Supplementary Information

Label-free Detection of Target Proteins Using Peptide Molecular

Wires as Conductive Supports

Mihaela Puiua, Lucian-Gabriel Zamfira,b, George Madalin Danilaa,c, Francesco Papid,

Cristina Nativid, Valentin Mirceskie,f and Camelia Balaa,g*
a
Laboratory for Quality Control and Process Monitoring, University of Bucharest, 4-12 Elisabeta Blvd.,
030018-Bucharest, Romania
b
ICUB, University of Bucharest, 36-46 Kogalniceanu Blvd., 050107 Bucharest, Romania
c
Romanian Doping Control Laboratory, 37-39 Basarabiei Blvd., 022103-Bucharest, Romania
d
Department of Chemistry “Ugo Schiff”, University of Florence, via della Lastruccia, 13, I-50019Sesto F.no
(FI), Italy
e
Institute of Chemistry, Faculty of Natural Sciences and Mathematics, “Ss Cyril and Methodius” University,
P.O. Box 162, Skopje, R. N. Macedonia
f
Department of Inorganic and Analytical Chemistry, University of Lodz, Tamka 12, Lodz, Poland
g
Department of Analytical Chemistry, University of Bucharest, 4-12 Elisabeta Blvd., 030018-Bucharest,
Romania

e-mail: camelia.bala@chimie.unibuc.ro; Tel./Fax: +40 214104888

1
Table of Contents

Figure S1 Chemical structure of the ligands selected for affinity pairing: (A) α-Tn_mimetic-HMDA
for anti - α-Tn antibody detection and (B) CJC-1295 agonist peptide for GHS-R1a detection……S3

Synthesis of α-Tn_mimetic-HMDA (compound A) starting from α-Tn_mimetic

(compound 1).……………………………………………………………………………………S4-6
Figure S2 Schematic representation of compound 2 synthesis……………………………………S4
Figure S3 Schematic representation of compound 3 synthesis……………………………………S5
Figure S4 Schematic representation of α-Tn_mimetic-HMDA (compound A) synthesis from
compound 3……………………………………………………………………………………..…S6
Figure S5 CD spectrum of 100 µM SP in acetonitrile………………………………………...…...S7
Figure S6 (A) Determination of ESA for 4 gold rod electrodes through CV measurements in 0.05
M H2SO4 (υ = 0.1 V s-1); (B) Roughness factors obtained after surface activation using the
combined procedures: CV scans in basic and acidic conditions followed by a triple-potential pulse
waveform in 100 mM PBS……………………………………………………………………….... S8

Figure S7 CV responses of the SP/MCH-modified electrodes in 1mM K3FeCN6 /1M KCl,

following an18-hour incubation of electrodes in SP/MCH solutions (scan rate 100mV/s) ………. S9

Figure S8 Estimation of MB/SP/MCH surface coverage through MB signaling in CV using the

peak current vs. scan rate linear dependence (voltammograms obtained in 100 mM PBS,
monitoring the anodic branch) ……………………………………………………………….…. S10

Figure S9 Estimation of MB-peptide surface coverage through MB signaling in ACV using the
slope of the average peak current vs. ACV frequency dependence. Here Iavg represents average ac
current is 2/𝜋𝜋 times the peak AC current………………………………………………….….…. S10

Table S1 Surface coverages of mixed MB/SP/MCH determined through the surface-confided MB

signaling in CV and ACV (triplicate experiments) ……………………………………….…… S11

Figure S10 Quasi-reversible maximum of MB in absence (curve (A), right ordinate) and in the
presence of 50 µM of the support peptide (curve (B), left ordinate).……………………….…… S12

Figure S11 Peak potential separation (ΔEp) of the forward and backward SW voltammetric
components as a function of the SW amplitude (Esw)…………………………………………… S12

Figure S12 Variation of peak current of peptide-confined MB vs. time (for the peptide film
obtained through immersion in 100 µM SP)…………………………………………………….. S13

