Modular synthesis, spectroscopic characterization and in situ functionalization using "click" chemistry of azide terminated amide containing self-assembled monolayers

Article abstract of DOI:10.1039/c3ra43415j

A general and modular synthetic route is developed for the synthesis of azidoalkylthiol molecules, which contain an amide functional group, from common and easily available precursors. SAMs (self-assembled monolayers) formed by these amide containing azidoalkylthiols are characterised by attenuated total reflectance FTIR spectroscopy, X-ray photoelectron spectroscopy, contact angle goniometry and surface enhanced Raman spectroscopy. Signature peaks are identified in the data which allows direct observation of all the functional groups on Au electrodes bearing these monolayers. The terminal azide group is functionalised with electroactive ferrocene group in situ, using "click" chemistry. An alkyne bearing heme derivative is covalently attached using the same technique resulting in O₂ reducing electrodes.

Full text of DOI:10.1039/c3ra43415j

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PAPER
Modular synthesis, spectroscopic characterization and
in situ functionalization using ‘‘click’’ chemistry of azide
terminated amide containing self-assembled
monolayers3
Cite this: RSC Advances, 2013, 3,
17174
Sabyasachi Bandyopadhyay, Sohini Mukherjee and Abhishek Dey*
A general and modular synthetic route is developed for the synthesis of azidoalkylthiol molecules, which
contain an amide functional group, from common and easily available precursors. SAMs (self-assembled
monolayers) formed by these amide containing azidoalkylthiols are characterised by attenuated total
reflectance FTIR spectroscopy, X-ray photoelectron spectroscopy, contact angle goniometry and surface
enhanced Raman spectroscopy. Signature peaks are identified in the data which allows direct observation
of all the functional groups on Au electrodes bearing these monolayers. The terminal azide group is
functionalised with electroactive ferrocene group in situ, using ‘‘click’’ chemistry. An alkyne bearing heme
derivative is covalently attached using the same technique resulting in O2 reducing electrodes.
Received 23rd May 2013,
Accepted 16th July 2013
DOI: 10.1039/c3ra43415j
www.rsc.org/advances
SAMs is that the technique used to attach the electroactive
1. Introduction
probe vary from case to case i.e. it lacks
approach.5,21,23,24
a general
Thiols bearing amide functionalities are known to form
organized SAMs on Au surfaces much alike those formed by
aliphatic thiols.1–13 These surfaces are very stable and they
show capacitance and electrochemical shielding of the under-
lying electrodes.14–16 These surfaces can be characterized
using surface sensitive tools such as X-ray photoelectron
spectroscopy (XPS),4–6,14,17–20 ellipsometry,5,6,9,21–23 contact
angle (CA) goniometry4–6,9,14,20,22,23 and reflectance mode
FTIR spectroscopy.4–6,9,14,15,20,22–24 The N–H bending and
CLO stretching modes of these SAMs show strong evidence
for hydrogen bonding.5,6,24 Because of their biocompatibility
and stability under aqueous conditions polypeptide based
SAMs have found applications in chemistry and biology.14,25–28
In particular, amide containing SAMs have been extensively
used to investigate charge-transfer (CT) properties of biomo-
lecules attached to these surfaces.29–32 Several groups have
investigated, in detail, the CT properties of these surfaces and
a general consensus has been built that CT through peptide
bonds are more facile.33–35 However, recent results from the
Whitesides group suggests otherwise.36 The problem of
investigating charge transfer through peptide bond bearing
‘‘Click’’ chemistry37,38 is a convenient method for covalent
attachment and has been used to functionalize azide
terminated alkylthiol SAMs on Au electrodes.11,39–43 Recently
‘‘click’’ chemistry has been used to attach peptides onto
poly(ethylene glycol) (PEG) or hydrocarbon monolayers.40–42
While there is a report where azide terminated peptide
containing molecules were clicked to acetylene terminated
SAM on silicon surface,39 so far there are no reports of azide
terminated amide containing alkylthiol SAM surfaces and
their in situ functionalization. This is mainly because the
synthesis of such linkers bearing azide, amide and thiol
groups are complicated and they are inherently unstable as the
thiol group can reduce the azide groups44–46 and catalyze the
hydrolysis of the amide groups.47,48 In this manuscript we
report a modular approach for synthesizing azide terminated
alkylthiols containing amide functionality designated as
azidoalkylamidethiol (Fig. 1 and Scheme 1). Using this
approach several linker (azidoalkylamidethiol) and diluent
(alkylthiols bearing amide functionality of appropriate length
designated as alkylamidethiol) pairs are synthesised and
characterized (Fig. 1b). Throughout the manuscript the
azidoalkylthiol, the azidoalkylamidethiol and the alkylami-
dethiol are abbreviated as AzAl, AzAm and AlAm, respectively.
These linker diluent pairs are used to form mixed SAMs on Au
electrodes. SAMs formed using these molecules are charac-
terised using XPS, attenuated total reflectance FTIR (ATR-
FTIR) spectroscopy, surface enhanced Raman spectroscopy
Department of Inorganic Chemistry, Indian Association for the Cultivation of Science,
2A & 2B Raja S. C. Mullick Road, Jadavpur, Kolkata-700032, India.
E-mail: icad@iacs.res.in; Fax: +91-33-2473 2805; Tel: +91-33-2473 4971 extn 1366
Electronic supplementary information (ESI) available: Details of the synthetic
3
procedures and schematic diagram of synthesis procedure of amidealkylthiol
diluents. SERS of N3C8NHCOC4SH SAM on Au disk. CA goniometry of all linker
SAMs on Au wafers. Characterization data (1H, 13C NMR, ESI-mass spectra) of
selective compounds. See DOI: 10.1039/c3ra43415j
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chromatography was performed on SiO2 (60–100 mesh).
