
)
(Ω–1cm
–2s
P)
CPE-P
a 140 623 25766 857 0.4642 8.9665 0.7276
b 192 66334 35487 806 0.3203 0.6317 0.8721
c 188 11580 20191 490 0.4919 1.0606 0.8476
d 203 22074 30467 887 0.3669 1.9423 0.8521
e 178 69420 21892 574 0.2599 3.2755 0.8315
a: bare gold electrode
b: imprinted electrode from DHAP and PMBA/PATP before HRP extraction
c: imprinted electrode from DHAP and PMBA/PATP after HRP extraction
d: imprinted electrode from DHAP and PMBA/PATP upon addition of HRP (18 μg/mL)
e: non-imprinted electrode
S-11
Fig. S7 CV curves of various kinds of HRP-bound gold electrodes toward H2O2 of
different concentrations at 100 mV/s in N2-saturated PBS solution (pH = 7.4): (A)
HRP-imprinted SAM coated gold electrode from DHAP and PMBA/PATP; (B)
HRP-bound SAM coated gold electrode from PMBA/PATP; (c) HRP-adsorbed gold
electrode.
-1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4-80
-70
-60
-50
-40
-30
-20
-10
0
10C
Cu
rre
nt / A
Potential / V (vs. SCE)
0 mM
0.5 mM
1.0 mM
2.0 mM
4.0 mM
5.0 mM
-1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4-80
-70
-60
-50
-40
-30
-20
-10
0
10B
Curr
ent / A
Potential / V (vs. SCE)
0 mM
0.1 mM
0.5 mM
1.0 mM
2.0 mM
4.0 mM
-1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4-80
-70
-60
-50
-40
-30
-20
-10
0
10A
Curr
ent / A
Potential / V (vs. SCE)
0 mM
0.5 mM
1.0 mM
2.0 mM
4.0 mM
5.0 mM
6.0 mM
S-12
-1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4
-40
-30
-20
-10
0
10
a
b
c
d
Curr
ent / A
Potential / V (vs. SCE)
Fig. S8 CV curves of various kinds of electrodes toward 2 mM H2O2 at 100 mV/s in
N2-saturated PBS solution (pH = 7.4): (a) bare gold electrode; (b) HRP-adsorbed gold
electrode; (c) HRP-bound SAM coated gold electrode from PMBA/PATP; (d)
HRP-imprinted SAM coated gold electrode from DHAP and PMBA/PATP.
S-13
-0.2 0.0 0.2 0.4 0.60
2
4
6
8
10B
C D
A
Cu
rre
nt
/ A
Potential / V (vs. SCE)
0 g/mL
120 g/mL
-0.2 0.0 0.2 0.4 0.60
2
4
6
8
10
Cu
rre
nt
/ A
Potential / V (vs. SCE)
0 g/mL
120 g/mL
-0.2 0.0 0.2 0.4 0.60
2
4
6
8
10
Cu
rre
nt
/ A
Potential / V (vs. SCE)
0 g/mL
120 g/mL
-5 0 5 10 15 200
2
4
6
i / A
Strorage Time / Day
Fig. S9 DPV cathodic peak currents of the HRP-imprinted SAM coated electrode
from DHAP and PMBA/PATP before and after addition of 120 μg/mL HRP in 10
mM PBS solution (pH = 7.4) of 2.5 mM Fe(CN)64−/3−
and 0.1 M KCl: (A) freshly
prepared; (B) stored for 7 days; (C) stored for 16 days. (D) Decrease of DPV cathodic
peak currents of the HRP-imprinted electrode upon addition of 120 μg/mL HRP for
different storage times.
S-14
-0.2 0.0 0.2 0.4 0.60
2
4
6
8
10
Cu
rre
nt
/ A
Potential / V (vs. SCE)
d
c
b
a
Fig. S10 DPV curves of the HRP-imprinted SAM coated electrode from MDTG and
PMBA/PATP before (a) and after (b) washing with aqueous acidic solution (pH = 3.1)
and water and the HRP-imprinted SAM coated electrode from DHAP and
PMBA/PATP before (c) and after (d) washing with aqueous acidic solution (pH = 3.1)
and water. The DPV curves were measured in 10 mM PBS solution (pH = 7.4) of 2.5
mM Fe(CN)64−/3−
and 0.1 M KCl.