2
Figure S13 AFM images taken in tapping mode of (A) bare gold, (B) gold surface functionalized
with MB/SP/MCH mixed layer, (C) gold surface functionalized with CJC-1295/ MB/SP/MCH
mixed layer and (D) CJC-1295/ MB/SP/MCH modified surface after GHS-R1a
binding………………….................................................................................................................S14
Figure S14 Evidence of ET capabilities of the functionalized sensor through CV measurements
following the modular surface modification………………………………………………….…...S15
Figure S15 Characterization of modular surface modification through EIS measurements……. S16
Figure S16 Decrease of SWV current at the binding of the GHS-Ra to the CJC-1295 immobilized
onto MB/SP/MCH electrode using two different linkers: ethylenediamine and hexamethylenediamine
…………………………………….………………………………………………………………S17

Figure S17 Calibration curve for GHS-R1a detection obtained in an ELISA sandwich assay…. S17
Figure S18. Anti-interference performance evaluated as signal suppression in SWV following the
incubation of the modified sensors the target (A) GHS-R1a and (B) anti-α-Tn antibody in the presence
of non-specific antibodies…………………………………………………………………………S18
Figure S19 Selectivity test for (A) GHS-R1a and- (B) Anti-α-Tn antibody detection in the presence
of non-specific antibodies…………………………………………………………………………S18

3
 A B

Figure S1 Chemical structure of the ligands selected for affinity pairing: (A) α-Tn_mimetic-HMDA
for anti - α-Tn antibody detection and (B) CJC-1295 agonist peptide for GHS-R1a detection

4
Synthesis of α-Tn_mimetic-HMDA (compound A) starting from α-Tn_mimetic
(compound 1)

OAc 6 OAc
AcO AcO
H 2N NHBoc 4 5
O
O 4
AcO AcO
TBTU 3 2 1
SO SO
NMM
O O
DMF
HN 86% HN 4'
5'
COOH C N NHBoc
O H 4
1 2

Figure S2 Schematic representation of compound 2 synthesis

To a solution of 1 (425 mg, 0.93 mmol) in dry DMF (6 mL), a fresh solution of TBTU (597 mg, 1.86
mmol) and NMM (204 µL, 1.86 mmol) in dry DMF (12 mL) was added. Subsequently, N-Boc-1,6-
hexanediamine (242 mg, 1.12 mmol) dissolved in dry DMF (2 mL) was added and the solution was
stirred at room temperature for 3 h. The solvent was evaporated and the residue purified by flash
chromatography (CH2Cl2/MeOH 20:1) to give 2 (526 mg, 86%).
[α]D22 = +77.7 (c 0.6, CHCl3); 1H NMR (500 MHz, CDCl3) δ: 6.96 (bs, 1H, NH), 6.84 (bs, 1H, NH),
5.70 (d, J1,2 = 2.7 Hz, 1H, H-1), 5.43-5.41 (dd, J4,3 = 3.1 Hz, J4,5 = 1.1 Hz, 1H, H-4), 5.02 (dd, J3,2 =
11.7 Hz, J3,4 = 3.1 Hz, 1H, H-3), 4.63 (bs, 1H, NH), 4.45-4.44 (m, 1H, H-5), 4.18-4.12 (m, 3H, H-5',
H-6a, H-6b), 3.63 (dd, J2,1 = 2.7 Hz, J2,3 = 11.7 Hz, 1H, H-2), 3.30-3.20 (m, 2H, CH2), 3.14-3.03 (m,
2H, CH2), 3.02-2.84 (m, H-4'a, H-4'b), 2.15 (s, 3H, COCH3), 2.05 (s, 3H, COCH3), 2.01 (s, 3H,
COCH3),1.53-1.41 (m, 13H, 2CH2, tBu ), 1.36-1.30 (m, 4H, 2CH2); 13C NMR (125 MHz, CDCl3) δ:
170.5 (Cq) 170.1 (Cq), 170.0 (Cq), 169.5 (Cq), 165.2 (Cq), 156.3 (Cq), 155.6 (Cq), 96.8 (Cq), 95.9
(CH, C-1), 79.2 (Cq, tBu), 69.0 (CH, C-5), 67.2 (CH, C-4), 65.9 (CH, C-3), 61.6 (CH2, C-6), 52.3
(CH, C-5'), 40.1 (CH2), 39.5 (CH2), 36.3 (CH, C-2), 30.8 (CH2, C-4'), 29.9 (CH2), 29.1 (CH2), 28.5
(3CH3, tBu), 26.0 (CH2), 25.8 (CH2) 20.8 (3CH3, Ac); ESI-MS m/z (%): 680.17 (100) [M+Na]+,
696.17 (45) [M+K]+.