Tetrahydrofuran (THF) and methanol were dried using
metallic sodium plates and dichloromethane (DCM) was dried
using calcium hydride powder. Au(111) wafers were bought
from Platypus Technologies (1000 Å of Au on 50 Å of Ti
adhesion layer on top of a Si(111) surface). The FTIR data of
compounds were measured on a Shimadzu FTIR 8400S
instrument. All NMR spectra were recorded on a Bruker
DPX-300 or DPX-500 spectrometer at room temperature. The
mass spectra were recorded by a QTOF Micro YA263 instru-
ment. All electrochemical experiments were performed using a
CH Instruments (CHI720D electrochemical analyzer). The
platinum counter electrodes, Ag/AgCl (satd. KCl solution)
reference electrodes, and the Teflon1 plate material evaluat-
ing cell (ALS Japan) were purchased from CH Instruments.
2.1. Formation of mixed SAMs
Gold wafers (Au) were cleaned by immersion in a Piranha
solution (1 : 3 H2O2/H2SO4) for 1 min (Piranha solution can be
explosive and needs to be handled with extreme caution),
followed by rinsing with copious amounts of deionized
water.11 This was followed by cleaning the wafers electro-
chemically by sweeping the voltage several times between 1.5
and 20.3 V in 0.5 M H2SO4 using Ag/AgCl reference electrode
which is reported to reduce the microscopic roughness of the
Au surface.52 After the electrochemical wash the substrate was
rinsed again with deionized water, followed by ethanol and
then dried with nitrogen gas.
For cyclic voltammetry experiments, cleaned Au wafers
purged with N2, were immersed into the deposition solution
and kept for at least 24 h. The deposition solution was
prepared by dissolving 1-octanethiol (diluent) and 1-azidoun-
decane-11-thiol (linker) in the desired ratio (typically 9 : 1) for
AzAl and C8SH mixed SAM and Cn+2NHCOC4SH (diluent) and
N3Cn+2NHCOC4SH (linker) in 9 : 1 ratio for AzAm-AlAm mixed
SAMs in ethanol (Fig. 2). The total thiol concentration of the
deposition solutions was always maintained at 0.4 mM for
AzAl–C8SH mixed SAM and 1 mM for AzAm-AlAm mixed SAMs
for CV experiment.
Fig. 1 (a) Representation of the basic structural backbone of AzAm molecules,
where n = 0, 2, 6 and m = 4. (b) Pairs of linkers and diluents, where AzAl pair up
with octanethiol and AzAm pair up with AlAm to form mixed SAMs on Au
surfaces; AzAl = azidoalkylthiol, AzAm = azidoalkylamidethiol and AlAm =
alkylamidethiol.
(SERS) and CA goniometry. These SAMs are then functiona-
lized with electroactive species such as ferrocene (Fc) (Fig. 2)
and electrocatalysts such as heme in situ using ‘‘click’’
chemistry.49–51 Cyclic voltammograms have been measured
as an evidence of covalent attachment on top of the surface.
The cyclic voltammogram of AzAl and octanethiol (C8SH)
mixed SAM has been used as a reference data point.11
2.2. X-Ray photoelectron spectroscopy (XPS)
The sample was prepared by dissolving the AzAm 1 and AzAm
3
separately in ethanol solvent; the concentration was
maintained at approximately 2 mM. After 48 h of keeping
the Au surface in the SAM solution, the surface was rinsed with
ethanol and was dried by blowing with N2. The XPS of AzAm 1
SAM was measured before and after clicking the EtnFc and
that of AzAm 3 SAM was measured only after clicking the
hemin-1Fe. The sample for X-ray photoelectron spectroscopy
(XPS) was kept under nitrogen until being transferred to the
vacuum chamber. The spectra were collected in a Omicron
(model: 1712-62-11) spectrometer using a high-resolution
monochromatic Al-Ka source at 1486.7 eV under 15 kV voltage
and 10 mA current maintaining a base pressure of 5 6 10210
mbar. The binding energies were calibrated to the Ag 3d5/2
peak at 368.2 eV. All peaks were fitted using a Gaussian–
Lorentzian (GL) peak fit using PeakFit-V4 software.
2. Experimental
2-Bromoethylamine hydrobromide and triethylamine were
purchased from Spectrochem Pvt. Ltd. and all other reagents
were purchased from Aldrich chemical company or Merck
Germany and used without any further purification. HPLC
grade methanol, dimethylformamide (DMF), and deuteriated
solvents were purchased from Aldrich. Dimethyl sulfoxide
(DMSO) and all other solvents were purchased from Merck
Germany or Rankem India. Unless otherwise mentioned all
reactions were performed at room temperature and column
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Scheme 1 Synthesis scheme of (A) 1-azidoethylamine, 1-azidobutylamine, 1-azidooctylamine, 1-azidoundecan-11-thiol (AzAl) and (B) azidoalkylamidethiols (AzAm).
Scheme for diluents are in ESI.3 Brown boxes show azidoalkylamine molecules. Phth = phthalimide.
2.3. ATR-FTIR spectroscopy
2.4. Surface-enhanced Raman spectroscopy (SERS)
The SAM deposited wafer was taken out from the solution and
thoroughly washed with ethanol. After drying the surface with
N2 flow the ATR-FTIR data were measured in a PerkinElmer-
400 instrument using DTGS detector at room temperature.
The Kr+ laser (Sabre Innova, model SBRC-DBW-K) from
Coherent and a spectrograph (model Trivista 555) fitted with
an electronically cooled Pixis CCD from Princeton Instruments
were used. The excitation wavelength used in the surface
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Fig. 2 The schematic representation of SAM formation and its in situ covalent modification.
Raman experiments was 676.4 nm and the power applied on
the sample was around 40 mW.
2.6. Covalent attachment of ethynylferrocene and hemin-1Fe
onto the SAMs
All Raman experiments were done using Au disks which
were mechanically polished with 1, 0.3 and 0.05 mm alumina,
respectively, and then rinsed with triple distilled or deionized
water. These surfaces were roughened following the reported
procedure53 and then immersed into the SAM solution. Two
different SAM solutions of the linker molecules such as AzAm
1 and AzAm 3 were prepared separately by dissolving them in
ethanol solvent maintaining the concentration of the ethanolic
solution at 2 mM.