S-15
-0.2 0.0 0.2 0.4 0.60
2
4
6
8
10
HRP-DHAP-PMBA/PATP
Fe(CN)6
4/3
Cu
rre
nt / A
Potential / V (vs. SCE)
0 g/mL
3
6
12
24
48
72
96
120
Fig. S11 DPV curves of the HRP-imprinted coated electrode from DHAP and
PMBA/PATP upon addition of HRP at different concentrations in 10 mM PBS
solution (pH = 7.4) of 2.5 mM Fe(CN)64−/3−
and 0.1 M KCl.
S-16
Fig. S12 (A) Fitting of the HRP-imprinted coated electrode from DHAP and
PMBA/PATP toward HRP of different concentrations using the Hill equation. (B)
Bilinear plots of DPV current response of the HRP-imprinted electrode from DHAP
and PMBA/PATP against logarithm of HRP concentration.
According to the Hill equation (eq S1),S2
y = Bmax xn/(x
n + Kd
n) (S1)
where Bmax is the maximum specific binding, Kd is the dissociation constant, and n is
Hill coefficient, the fitted Kd value was 5.6 × 10−7
M or 22.4 μg/mL (R2 = 0.98, Bmax =
6.51, and n = 0.88). The limit of detection of HRP was 1.18 μg/mL (2.95 × 10−8
M) at
S/N = 3. Then, the complex stability constant (Ka) of the imprinted SAM for HRP was
obtained to be 1.78 × 106 M
–1 (44.6 mL/ng).
0 20 40 60 80 100 1200
1
2
3
4
5
6
7
A
y = 6.51 x0.88
/(x0.88
+ Kd
0.88)
R2 = 0.98
Kd
Bmax
HRP Concentration / g/mL
i / A
1 10 100
0
1
2
3
4
5
6
7B
y = 2.648 + 3.993 log x
x = 36120 g/mL
R2 = 0.9959
y = 0.1285 + 2.080 log x
x = 036 g/mL
R2 = 0.9842
i / A
HRP Concentration / g/mL
S-17
1 10 100
0
1
2
3
4
5
6
7
y = 0.1670 + 0.4042 log x
x = 0120 g/mL
R2 = 0.9909
y = 2.648 + 3.993 log x
x = 36120 g/mL
R2 = 0.9959
y = 0.1285 + 2.080 log x
x = 036 g/mL
R2 = 0.9842
i / A
HRP Concentration / g/mL
HRP-imprinted
non-imprinted
Fig. S13 Bilinear plots of DPV current response of the HRP-imprinted SAM coated
electrode from DHAP and PMBA/PATP and linear calibration plot of the
non-imprinted counterpart against logarithm of HRP concentration.
The imprinting factor (IF) is defined as the ratio of the slope of the calibration plot
for the imprinted SAM to that for the non-imprinted SAM. The IF of the
HRP-imprinted SAM relative to the non-imprinted one was calculated to be 5.1 taking
into account their calibration plots in the concentration range of 0–36 μg/mL.
S-18
0 20 40 60 80 100 1200
1
2
3
4
5
6
7
Kd
Bmax
i / A
Lac Concentration / g/mL
Fig. S14 Fitting of the HRP-imprinted SAM coated electrode from DHAP and
PMBA/PATP toward Lac of different concentrations using the Hill equation.
According to the Hill equation,S2
the fitted Kd value was 1.48 × 10−6
M or 118
μg/mL (R2 = 0.95, Bmax = 3.2, and n = 0.233). Then, the complex stability constant (Ka)
of the HRP-imprinted SAM for Lac was obtained to be 6.76 × 105 M
–1 (8.47 mL/ng).
S-19
0 20 40 60 80 100 1200
1
2
3
4
5
6
7
Bmax
Kd
i / A
Mb Concentration / g/mL
Fig. S15 Fitting of the HRP-imprinted SAM coated electrode from DHAP and
PMBA/PATP toward Mb of different concentrations using the Hill equation.
According to the Hill equation,S2
the fitted Kd value was 6.2 × 10−6
M or 110
μg/mL (R2 = 0.98, Bmax = 2.2, and n = 0.358). Then, the complex stability constant (Ka)
of the HRP-imprinted SAM for Mb was obtained to be 1.61 × 105 M
–1 (9.09 mL/ng).
S-20
0 20 40 60 80 100 1200
1
2
3
4
5
6
7
Kd
Bmax
i / A
BHb Concentration / g/mL
Fig. S16 Fitting of the HRP-imprinted SAM coated electrode from DHAP and
PMBA/PATP toward BHb of different concentrations using the Hill equation.