5
 AcO OAc 6 OH
OH
O 5
4 O
AcO NH3 HO
2 1
SO 3 SO
MeOH
O O
>95 %
HN HN 4'
5'
C N NHBoc C
O O N NHBoc
H 4 H 4
2 3

Figure S3 Schematic representation of compound 3 synthesis

Compound 2 (470 mg, 0.76 mmol) was dissolved in MeOH (8 mL), and NH3 in MeOH 2M (4.5 mL)
was added. After stirring for 2 h, the reaction mixture was concentrated under vacuum to give
derivative 3 (410 mg, >95 %), which was used in the next step without further purification.
[α]D22 = + 31.2 (c 0.345, CH3OH); 1H NMR (500 MHz, CD3OD) δ: 5.67 (d, J1,2 = 2.8 Hz, 1H, H-1),
4.14-4.10 (m, 1H, H-5’), 4.03-3.99 (m, 1H, H-5), 3.96-3.94 (m, 1H, H-4), 3.80-3.70 (m, 2H, H-6a,
H-6b), 3.61 (dd, J3,2 = 11.0 Hz, J3,4 = 2.9 Hz, 1H, H-3), 3.46 (dd, J2,1 = 2.8 Hz, J3,2 = 11.0 Hz, 1H, H-
2), 3.28-3.16 (m, 2H, CH2), 3.04 (m, 2H, CH2), 2.93 (dd, J4’a,4’b = 16.7 Hz, J4 a’,5’ = 6.8 Hz, 1H, H-
4'a), 2.78 (dd, J4’b ,4’a 16.7 Hz, J4b’,5’ = 6.5 Hz, 1H, H-4'b), 1.43 (s, 13H, 2CH2, tBu), 1.36-1.30 (m, 4H,
2CH2); 13C NMR (125 MHz, CD3OD) δ: 172.7 (Cq), 167.6 (Cq), 158.6 (Cq), 157.8 (Cq), 98.3 (CH,
C-1), 96.8 (Cq), 79.8 (Cq), 75.0 (CH, C-5), 70.2 (CH, C-4), 67.2 (CH, C-3), 62.6 (CH2, C-6), 53.2
(CH, C-5'), 41.2 (CH2), 40.5 (CH2), 40.4 (CH, C-2), 32.1 (CH2, H-4'), 30.9 (CH2), 30.2 (CH2), 28.8
(3CH3, tBu), 27.4 (2CH2); ESI-MS m/z (%): 554.42 (70) [M+Na]+, 570.42 (100) [M+K]+.