Gold wafers immersed in the deposition solution were taken
out and rinsed with ethanol and then deionized water
successively to remove any excess adsorbate and then dried
with argon or nitrogen gas to remove any residual solvent. The
wafers were then inserted into a plate material evaluating cell
(ALS Japan). After the background current (BK) has been
measured (details in CV section) the surface was cleaned with
deionized water, then 60 ml of 100 mM Cu2SO4?5H2O (aq
solution), 300 ml of 100 mM ascorbic acid (aq solution), and 30
ml of 10 mM ethynylferrocene (EtnFc) (DMSO solution) were
added to 1 ml 3 : 2 DMSO–water mixture11,50 (Fig. 2). For
attachment of Hemin-1Fe, 100 ml of 100 mM Cu2SO4?5H2O,
200 ml of 100 mM ascorbic acid and 40 ml of 10 mg/ml Hemin-
1Fe (MeOH solution) were added to 1 ml 3 : 2 dimethyl
sulfoxide (DMSO)–water mixture.49 Then the thoroughly mixed
solution was poured onto the gold surface. The reaction was
allowed to proceed for 1–2 h at room temperature in the dark.
Then the resultant surface was thoroughly rinsed successively
with DMSO, ethanol and water.
2.5. Contact angle (CA) goniometry
Static CA of monolayer based films were measured on a
webcam-based semiautomatic contact angle meter at 25 uC
using Milli-Q water as a probe liquid. Drops of liquid having a
volume of 5 ml were added on each monolayer to measure the
CA. Piranha-washed and electrochemically cleaned surfaces
were used for monolayer deposition. Four different SAM
solutions of four different linker molecules were prepared by
dissolving them separately in ethanol maintaining a solution
concentration of 2 mM.
2.7. Cyclic voltammetry (CV) experiments
The Au wafer was fitted within the plate testing material by
using a viton O-ring of inner diameter 7.6 mm. pH 7 buffer
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containing 100 mM disodium hydrogen phosphate and 100
mM potassium hexafluorophosphate was used as the electro-
lytic solution for all cyclic voltammetric experiments. Ag/AgCl
was used as a reference electrode and Pt wire as a counter
electrode. The background current of SAMs were recorded
(before clicking the electroactive species) within a range from
+500 to 2500 mV at a scan rate of 50 mV s21 and 1 V s21. After
covalent attachment of the electroactive species the cyclic
voltammogram was recorded at 1 V s21 scan rate for both
EtnFc (Fig. 7) and Hemin-1Fe (Fig. 9b).
2.8.2.2. 1-Azidobutylamidebutylthiol [N3C4NHCOC4SH] (AzAm 2).
The product was purified by column chromatography on silica
gel with DCM as the eluent. A light yellow liquid compound
was obtained (thiol and disulfide mixture).
Yield: 75%. 1H NMR (CDCl3, 500 MHz): d 5.8, 6.1 (s, 1H),
3.55 (t, 2H, J = 6 Hz), 3.26–3.33 (m, 4H), 2.19 (t, 2H, J = 7.5 Hz),
1.58–1.8 (m, 8H). 13C NMR (CDCl3): d 172.55–172.87, 51, 44.6,
38.9, 38.7, 36.1, 36, 35.7, 33.5, 32, 28.7, 27, 26.3, 24.5, 24.4,
24.3, 23.02. IR spectra (cm21): 2550 (SH stretching), 2096 (N3
stretching), 1647, 1554 (amide stretching and bending). ESI-
MS (+ve ion mode, MeOH) m/z = 253.1 (20%) [MNa+], 459.27
(98%) [M1H+], 481.25 (100%) [M1Na+], where M1 is the
disulfide compound. Elemental analysis for C9H18N4OS: C,
46.93; H, 7.88; N, 24.32. Found: C, 47.18; H, 7.92; N, 24.18%.
2.8. Synthesis
Azidoalkylamines (brown boxes in Scheme 1A) are key
moieties used for the synthesis of the respective AzAms.
Scheme 1A summarizes the synthetic procedures for all the
azidoalkylamines used in this study. While 1-azidoethylamine
was formed in a single step by the addition of 3 equivalents of
sodium azide with 1-bromoethylamine in water, 1-azidobuty-
lamine was synthesized by treating 1-bromobutylphthallimide
with sodium azide followed by the addition of hydrazine
hydrate to the product obtained from the first step of the
reaction. To synthesize 1-azidooctylamine, 1-bromooctanol
was first treated with potassium phthalimide followed by
methanesulfonyl chloride, sodium azide and hydrazine
hydrate in the next three consecutive steps. The detailed
procedures of all these steps and that of the intermediate steps
for the thiol formation from all the above mentioned
azidoalkylamines are discussed in the supporting information
2.8.2.3. 1-Azidooctylamidebutylthiol [N3C8NHCOC4SH] (AzAm 3).
The product was purified by column chromatography on silica
gel with 1.5% MeOH–DCM mixture as the eluent. A light
yellow viscous compound was obtained.
1
Yield: 72%. H NMR (CDCl3, 300 MHz): d 5.7 (s, 1H), 3.5 (t,
2H, J = 6 Hz), 3.16–3.24 (m, 4H), 2.17 (t, 2H, J = 6.3 Hz), 1.68–
1.78 (m, 8H), 1.1–1.5 (m, 8H). 13C NMR (CDCl3): d 172.4, 51.5,
44.7, 39.6, 35.8, 32, 29.7, 29.2, 29.1, 28.8, 26.8, 26.7, 23.1. IR
spectra (cm21): 2513 (SH stretching), 2095 (N3 stretching),
1645, 1550 (amide stretching and bending). ESI-MS (+ve ion
mode, MeOH) m/z = 309 (75%) [MNa+], 571 (20%) [M1H+], 593
(100%) [M1Na+]. M1 is disulfide compound. Elemental analysis
for C13H26N4OS: C, 54.51; H, 9.15; N, 19.56. Found: C, 54.38; H,
9.32; N, 19.58%.