According to the Hill equation,S2
the fitted Kd value was 1.6 × 10−6
M or 102
μg/mL (R2 = 0.95, Bmax = 2.1, and n = 0.308). Then, the complex stability constant (Ka)
of the HRP-imprinted SAM for BHb was obtained to be 6.25 × 105 M
–1 (9.80 mL/ng).
S-21
Table S2 Repeatability of HRP-imprinted coated electrodes at different batches after
HRP extraction and upon rebinding of HRP at different concentrations.
electrode DPV peak current (i) or change (i)
(A)
mean
value
(A)
standard
deviation
relative
standard
deviation
(%) electrode 1 electrode 2 electrode 3
imprinted
electrode after
HRP removal
i = 7.268 i = 7.556 i = 8.080 7.635 0.4117 5.4
imprinted
electrode upon
rebinding of
HRP (6 g/mL)
i = 1.740 i = 1.625 i = 1.680 1.682 0.0814 4.8
imprinted
electrode upon
rebinding of
HRP (120
g/mL)
i = 6.093 i = 5.001 i = 5.547 5.547 0.7716 13.9
S-22
Fig. S17 DPV curves of the HRP-imprinted coated electrodes from DHAP and
PMBA/PATP upon addition of the mixed proteins of (A) HRP, Lac, Mb, and BHb
(1:1:1:1 in weight) and (B) Lac, Mb, and BHb (1:1:1 in weight) with different
concentrations of each protein in 10 mM PBS solution (pH = 7.4) of 2.5 mM
Fe(CN)64−/3−
and 0.1 M KCl.
-0.2 0.0 0.2 0.4 0.60
2
4
6
8
10B HRP-DHAP-PMBA/PATP
Fe(CN)6
4/3
Cu
rre
nt
/ A
Potential / V (vs. SCE)
0 g/mL
6
36
48
72
96
-0.2 0.0 0.2 0.4 0.60
2
4
6
8
10A HRP-DHAP-PMBA/PATP
Fe(CN)6
4/3
Cu
rre
nt
/ A
Potential / V (vs. SCE)
0 g/mL
1.5
3
12
24
48
72
S-23
Fig. S18 (A) Decrease of DPV cathodic peak current of the HRP-imprinted coated
electrodes from DHAP and PMBA/PATP as a function of concentration of each
protein of the mixed proteins of (a) HRP, Lac, Mb, and BHb (1:1:1:1 in weight) and
(b) Lac, Mb, and BHb (1:1:1 in weight) and (B) Comparison of the difference
between (a) and (b) with the decrease of DPV cathodic peak current of the
HRP-imprinted coated electrode from DHAP and PMBA/PATP as a function of
concentration of HRP only shown in Fig. 4A in 10 mM PBS solution (pH = 7.4) of
2.5 mM Fe(CN)64−/3−
and 0.1 M KCl.
0 20 40 60 80 100 120
0
1
2
3
4
5
6
7B
i / A
HRP Concentration / g/mL
HRP (only)
a b
0 20 40 60 80 100 120
0
1
2
3
4
5
6
7A
i / A
Protein Concentration / g/mL
a (HRP, Lac, Mb, and BHb)
b (Lac, Mb, and BHb)
S-24
-0.2 0.0 0.2 0.4 0.60
2
4
6
8
10
HRP-DHAP-PMBA/PATP
Fe(CN)6
4/3
Cu
rre
nt
/ A
Potential / V (vs. SCE)
0 g/mL
1.5
6
24
48
84
120
Fig. S19 DPV curves of the HRP-imprinted coated electrode from DHAP and
PMBA/PATP as a function of concentration of HRP in the presence of 1.17 mg/mL
(6.5 mM) glucose in 10 mM PBS solution (pH = 7.4) of 2.5 mM Fe(CN)64−/3−
and 0.1
M KCl.
S-25
Fig. S20 Decrease of DPV cathodic peak current of the HRP-imprinted coated
electrode from DHAP and PMBA/PATP as a function of concentration of HRP in the
presence of 1.17 mg/mL (6.5 mM) glucose in 10 mM PBS solution (pH = 7.4) of 2.5
mM Fe(CN)64−/3−
and 0.1 M KCl: (A) linear scale; (B) logarithmic scale.
