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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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-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


PMBA/PATP toward Mb of different concentrations using the Hill equation.



M or 110


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


PMBA/PATP toward BHb of different concentrations using the Hill equation.



M or 102


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


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


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 (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


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


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


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


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


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


0 20 40 60 80 100 120

0

1

2

3

4

5

6

7A

HRP

Lac

Mb

BHb


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


0 20 40 60 80 100 120

0

1

2

3

4

5

6

7A

HRP

Lac

Mb

BHb


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


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


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


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


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


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


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


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.



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


0 20 40 60 80 100 1200

1

2

3

4

5A

Kd

Bmax

i / A


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-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


Fig. S30 Fitting of the Lac-imprinted SAM coated electrode from DHAP and

PMBA/PATP toward HRP of different concentrations using the Hill equation.



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


PMBA/PATP toward Mb of different concentrations using the Hill equation.



M or 67.1


(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


PMBA/PATP toward BHb of different concentrations using the Hill equation.



M or 37.1


(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

References

(S1) X. Zhang, X. Du, X. Huang and Z. Lv, J. Am. Chem. Soc., 2013, 135, 9248−9251.

(S2) S. Goutelle, M. Maurin, F. Rougier, X. Barbaut, L. Bourguignon, M. Ducher and

P. Maire, Fundam. Clin. Pharmacol., 2008, 22, 633−648.

(S3) O. Chailapakul and R. M. Crooks, Langmuir, 1995, 11, 1329−1340.

(S4) T. Y. Saji and S. Aoyagui, J. Electroanal. Chem., 1975, 61, 147–153.

(S5) J. Guo and S. Amemiya, Anal. Chem., 2005, 77, 2147–2156.

(S6) C. Amatore, J.-M. Savéant and D. Tessier, J. Electroanal. Chem., 1983, 147,

39–51.

(S7) O. Chailapakul and R. M. Crooks, Langmuir, 1993, 9, 884−888.

(S8) T. Saeki, H. Sunayama, Y. Kitayama and T. Takeuchi, Langmuir, 2019, 35,

1320−1326.

(S9) A. Stephenson-Brown, A. L. Acton, J. A. Preece, J. S. Fossey and P.M. Mendes,

Chem. Sci., 2015, 6, 5114−5119.

(S10) J. Huang, Y. Wu, J. Cong, J. Luo and X. Liu, Sens. Actuators B, 2018, 259,

1−9.

(S11) X. Bi and Z. Liu, Anal. Chem., 2014, 86, 959−966.

creation of glycoprotein imprinted self-assembled monolayers … · 2020. 3. 4. · s-1 supporting...

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