(ESI ). The final thiols (AzAl and AzAms, Scheme 1A and B)
3
from thioacetate were synthesised following the reported
procedures.11
2.8.1. 1-Azidoundecan-11-thiol [N3C11SH] (AzAl). 1-
Azidoundecan-11-thiol was synthesised as according to the
reported procedure.11
3. Results
3.1. Synthesis
1
Yield of the final product: 78%. H NMR (CDCl3, 500 MHz):
d 3.16 (t, 2H, J = 6.9 Hz), 2.58 (t, 1H, J = 7.5 Hz), 2.4 (q, 2H, J =
7.5 Hz), 1.45–1.57 (m, 4H), 1.18–1.26 (m, 14H). 13C NMR
(CDCl3): d 51.4, 34, 29.35, 29.12, 29, 28.9, 28.74, 28.41, 28.26,
26.61 24.6. IR spectra (cm21): 2555 (SH stretching), 2102 (N3
stretching). Elemental analysis for C11H23N3S: C, 57.6; H,
10.11; N, 18.32. Found: C, 57.38; H, 10.32; N, 17.98%.
In this report AzAm moieties i.e. N3Cn+2NHCOCmSH (n = 0, 2, 6
and
N3Cn+2NHCOCmSH acts as the linker (constitutes 10% of the
SAM deposition solution, Fig. 2) then molecule
n+2NHCOCmSH acts as the diluent (constitutes 90% of the
m
=
4; Fig. 1) are prepared. If
a
molecule
a
C
SAM deposition solution). The synthesis of azidoalkylamines
i.e. N3Cn+2NH2 (n = 0, 2, 6; brown boxes in Scheme 1) can be
performed using several routes reported in the literature.54–56
The selective reduction of a symmetric diazide to form an
azidoalkylamine under biphasic conditions, while convenient,
is complicated by the need to separate the triphenylphosphine
oxide product.56 In our experience this method does not work
well for longer chains due to limited solubility of monoamines
in aqueous acid. Alternatively we found that one can attain
high yields at the azidoalkylamine step by using a bromoalk-
ylphthalimide (Scheme 1A) or preparing a hydroxyalkylphtha-
limide (Scheme 1A) derivative from a dibromoalkane or
bromoalkanol and then substituting the terminal leaving
group with an azido group followed by reduction of the
phthalimide group to an amine (Scheme 1A). The azidoalk-
ylamine thus synthesized can be coupled to a haloalkyl acid
halide (XCmCOX; X = Cl, Br and m = 4) followed by substitution
of the terminal halide group with thioacetate followed by acid
hydrolysis to obtain a terminal thiol (Scheme 1B). It is
2.8.2. Azidoalkylamidethiol [N3Cn+2NHCOCmSH] (AzAm).
See Scheme 1B, the details of first two steps are given in the
supporting information. The thiol compounds (AzAms),
3
shown as final product in Scheme 1B were synthesised as
according to the reported procedure.11
2.8.2.1. 1-Azidoethylamidebutylthiol [N3C2NHCOC4SH] (AzAm 1).
The product was purified by column chromatography on silica
gel with a 1.5% MeOH–DCM mixture as eluent. A light yellow
viscous compound was obtained.
1
Yield: 80%. H NMR (CDCl3, 500 MHz): d 6 (s, 1H), 3.53 (t,
2H, J = 6 Hz), 3.39–3.44 (m, 4H), 2.22 (t, 2H, J = 7 Hz), 1.75–1.83
(m, 4H), 1.2 (s, 1H). 13C NMR (CDCl3): d 173, 51, 44.6, 39, 35.6,
32, 23. IR spectra (cm21): 2519 (SH stretching), 2102 (N3
stretching), 1647, 1550 (amide stretching and bending). ESI-
MS (+ve ion mode, MeOH) m/z = 204.1 (100%) [MH+].
Elemental analysis for C7H14N4OS: C, 41.56; H, 6.98; N,
27.70. Found: C, 41.38; H, 7.22; N, 27.58%.
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advisable to avoid using basic conditions where a thiol can be
deprotonated to thiolate which is a much stronger reducing
agent and can reduce the azide group or even O2.44–46
transfer rate through the SAMs33,36 as well as the formal
potential of the species.
3.2. X-Ray photoelectron spectroscopy
The synthesis strategy discussed uses relatively inexpensive
starting materials and high yielding steps. The synthesis is
highly modular as the spacer regions (n or m) (Fig. 1) between
the terminal azide or thiol and the amide functional group can
be easily varied. This is an important attribute as this allows
controlling the distance between the electrode (bound to the
thiol end) and the electroactive species (to be attached to the
azide end) which is known to allow the tuning of the electron
X-Ray photoelectron spectroscopy5,6,17,18,51,57 is an useful
spectroscopic technique to determine functional groups
present on the surface as well as the elemental composition
of the surface. The XPS data obtained, clearly indicate that the
SAM formed by AzAm 1 on the Au surface contains carbon,
nitrogen, oxygen and sulfur (Fig. 3a–d, Table 1). Moreover after
clicking the EtnFc onto the SAM the XPS data clearly shows the
presence of Fe (Fig. 3e). The major C 1s peak shown by the
Fig. 3 High-resolution XP spectra for N3C2NHCOC4SH (AzAm 1) SAM fabricated on gold wafer. Blue and brown spectra represents the SAM before and after EtnFc
click, respectively; C 1s, N 1s, O 1s, S 2p and Fe 2p regions are shown. The peak fittings are represented by pink, red, light green and sky-blue colour codes. The EtnFc
spectra belong to clicked surfaces. The binding energy values are in eV.