0 20 40 60 80 100 120
0
1
2
3
4
5
6
7A
HRP-DHAP-PMBA/PATP
Fe(CN)6
4/3
i / A
HRP Concentration / g/mL
0 mM glucose
6.5 mM glucose
1 10 100
0
1
2
3
4
5
6
7B
HRP-DHAP-PMBA/PATP
Fe(CN)6
4/3
i / A
HRP Concentration / g/mL
0 mM glucose
6.5 mM glucose
S-26
-0.2 0.0 0.2 0.4 0.6-6
-4
-2
0
2
4
6
Cu
rre
nt / A
Potential / V (vs. SCE)
a
b
Fig. S21 CV curves of the HRP-imprinted SAM coated electrode from DHAP and
PMBA/PATP (a) after washing with aqueous acidic solution (pH = 3.1) and water and
(b) upon addition of HRP (60 μg/mL) in 10 mM PBS solution (pH = 7.4) of 2.5 mM
FcDM and 0.1 M KCl.
S-27
-0.2 0.0 0.2 0.4 0.60
2
4
6
8
10
HRP-DHAP-PMBA/PATP
FcDM
Cu
rre
nt / A
Potential / V (vs. SCE)
0 g/mL
1.5
6
24
48
72
96
120
Fig. S22 DPV cathodic peak currents of the HRP-imprinted SAM coated electrode
from DHAP and PMBA/PATP upon addition of HRP of different concentrations in 10
mM PBS solution (pH = 7.4) of 2.5 mM FcDM and 0.1 M KCl.
S-28
0 20 40 60 80 100 120
0
1
2
3
4B
FcDM
Fe(CN)6
4/3
i / A
Protein Concentration / g/mL
0 20 40 60 80 100 120
0
1
2
3
4
5
6
7A
HRP
Lac
Mb
BHb
Protein Concentration / g/mL
i / A
Fig. S23 Decrease of DPV cathodic peak current of the HRP-imprinted SAM coated
electrodes from DHAP and PMBA/PATP as a function of concentration of different
proteins in 10 mM PBS solution (pH = 7.4) of 0.1 M KCl with different electroactive
probes at 2.5 mM: (A) Fe(CN)64−/3−
; (B) FcDM.
S-29
0 20 40 60 80 100 120
0.00
0.05
0.10
B
Fe(CN)6
4/3
E
/ V
Protein Concentration / g/mL
0 20 40 60 80 100 120
0
1
2
3
4
5
6
7A
HRP
Lac
Mb
BHb
Protein Concentration / g/mL
i / A
Fig. S24 (A) Decrease of DPV cathodic peak current of the HRP-imprinted SAM
coated electrodes from DHAP and PMBA/PATP as a function of concentration of
different proteins in 10 mM PBS solution (pH = 7.4) of 2.5 mM Fe(CN)64−/3−
and 0.1
M KCl. (B) Change of the open-circuit potential of the HRP-imprinted SAM coated
electrodes from DHAP and PMBA/PATP as a function of concentration of different
proteins in 10 mM PBS solution (pH = 7.4) of 0.1 M KCl.
S-30
Fig. S25 CV curves of the (A) PMBA-modified electrode, (B) PATP-modified
electrode, (C) modified electrode from the equimolar mixture of PMBA and PATP,
and (D) imprinted SAM coated electrode from DHAP and PMBA/PATP after HRP
extraction as a function of pH in 10 mM PBS solution of 2.5 mM Fe(CN)64–/3–
and 0.1
M KCl.
-0.2 0.0 0.2 0.4 0.6-40
-30
-20
-10
0
10
20
30
40
(A) PMBA
C
urr
en
t / A
Potential / V (vs. SCE)
pH 2
pH 3
pH 4
pH 5
pH 6
pH 7
pH 8
pH 9
pH 10
pH 11
-0.2 0.0 0.2 0.4 0.6-40
-30
-20
-10
0
10
20
30
40
(B) PATP
Curr
en
t / A
Potential / V (vs. SCE)
pH 2
pH 3
pH 4
pH 5
pH 6
pH 7
pH 8
pH 9
pH 10
pH 11
-0.2 0.0 0.2 0.4 0.6-40
-30
-20
-10
0
10
20
30
40
(C) PMBA/PATP
Curr
en
t /
A
Potential / V (vs. SCE)
pH 2
pH 3
pH 4
pH 5
pH 6
pH 7
pH 8
pH 9
pH 10
pH 11
-0.2 0.0 0.2 0.4 0.6-40
-30
-20
-10
0
10
20
30
40
(D) DHAP-PMBA/PATP
Cu
rre
nt / A
Potential / V (vs. SCE)
pH 3
pH 4
pH 5
pH 6
pH 7
pH 8
pH 9
pH 10
pH 11
S-31
-0.2 0.0 0.2 0.4 0.6-20
-15
-10
-5
0
5
10
15
20
(B)
Cu
rre
nt
/ A
Potential / V (vs. SCE)
a
b
-0.2 0.0 0.2 0.4 0.6-20
-15
-10
-5
0
5
10
15
20
(A)
Cu
rre
nt
/ A
Potential / V (vs. SCE)
a
b
c
Fig. S26 (A) CV curves of the Lac-imprinted SAM coated electrode from DHAP and
PMBA/PATP (a) before and (b) after washing with aqueous acidic solution and water
and (c) upon addition of Lac (36 μg/mL). (B) CV curves of the non-imprinted
electrode from DHAP and PMBA/PATP (a) before and (b) after addition of Lac (36
μg/mL). The electrolyte solution used was 10 mM PBS solution (pH = 7.4) of 2.5 mM
Fe(CN)64−/3−
and 0.1 M KCl.