6
 OH OH 6 OH
OH
O 5
4 O
HO TFA HO
2 1
SO 3 SO
CH2Cl2
O O
HN HN 4' CF3COO
5'
C N NHBoc C
O O N NH3
H 4 H 4
3 A

Figure S4 Schematic representation of α-Tn_mimetic-HMDA (compound A) synthesis from

compound 3
To a suspension of 3 (410 mg, 0.77 mmol) in dry CH2Cl2 (13 mL), TFA (2 mL, 26.12 mmol) was
added. After 2 h, the reaction mixture was concentrated under vacuum to give crude A (550 mg)
which was used without further purification.
1
H NMR (500 MHz, CD3OD) δ: 5.66 (d, J1,2 = 2.8 Hz, 1H, H-1), 4.14-4.09 (m, 1H, H-5'), 4.04-3.99
(m, 1H, H-5), 3.95-3.93 (m, 1H, H-4), 3.80-3.71 (m, 2H, H-6a, H-6b), 3.61 (dd, J3,2 = 10.9 Hz, J3,4 =
2.9 Hz, 1H, H-3), 3.46 (dd, J2,1 = 2.8 Hz, J2,3 = 10.9 Hz, 1H, H-2), 3.29-3.17 (m, 2H, CH2), 3.02-2.96
(m, 1H, H-4'a), 2.94-2.89 (m, 2H, CH2), 2.80-2.74 (m, 1H, H-4'b), 1.70-1.62 (m, 2H, CH2), 1.58-1.50
(m, 2H, CH2) 1.47-1.32 (m, 4H, 2CH2).

7
 0.00
MRE (deg cm2 dmol-1)

-1.50x104

-3.00x104

-4.50x104

200 220 240 260 280

λ (nm)

Figure S5 CD spectrum of 100 µM SP in acetonitrile. The negative peaks at 207 and 222 nm sustain
the hypothesis of an α-helix configuration

8
 A B

Figure S6 (A) Determination of ESA for 4 gold rod electrodes through CV measurements in 0.05 M
H2SO4 ( υ = 0.1 V s-1); (B) Roughness factors obtained after surface activation using the combined
procedures: CV scans in basic and acidic conditions followed by a triple-potential pulse waveform in
100 mM PBS

Figure S7 CV responses of the SP/MCH-modified electrodes in 1mM K3FeCN6 /1M KCl, following
an18-hour incubation of electrodes in SP/MCH solutions (scan rate 100 mV/s)

9
 3.0

100 µM SP
2.5
50 µM SP
10 µM SP
2.0 without SP

1.5
Ip(µA)

1.0

0.5

0.0
0 200 400 600 800 1000

ν(mV/s)

Figure S8 Estimation of MB/SP/MCH surface coverage through MB signaling in CV using the

peak current vs. scan rate linear dependence (voltammograms obtained in 100 mM PBS,
monitoring the anodic branch)

100 µM SP
2.5 50 µM SP
10 µM SP
withoutSP
2.0

1.5
Iavg(µA)

1.0

0.5

0.0
0 50 100 150 200
fACV(Hz)

Figure S9 Estimation of MB-peptide surface coverage through MB signaling in ACV using the
slope of the average peak current vs. ACV frequency dependence. Here Iavg represents average ac
current is 2/𝜋𝜋 times the peak AC current
10
Table S1 Surface coverages of mixed MB/SP/MCH determined through the surface-confided MB
signaling in CV and ACV (triplicate experiments)

[SP] (µM) Surface coverage of bound MB

Γ× 1011 mol/cm2

CV ACV
100 2.52±0.11 3.78±0.16
50 3.61±0.31 4.91±0.22
10 0.911±0.047 1.063 ± 0.034
0 0.488±0.035 0.504±0.035

11
Figure S10 Quasi-reversible maximum of MB in absence (curve (A), right ordinate) and in the
presence of 50 µM of the support peptide (curve (B), left ordinate). The other parameters are SW
amplitude Esw = 25 mV and step potential ΔEstep = 1.5 mV.