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Table 1 XPS data and qualitative elemental analysis of AzAm 1 SAMs on Au wafers before and after the click reaction with EtnFc and also the XPS data of Hemin-1Fe
clicked AzAm 3 SAMs
BE before click
Atom% by XPS
BE after click
Atom% by XPS
N3C2NHCOC4SH SAMs (AzAm 1) clicked by EtnFc
C 1s (alkyl, CLO)
285.2, 287.6
399.9, 405
532, 534.1
161.6, 163.2
51.7 (53.8)
28.2 (30.8)
8.4 (7.7)
284.6, 287.6
400
531.8
161.7, 163.2
707.8, 720.7
68.9 (71.0)
14.4 (16.4)
5.1 (4.2)
7.2 (4.2)
4.4 (4.2)
N 1s
O 1s (CLO, OH)
S 2p3/2, S 2p1/2
Fe 2p3/2, Fe 2p1/2
11.7 (7.7)
N3C8NHCOC4SH SAMs (AzAm 3) clicked by Hemin-1Fe
C 1s (alkyl, CLO)
N 1s
O 1s (CLO, OH)
S 2p3/2, S 2p1/2
Fe 2p3/2, Fe 2p1/2
284.7, 287.6
398.3, 399.8, 405.2
531.8, 533.6
161.6, 163.2
712.8, 723.0
75.5 (78.0)
16.4 (14.7)
4.5 (4.4)
2.4 (1.5)
1.2 (1.4)
a
BE = Binding energy. Values shown within parentheses are the atom percentages estimated from the molecular formula of the respective
SAM forming molecule.
ordinary SAM (before click) at 285.2 eV is due to the alkyl
carbon present in the film (Fig. 3a). The less intense C 1s peak
at a slightly higher binding energy of 287.6 eV is due to the
presence of carbonyl carbon of the amide group.5,17,42,58,59 An
additional peak appearing at 286.3 eV, resolved by Gaussian–
Lorentzian fitting of the data, possibly originates from the C
atoms directly bound to the amide and azide N.58 Both the
amide carbon and the carbon directly bound to the N of azide
group or amide N are electron deficient and hence they have
higher binding energy than the alkyl carbon atoms. After
clicking the EtnFc, the C 1s shifted to slightly lower energy
such as 284.6 eV which may be because of the presence of
electron rich ferrocenyl C. Furthermore the intensity of the
C 1s peak is also increased, clearly indicating the presence of
more C on the clicked surface (Fig. 3a, Table 3). In ordinary
amide SAM the N 1s peak for amide and azide functional
groups appear at two distinct regions, ca. 399.9 and 405 eV.
The lower binding energy peak is due to the amide N and the
two terminal N atoms of the azide group and the higher
binding energy peak originates from the electron deficient
central N atom of the azide group.51,58–60 On the other hand in
the clicked surface, the N 1s peak of higher binding energy is
absent whereas a broad peak appearing at 400 eV indicates the
formation of 1,2,3-triazole (Fig. 3b).60,61 The O 1s peak due to
the presence of a carbonyl group on both the normal and
clicked surfaces appear at 532 and 531.8 eV, respectively
(Fig. 3c).5,17,58 The broadening of the peak of the normal
surface may be due to the presence of hydroxyl oxygen of
ethanol (from the deposition solution).17,58 The S 2p3/2 and
S 2p1/2 peaks appear at 161.6 and 163.2 eV, respectively, for
both the normal and clicked surfaces in 2 : 1 ratio from the
sulfur bound to the gold.5,17,18,59 Thus the XPS data of the
ordinary SAM is consistent with immobilization of the amide
containing, azide terminated thiols on the Au electrodes. An
additional signal appearing at 707.8 and 720.7 eV for Fe 2p3/2
and Fe 2p1/2, respectively (Fig. 3e), is further an indication for
the formation of a 1,2,3-triazole moiety after EtnFc is clicked
on the AzAm 1 surface.60,62
To confirm the attachment of the electrocatalyst hemin-1Fe
onto the surface, the XPS data of the hemin-1Fe clicked AzAm
3 surfaces have been collected. The peaks for C 1s, O 1s and
S 2p (Table 1) occur in the same region as observed in AzAm 1
SAM (Fig. 3) indicating the formation of AzAm 3 monolayer.
On the other hand peaks appearing for N 1s and Fe 2p regions
in AzAm 3 SAMs (Fig. 4) are very different from those of the
AzAm 1 SAMs (Fig. 3). An additional N 1s appeared at 398.3 eV
due to the presence of porphyrin N which is at lower energy
relative to the amide nitrogens due to their anionic nature.60,63
The appearance of a very weak peak at 405.2 eV (in the noise
level) is due to the presence of free azide on the surface. The
Fe 2p3/2 and Fe 2p1/2 peaks appearing at 712.8 and 723.0 eV
clearly indicate the presence of Fe(III) porphyrin on the
surface.64,65
The elemental composition of the surface has been
determined by XPS. The binding energy of each peak before
and after the click reaction and the surface composition are
summarized in Table 1. This is a qualitative analysis23 of the
surface composition and is estimated from the area under the
peaks.5,17 The atom percentage determined by XPS (Table 1) is
the ratio of the peak area of a particular element with the total
area obtained for all the elements of the molecule. The values
shown in parentheses are the atom percentages estimated
from the molecular formula of the species used to prepare the
SAM.
3.3. ATR-FTIR
ATR-FTIR data show the presence of amide functionalities on
the surface consistent with previous reports17,18,20,24 (Fig. 5). In
organic self-assembled monolayers on reflective substrates,
specific information about the alkyl chain can be obtained
from the relative intensities of the methylene stretching
bands.5,6 Fig. 5 shows the ATR-FTIR spectra of AzAl SAM,
AzAm 1 SAM and AzAm 3 SAM on Au wafers. The CH2 (asym)
and CH2 (sym) stretching frequencies are obtained at 2850 and
2918 cm21, respectively (Table 2), suggesting the formation of
close-packed alkyl chains on gold surfaces (Fig. 5b). The amide
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Fig. 4 High-resolution XP spectra of Hemin-1Fe clicked N3C8NHCOC4SH (AzAm 3) SAM fabricated on gold wafer. C 1s, N 1s, O 1s, S 2p and Fe 2p regions are
represented here. The peak fittings are shown by pink, red, light green and green colour codes. The binding energy values are in eV.