S-32
-0.2 0.0 0.2 0.4 0.60
2
4
6
8
10
Lac-DHAP-PMBA/PATP
Fe(CN)6
4/3
Cu
rre
nt / A
Potential / V (vs. SCE)
0 g/mL
1.5
6
24
48
72
96
120
Fig. S27 DPV cathodic peak currents of the Lac-imprinted SAM coated electrode
from DHAP and PMBA/PATP upon addition of Lac of different concentrations in 10
mM PBS solution (pH = 7.4) of 2.5 mM Fe(CN)64−/3−
and 0.1 M KCl.
S-33
Fig. S28 (A) Fitting of the Lac-imprinted SAM coated electrode from DHAP and
PMBA/PATP toward Lac of different concentrations using the Hill equation. (B)
Bilinear plots of DPV current response of the Lac-imprinted electrode from DHAP
and PMBA/PATP against logarithm of Lac concentration.
According to the Hill equation,S2
the fitted Kd value was 4.0 × 10−7
M or 31.9
μg/mL (R2 = 0.96, Bmax = 4.90, and n = 0.87). The limit of detection of Lac was 1.43
μg/mL (1.79 × 10−8
M) at S/N = 3. Then, the complex stability constant (Ka) of the
imprinted SAM for Lac was obtained to be 2.50 × 106 M
–1 (31.3 mL/ng).
1 10 100
0
1
2
3
4
5B
y = 0.2096 + 1.245 log x
x = 036 g/mL
R2 = 0.9961
y = 3.655 + 3.709 log x
x = 36120 g/mL
R2 = 0.9761
i / A
Lac Concentration / g/mL
0 20 40 60 80 100 1200
1
2
3
4
5A
Kd
Bmax
i / A
Lac Concentration / g/mL
S-34
1 10 100
0
1
2
3
4
5
y = 0.1503 + 0.3477 log x
x = 0120 g/mL
R2 = 0.9974
y = 3.655 + 3.709 log x
x = 36120 g/mL
R2 = 0.9761
y = 0.2096 + 1.245 log x
x = 036 g/mL
R2 = 0.9961
i / A
Lac Concentration / g/mL
Lac-imprinted
non-imprinted
Fig. S29 Bilinear plots of DPV current response of the Lac-imprinted SAM coated
electrode from DHAP and PMBA/PATP and linear calibration plot of the
non-imprinted counterpart against logarithm of Lac concentration.
The IF of the Lac-imprinted SAM relative to the non-imprinted one was
calculated to be 3.6 taking into account their calibration plots in the concentration
range of 0–36 μg/mL.
S-35
0 20 40 60 80 100 1200
1
2
3
4
5
Kd
Bmax
i / A
HRP Concentration / g/mL
Fig. S30 Fitting of the Lac-imprinted SAM coated electrode from DHAP and
PMBA/PATP toward HRP of different concentrations using the Hill equation.
According to the Hill equation,S2
the fitted Kd value was 1.25 × 10−6
M or 50.1
μg/mL (R2 = 0.99, Bmax = 3.61, and n = 0.373). Then, the complex stability constant
(Ka) of the Lac-imprinted SAM for HRP was obtained to be 8.00 × 105 M
–1 (20.0
mL/ng).
S-36
0 20 40 60 80 100 1200
1
2
3
4
5
Kd
Bmax
i / A
Mb Concentration / g/mL
Fig. S31 Fitting of the Lac-imprinted SAM coated electrode from DHAP and
PMBA/PATP toward Mb of different concentrations using the Hill equation.