Figure S11 Peak potential separation (ΔEp) of the forward and backward SW voltammetric
components as a function of the SW amplitude (Esw). The best fit between experimental and
theoretical data for the experiment conducted with 50 µM SP. The other conditions for both
experiments and simulations are SW frequency f = 25 Hz, step potential ΔEstep = 1.5 mV and
temperature 298 K. The simulations have been conducted with the model for the two-step surface
EE electrode mechanism 1 with the rate constant of the first step ksur,1 = 20 s-1 and the cathodic ET
coefficient α1 = 0.5. The second electron transfer step is assumed to proceed with the standard rate
constant of ksur,2 > 1000 s-1, and α2 = 0.5.

12
 100

80

60
Ip/Ip0 x 100

40

20

0
0 10 20 30
Time (days)

Figure S12 Variation of peak current of peptide-confined MB vs. time (for the peptide film obtained
through immersion in 100 µM SP). All experiments were performed in 100 mM PBS buffer, pH =
7.4 and 25oC

13
Figure S13 AFM images taken in tapping mode of (A) bare gold, (B) gold surface functionalized with MB/SP/MCH mixed layer, (C) gold surface
functionalized with CJC-1295/ MB/SP/MCH mixed layer and (D) CJC-1295/ MB/SP/MCH modified surface after GHS-R1a binding. (B) – (D) images
also contain the corresponding height profiles.
14
Figure S14 Evidence of ET capabilities of the sensor through CV measurements following the
modular surface modification in 2.5 mM [Fe(CN)6]3-/4-/0.1M KCl (scan rate 100 mV/s). As the
deposition of SP and the further backfilling with MCH clearly enhance the insulating properties of
the mixed layer, the covalent binding of MB and the subsequent attachment of HDMA facilitate the
electron transfer between the redox probe and the modified surface. Finally, the immobilization of
CJC-1295 ligand hinders ET, thus increasing the insulating properties of the functionalized layer.

15
Figure S15 Characterization of the modular surface modification through EIS measurements: first, a
significant increase of charge transfer resistance (Rct) after the deposition of 100 µM SP and
backfilling with 1mM MCH was noticed; the attachment of the MB tag and the subsequent binding
of the HMDA linker led to a gradual decrease of Rct; finally, the immobilization of the CJC-1295
agonist peptide increased the insulating properties of the sensing layer. Impedance spectra were
recorded from 100 MHz to 100 kHz and obtained in the presence of the redox probe, at a formal
potential of 250 mV vs. Ag/AgCl, and AC amplitude of 10 mV. The inset shows the equivalent circuit
model: RS is the solution resistance, CPE is the constant phase element, and ZW is the Warburg
impedance.

16
 without GHS-R1a
with 50 ng/mL GHS-R1a, EDA linker
4 with 50 ng/mL GHS-R1a, HDMA linker

I (µA)

0
-0.4 -0.2 0.0
E vs. Ag/AgCl

Figure S16 Decrease of SWV current at the binding of the GHS-Ra to the CJC-1295 immobilized
onto MB/SP/MCH electrode using two different linkers: ethylenediamine and hexamethylenediamine
(SWVs recorded in 100 mM PBS, ESW = 25 mV, ΔEstep = 1.5 mV, and f = 25 Hz).

2.5

2.0

1.5
Abs

1.0

0.5

0.0

0 5 10 15 20 25 30
GHS-R1a (ng/mL)

Figure S17 Calibration curve for GHS-R1a detection obtained in an ELISA sandwich assay. All
experiments were performed in triplicate
17
Figure S18 Anti-interference performance evaluated as SS in SWV following the incubation of the
modified sensors with the target (A) GHS-R1a and (B) anti-α-Tn antibody in the presence of non-
specific antibodies (at saturation concentration). All experiments were performed in triplicate.

Figure S19 Selectivity test for (A) GHS-R1a and- (B) Anti-α-Tn antibody detection in the presence
of non-specific antibodies

Reference

1.Mirčeski, V.; Gulaboski, R., A Theoretical and Experimental Study of a Two-step Quasireversible
Surface Redox Reaction by Square-wave Voltammetry. Croat. Chem. Acta 2003, 76 (1), 37-48.

18