I (carbonyl stretching, nCLO) and the amide II (in plane N–H
bending, bN–H) bands appear at 1645 and 1536 cm21
–CLO and the –N–H bonds are nearly parallel to the surface
plane.14,24 Peaks appearing at 1450 and 1492 cm21 are due to
the symmetrical bending motion of the alkyl group.66 The IR
peak intensity increases when the dipole moment of the
absorber is perpendicular to the reflective surface plane, and
decreases when the dipole moment of the absorber is parallel
to the reflective surface plane.20 These results are comparable
to the results reported by Clegg et al.5 and Tam-Chang et al.6
where the amide functionalities are close to the surface (one or
two C atoms apart from surface) and Sabapathy et al. where
,
respectively (Fig. 5c, Table 2). The amide (I and II) bands shift
to higher energies relative to the values obtained of the pure
product suggesting the presence of interchain hydrogen
bonding.5,6,24 Hutchison proposed that any shift of the amide
II peaks to higher energies indicate an increasing restriction
imposed on the N–H bending motion and due to the
intermolecular H-bonding interactions within the mono-
layer.14 The broad shape of amide I peak suggests that the
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Fig. 5 Attenuated total reflectance spectroscopy of AzAm 1, AzAm 3 and AzAl surfaces in (a) azide region (1900–2280 cm21) and (b) methylene region (2780–3000
cm21). (c) Amide region of AzAm surfaces (1300–1900 cm21) on gold (Au) wafer.
the amide functionality was five carbon atoms away from the
surface.24
nC–N).57,69–71 The Raman spectra of the AzAm 1 SAM covered
surface was recorded before and after covalent attachment
with EtnFc (Table 2, Fig. 6). The carbonyl stretching frequency
(nCLO), i.e. amide I, appears at 1652 cm21 (Fig. 6b, green and
red)57 and is much broader and weaker than amide II which
appears at 1532 cm21 (Fig. 5b, green and red).57,69 The amide
III (out-of-plane C–N stretching mode in combination with in-
plane N–H bending) appears at 1303 and 1306 cm21 as a
doublet (Fig. 6a, green and red) in this surface, consistent with
previous reports.17,69 The naz-asym which is intense in IR, is
weak in Raman.72,73 Conversely, the symmetric stretching
frequency of the azide group (naz-sym) can be observed using
SERS.69,72 The SERS data clearly indicate the naz-sym at 1340
cm21. Note that the naz-sym which appears at 1340 cm21
(Fig. 6a, green), totally disappears after covalent attachment of
the EtnFc (red). Instead new peaks appear at 1291, 1371 and
1538 cm21 (Fig. 5a and b, red) which represents the stretching
frequencies of the triazole ring (ntrz) formed via Huisgen 1,3-
dipolar cycloaddition of the azide and alkyne (Table 2).70,71,74
This provides a direct spectroscopic evidence for the covalent
attachment on these surfaces.
The asymmetric stretching of the azide group (naz-asym
)
appears at 2100 cm21 51,67,68
(Fig. 5a, Table 2) which is much
,
broader40,41 and weaker relative to the amide I. This may be
because of the much weaker interaction of the azide terminal
dipoles with the surface plasmons due to the significant
separation between the terminal azide group and the surface.
3.4. Surface-enhanced Raman spectroscopy (SERS)
Amide bearing SAMs are characterized by three vibrations in
SERS. These are amide I (carbonyl stretching, nCLO), amide II
(in plane N–H bending, bN–H) and amide III (C–N stretching,
Table 2 Designated peaks of ATR-FTIR spectra and SERS of AzAm 1 SAM
Wavenumber/cm21
ATR-FTIR
SERS
Designated peak
y1645
y1652
y1532
Amide I
Amide II
Alkyl bending
Amide III
Azide group
Triazole ring stretch
Triazole ring stretch
Triazole ring stretch
Methylene group
Methylene group
y1536
The SERS spectrum of AzAm 3 SAM (Figure in ESI ) is
3
y1450, 1492
qualitatively the same as that of the AzAm 1 SAM with the
amide I, amide II and amide III peaks at 1654, 1531 and 1305
cm21, respectively. The symmetric stretching frequency for the
y1303, 1306
y1340 (sym)
y1291
y1371
y1538
y2100 (asym)
azide group appears at 1341 cm21
.
y2850
y2918
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3.6. Covalent attachment
3.6.1. Double-layer capacitance. Double-layer capaci-
tance14,15,29 (Cdl) is an useful parameter to describe the
electrochemical shielding of the electrode by the SAMs, since
a well-ordered SAM in the absence of any attached redox
species behaves like an ideal capacitor.14 From the Helmholtz
parallel plate capacitor model, Cdl in well-ordered SAMs varies
inversely with film thickness d. According to Clegg and
Hutchison,14 Cdl can also be determined from the charging
current of the SAMs ic as:
Cdl = ic/nA
(1)
where n is scan rate and A is electrode surface area. For AzAl
and C8SH mixed SAMs, Cdl is estimated to be 4 ¡ 0.03 mF
cm22, consistent with a previous report (Table 3).11 For an
AzAm mixed SAM having the same chain length as AzAl mixed
SAM, Cdl is estimated to be 21 ¡ 2 mF cm22. The Cdl decreases
on increasing the chain length of AzAm mixed SAMs from 21
¡ 2 mF cm22 (for AzAm 1 SAM) to 14 ¡ 0.7 mF cm22 (for AzAm
3 SAM) (Table 3, Fig. 7).33 Similar decrease of Cdl with
increasing chain length has been previously reported.14 Note
that, even for AzAm 3 mixed SAM, where there are 14 atoms in
the chain the Cdl is more than the octanethiol SAM where only
8 atoms in the chain, i.e. a peptide bond containing SAM has
higher Cdl than an aliphatic SAM, consistent with previous
reports.14,33
Fig. 6 Surface-enhanced Raman spectrum of AzAm 1 SAM on Au disks – (a) low
range and (b) high range. * represents the plasma lines. ntrz and naz-sym
represents triazole stretching frequency and azide symmetric stretching
frequency, respectively. Spectrum of AzAm 3 SAM (full range) is shown in ESI.3
3.6.2. Cyclic voltammetry (CV). Gold electrodes modified
with mixed monolayers containing AzAm SAMs and AzAl SAMs
with their respective diluents were functionalised in situ using
‘‘click’’ chemistry.38,75 Fig. 7 shows a typical cyclic voltammo-
gram obtained for EtnFc functionalized mixed SAMs. Here, the
electrochemical properties of the AzAl and C8SH mixed SAM
surface has been used as a reference system and it reproduces
earlier reports.11,50,51 A clear Fc+/Fc process was observed
around 330 ¡ 4 mV vs. Ag/AgCl when EtnFc was ‘‘clicked’’ on
the mixed monolayer (Fig. 7 and Table 3). This provides direct
evidence for covalent attachment of EtnFc units to the azide
terminus of the SAMs. In the AzAm-AlAm mixed SAM surfaces
of the same chain length the Fc+/Fc process occurs at 332 ¡ 6
mV (Table 3). A slight shift of redox potential of Fc clicked
AzAm-AlAm mixed SAMs is known to occur when the surface
was fully covered with amide diluent rather than alkyl
diluent.33 Collman et al.11 reported that on covalent attach-
ment of EtnFc to AzAl–C8SH SAM the charging currents at
relatively positive potentials (i.e. .0.4 V) increases (Fig. 7).