According to the Hill equation,S2
the fitted Kd value was 1.04 × 10−6
M or 67.1
μg/mL (R2 = 0.99, Bmax = 1.99, and n = 0.448). Then, the complex stability constant
(Ka) of the Lac-imprinted SAM for Mb was obtained to be 9.61 × 105 M
–1 (14.9
mL/ng).
S-37
0 20 40 60 80 100 1200
1
2
3
4
5
Kd
Bmax
i / A
BHb Concentration / g/mL
Fig. S32 Fitting of the Lac-imprinted SAM coated electrode from DHAP and
PMBA/PATP toward BHb of different concentrations using the Hill equation.
According to the Hill equation,S2
the fitted Kd value was 2.08 × 10−6
M or 37.1
μg/mL (R2 = 0.99, Bmax = 2.88, and n = 0.503). Then, the complex stability constant
(Ka) of the Lac-imprinted SAM for BHb was obtained to be 4.81 × 105 M
–1 (26.9
mL/ng).
S-38
Estimation of number density of imprinted cavities
If the imprinted cavities are assumed to act as independent disk-shaped
nanoelectrodes and have average radii of template proteins estimated from their
dimensions, approximate number density of imprinted cavities can be calculated using
eq S2,S3
*
04
jN
nFDC r (S2)
where N is the number density of imprinted cavities, j is the current density and is
obtained from CV data, r0 is the surface defect radius and is obtained from average
radius of protein templates, F is the Faraday constant, D (D = 8.3 × 10–6
cm2/s for
Fe(CN)64−/3−
;S4
D = 7.0 × 10–6
cm2/s for FcDM
S5) and C
* are the diffusion coefficient
and bulk concentration of electroactive probes, respectively, and n is the number of
electrons transferred per probe.
The application of eq S2 for determination of the number density of cavities
imprinted in the SAM (N) assumes that the diffusion coefficients of the redox probes
in the aqueous solution and in the imprinted SAM were the same. However, typically,
the latter is much lower. Therefore, it is clearly pointed out that the determined N
value is at least the lowest possible limit of N.
Estimation of fractional surface coverage of imprinted cavities
According to the theoretical model developed by Amatore et al.,S6
the surface
coverage of imprinted cavities is calculated approximately. The model is suitable for
the chemical systems if diffusion is radial and the diffusion layers of the individual
ultra-microelectrodes do not overlap. Fractional surface coverage of imprinted
cavities, θ, can be approximately calculated using eq S3,S6,S7
*
lim 0/(0.6 )i nFSC D r (S3)
where ilim is the maximum limiting current and is obtained from CV data, n is the
number of electrons transferred per electroactive probe, F is the Faraday constant, S is
the geometrically projected surface area of the Au electrode (S = 0.0314 cm2), C
* and
D are the bulk concentration and diffusion coefficient of electroactive probes,
respectively, and r0 is the average radius of imprinted cavities.
S-39
Table S3 Comparison of the preparation parameters and performance of glycoprotein imprinted sensors based on boronate affinity.
glycoprotein imprinted sensor glycoprotein pH glycoprotein
assembly
initiator method Ka (M−1
) linear range
(g/mL)
limit of
detection
(g/mL)
selectivity reference
imprinted SAM HRP 7.4 coassembly no DPV 1.78 × 106 0−120 1.18 good this work
imprinted SAM Lac 7.4 coassembly no DPV 2.50 × 106 0−120 1.43 good this work
SAM & fluorinated
phenylboronic acid & surface
imprinting
ovalbumin 7.4 pre-assembly yes SPR 4.7 × 106 0−100 1.32 good S8
SAM & phenylboronic acid &
surface imprinting
PSA 8.5 pre-assembly yes SPR 5.6 × 105 N/A N/A good S9
SAM & phenylboronic acid &
surface imprinting
RNase B 8.5 pre-assembly yes SPR 3.2 × 105 N/A N/A good S9
graphene & phenylboronic
acid & surface imprinting
ovalbumin 8.5 pre-assembly NH3H2O
(pH = 9.3)
DPV 1.78 × 106 1.0 × 10
−7
−0.1
2.0 × 10−8
good S10
microplate & phenylboronic
acid & surface imprinting
HRP 8.5 pre-assembly yes ELISA 8.3 × 108 0−0.1 N/A good S11
SAM: self-assembled monolayer
DPV: differential pulse voltammetry
SPR: surface plasmon resonance
PSA: prostate specific antigen
ELISA: enzyme-linked immunosorbent assay
N/A: not available
S-40
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