3.5. Contact angle (CA) goniometry
The changes in surface wettability after monolayer deposition
were investigated by measuring the static CAs of a water
droplet.4,6,20,57 The CA of a bare gold wafer is determined to be
50.5 ¡ 1.5u. Upon N3C11SH SAM formation on the surface, the
CA increases to 77 ¡ 1.8u, consistent with previous reports.51
The CAs were measured for AzAm 1 SAM, AzAm 2 SAM and
AzAm 3 SAM to be 65 ¡ 1.7, 70 ¡ 1.5 and 84 ¡ 1.6u,
respectively. These values indicate an increased hydrophilicity
of amide-containing surfaces relative to AzAl surfaces.
Furthermore the increasing CA with increasing chain length
indicates that the hydrophobicity of the surface increases as
the number of methylene units on the AzAm SAMs are
increased.
Table 3 Electrochemical characteristic of SAMs
Linker (Lin)
Diluent (Dil)
Lin : Dil
Chain length
Ea/mV
Cdlb/mF cm22
N3C11SH
C8SH
1 : 9
1 : 9
1 : 9
1 : 9
8
8
9
14
330 ¡ 4
332 ¡ 6
329 ¡ 6
330 ¡ 5
4 ¡ 0.03
21 ¡ 2
18 ¡ 1.2
14 ¡ 0.7
N3C2NHCOC4SH
N3C4NHCOC4SH
N3C8NHCOC4SH
C2NHCOC4SH
C3NHCOC4SH
C8NHCOC4SH
a
b
Formal potential of redox active species vs. Ag/AgCl. Double layer capacitance.
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Fig. 7 Cyclic voltammogram of the mixed SAMs of AzAl and the mixed SAMs of
AzAms using Ag/AgCl as a reference electrode and Pt wire as a counter
electrode at 1 V s21 scan rate. The coverage maintained here is around 10% in
all SAMs. pH 7 buffer was used as electrolyte. The background current is the
cyclic voltammogram of AzAl + C8SH SAM before covalent modification with
ferrocene.
In the case of AzAm-AlAm SAMs the same electrochemical
behaviour was observed when either of the EtnFc (Fig. 7) or
Hemin-1Fe (Fig. 9a) was attached to the SAM. This indicates
that covalent modification of AzAm-AlAm mixed SAMs with
EtnFc or Hemin-1Fe leads to little disruption of the monolayer
packing.11
Fig. 9 (a) Background current (BK) and oxygen reduction current of Hemin-1Fe
clicked azidoalkylamidethiol mixed SAMs at 50 mV s21 scan rate. (b) Cyclic
voltammogram of FeIII/FeII reduction process of Hemin-1Fe at 1 V s21 scan rate.
The voltammogram were recorded in the presence of silver wire reference
electrode. BK is the cyclic voltammogram of N3C2NHCOC4SH + C2NHCOC4SH
SAM before clicking the electrocatalyst Hemin-1Fe.
Integration of the charge under the cathodic or anodic wave
provides an estimation of the number of electroactive species
present on the surface. For these azidoamide containing
mixed monolayers the integrated current indicates that the
surface coverage is around 6–7% (where the mole fraction of
deposition solution was maintained at 10%) and the number
of molecules adsorbed onto the surface was y4.0 6 1013
from integrated Fc+/Fc current) increases with increasing mole
fraction of the linker molecule in the deposition solution
(xsoln). The coverage initially increases linearly with increasing
mole fraction of the linker, and then saturates at (24 ¡ 1.7) 6
10211 mol cm22 and (19 ¡ 1.3) 6 10211 mol cm22 i.e. even
when x = 1, no further increase in Fc/Fc+ current was observed
for AzAm 1-AlAm 1 mixed SAMs and AzAm 3-AlAm 3 mixed
SAMs, respectively (Fig. 8). Alternatively, when x = 1 was used,
molecules cm22, i.e. 6.7 6 10211 mol cm22 11 The coverage of
.
electroactive species covalently bound to the monolayer can be
controlled by changing the ratio of linker and diluent thiols in
the monolayer deposition solution.51 This was demonstrated
on a mixed AzAl–C8SH SAM, AzAm 1-AlAm 1 SAM and AzAm
3-AlAm 3 SAM by using different ratios of linker and diluent in
the deposition solution followed by covalent attachment of
EtnFc to them. For these mixed SAMs the coverage (estimated
i.e. when the full surface was covered with AzAl SAM51
a
coverage of (36 ¡ 2.7) 6 10211 mol cm22 was observed
(Fig. 8). This implies that the surface packing of AzAm SAMs
are less dense than that of the AzAl SAM. This could be due to
the H-bonding network which separates the adjacent thiol
molecules on the surface.6
Apart from ferrocene, the gold electrodes modified with the
mixed AzAm-AlAm SAMs have also been covalently functiona-
lized with alkyne modified hemin. Hemin is known to catalyze
electroreduction of O2.76–79 In the presence of O2 in the buffer,
a clear well developed catalytic O2 reduction was observed at
2450 ¡ 8 mV vs. Ag/AgCl (Fig. 9a). Thus the alkyne-modified
hemin moiety can be covalently attached to the AzAm SAM and
its exhibits electrocatalytic O2 reduction. In Ar atmosphere it
shows a FeIII/II process at 2340 ¡ 5 mV vs. Ag/AgCl (Fig. 9b)
consistent with our previous report.49
Thus the AzAm SAM is also able to covalently attach an
electrocatalyst such as Hemin-1Fe. The data shows that the
electrochemical and electrocatalytic behaviour of this cova-
Fig. 8 The coverage effect of N3C11SH + C8SH mixed SAM, N3C2NHCOC4SH +
C2NHCOC4SH mixed SAM and N3C8NHCOC4SH + C8NHCOC4SH mixed SAM.
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lently attached Hemin-1Fe is quite comparable to the data
obtained on an aliphatic SAM surface.49
lization of the ferrocene group and iron porphyrin electro-
catalyst are used as examples.
These mixed azide terminated and methyl terminated
amide groups bearing monolayers can be functionalized in
situ using electroactive species. The fraction of the surface
covered by these electroactive species can be effectively
controlled by regulating the proportion of the azide termi-
nated linker molecules in the deposition solution. The
electrodes functionalized with EtnFc and Hemin using ‘‘click’’
chemistry behave very similarly to the alkylthiol SAM counter-
parts suggesting that these electrodes are amenable towards
functionalization with different types of species.
In summary we report a general synthetic strategy and
surface characterization for azide terminated amide contain-
ing azidoalkylthiols. The modular approach developed allows
easy variation of the intervening chain lengths. These
molecules can be immobilized on Au electrodes forming
monolayers with H-bonding networks. These azide groups are
functionalized in situ, using ‘‘click’’ reaction, with ferrocene
and hemin groups. The resultant functionalized surfaces
exhibit the electrochemical properties of the parent com-
plexes. This is the first report of in situ ‘‘click’’ reaction on
amide containing SAMs. The electron transfer rate through
these azidoalkylamidethiol mixed SAMs will be investigated
further in the near future.
4. Discussion
In this manuscript we describe a general synthetic route for
amide containing azidoalkylthiol SAMs. The modular
approach used allows systematic variation of the spacer group
between the thiol (tethered to the Au electrode) and the amide
functionality as well as the spacer group between the amide
and the azide (exposed to the solvent) functional group. These
functional groups can, in principle, be functionalized by any
electroactive species bearing a terminal alkyne group, in situ,
using ‘‘click’’ chemistry.11,50 We illustrate this approach by
covalently attaching a ferrocene and heme residue.11,49,59 The
spacer between the azide and the amide functional group is
systematically varied which, after in situ covalent attachment
of ferrocene, leads to systematic variation of the distance
between the electroactive ferrocene group and the amide
(Fig. 1). Sabapathy et al. had previously reported ferrocene-
functionalized amidothiol SAMs where the spacing between
the electroactive ferrocene group and the amide was varied by
varying the substituent on the ferrocene.24 While this
approach worked well, it may be limited by the availability of
the synthesis of appropriately substituted ferrocenes. The
modular approach used here circumvents this, as azidoalk-
ylamines of varying chain length can be synthesized from
easily available starting material.
Acknowledgements
This work has been funded by Department of Science and
Technology, India, SR/S1/IC-35/2009. Mr. Kaustabh Dan and
Prof. Alokmay Datta (SINP, Kolkata) are thanked for their help
in obtaining the ATR-FTIR data. Prof. G. B. Talapatra (IACS,
Kolkata) is thanked for his help to collect the CA goniometry
data.
The successful immobilization of the SAM is established
using XPS, ATR-FTIR, SERS and CA goniometry data.1,4,5,9,18
The XPS data shows a C 1s ionization at 287.6 eV5,6,17 and a
N 1s ionization at 399.9 eV corresponding to the amide group
(Fig. 3).4–6,17 The S 2p region indicates the formation of a
monolayer on Au.5,17,59 Furthermore XPS provides direct
evidence for the ‘‘click’’ reaction as the Fe 2p1/2 and 2p3/2
transitions of the ferrocene and iron porphyrins could be
identified and the N 1s transition from the central azide
nitrogen atom at 405 eV is no longer observed. Simultaneously
the ATR-FTIR data indicates the presence of amide I and
amide II vibrations at 1645 and 1536 cm21 (Table 2 and Fig. 5),
respectively, consistent with the presence of an amide
group.9,13,24 The same set of vibrations are also observed in
the SERS data where amide I, amide II and amide III vibrations
appear at 1652, 1532 and 1303 cm21 (as a doublet; Table 2 and
Fig. 6), respectively.57,69,71
The presence of an azide group is indicated by the presence
of N 1s peaks at 399.9 and 405 eV (Fig. 3) corresponding to the
ionization of the terminal and central N atoms of the azide
group, respectively.51,58 While the FTIR data revealed the N–N
asymmetric vibration (IR allowed) of the azide group, the SERS
data clearly showed the N–N symmetric vibration (Raman
allowed) at 1340 cm21 (Fig. 6 and Table 2).69,72 This feature is
replaced by bands at 1291, 1371 and 1538 cm21 (Fig. 6) in
SERS for a triazole ring as the azide groups are functionalized
using ‘‘click’’ chemistry.70,71,74 In this case covalent immobi-
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