Monitoring Solid-Phase Reactions in Self-Assembled Monolayers by Surface-Enhanced Raman Spectroscopy

Article abstract of DOI:10.1002/anie.202102319

Nanopatterned surfaces enhance incident electromagnetic radiation and thereby enable the detection and characterization of self-assembled monolayers (SAMs), for instance in surface-enhanced Raman spectroscopy (SERS). Herein, Au nanohole arrays, developed

Full text of DOI:10.1002/anie.202102319

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: Monitoring Solid-phase Reactions in Self-assembled Monolayers
by Surface-enhanced Raman Spectroscopy
Authors: Dominik Scherrer, David Vogel, Ute Drechsler, Antonis
Olziersky, Christof Sparr, Marcel Mayor, and Emanuel Marc
Lörtscher
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10.1002/anie.202102319
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RESEARCH ARTICLE
Monitoring Solid-phase Reactions in Self-assembled Monolayers
by Surface-enhanced Raman Spectroscopy
Dominik Scherrer,[a, b] David Vogel,[b] Ute Drechsler, [a] Antonis Olziersky,[a] Christof Sparr,[b] Marcel
Mayor,[b,c,d] and Emanuel Lörtscher*[a]
[a]
[b]
D. Scherrer, U. Drechsler, Dr. A. Olziersky, Dr. E. Lörtscher
Science and Technology Department, IBM Research Europe
Säumerstrasse 4, CH-8803 Rüschlikon (Switzerland)
E-mail: eml@zurich.ibm.ch
D. Scherrer, D. Vogel, Prof. C. Sparr, Prof. M. Mayor
Department of Chemistry, University of Basel
St. Johanns-Ring 19, CH-4056 Basel (Switzerland)
E-mail: christof.sparr@unibas.ch; marcel.mayor@unibas.ch
[c]
[d]
Institute for Nanotechnology (INT), Karlsruhe Institute of Technology (KIT), P. O. Box 3640, 76021 Karlsruhe, Germany
Lehn Institute of Functional Materials (LIFM), School of Chemistry, Sun Yat-Sen University (SYSU), Guangzhou 510275, P.R. of China
Supporting information for this article is given via a link at the end of the document.
surface, many of them lack the ability to characterize the chemical
composition of a surface, e.g., to detect functional groups within
the SAM. In that respect, infrared (IR) spectroscopy and variants
thereof including Fourier-transform IR (FTIR) spectroscopy,
Abstract: Nanopatterned surfaces enhance incident electromagnetic
radiation and thereby enable the detection and characterization of
self-assembled monolayers (SAMs), for instance in surface-enhanced
Raman spectroscopy (SERS). In this work, Au nanohole arrays,
developed and characterized as SERS substrates, are exemplarily
used for monitoring a solid-phase deprotection and a subsequent
copper(I)-catalyzed alkyne-azide cycloaddition “click” reaction,
performed directly on the corresponding SAMs. The SERS substrate
was found to be highly reliable in terms of signal reproducibility and
chemical stability. Furthermore, the intermediates and the product of
the solid-phase synthesis were identified by SERS. The spectra of the
immobilized compounds showed minor differences compared to
spectra of the microcrystalline solids. With its uniform SERS signals
and the high chemical stability, the platform is paving the way to
monitor molecular manipulations in surface functionalization
applications.
polarization-modulation
infrared
reflection-absorption
spectroscopy (PM-IRRAS) are sensitive enough to measure solid
thin films to provide information about the chemical functional
groups present in a SAM.[17–20] Raman spectroscopy is an optical
characterization method that relies on inelastic scattering of
incident, monochromatic light at chemical bonds. It can therefore
provide substantial information about the chemical composition of
the specimen,[21] at least for Raman-active bonds. Contrary to IR
spectroscopy, where bonds with large dipole moments exhibit the
highest signal intensities, bonds that are non-polar and
polarizable are generally highly Raman-active. IR and Raman
spectroscopy are therefore used mostly complementary, as (with
exceptions) many IR-inactive (or weakly active) molecular
vibrations are Raman-active and vice versa. An advantage of
Raman over IR spectroscopy is that it can be used in aqueous
solutions as well, while IR suffers from the strong IR self-
absorption of H2O which may mask other bonds of interest.[22] In
return, Raman spectroscopy is a second order and therefore an
inherently weak process (with Raman scattering probabilities of
10–14 to 10–7, and cross-sections of 10–30 to 10–25 cm2 for a single
molecule). This is usually compensated for by probing multiple
thousands of molecules in parallel to obtain an adequate signal
intensity, using high laser intensities or long acquisition times.
Another method to increase Raman signal intensities is to locally
Introduction
The functionalization of solid surfaces with self-assembled
monolayers (SAMs) is a widely applied method for surface
modifications with applications in catalysis,[1–3] chemical and
biochemical sensing,[4–6] molecular electronics,[7–9] wettability,[10]
corrosion
prevention,[11]
and
many
others.
Various
functionalization methods rely on a stepwise assembly or a
modification of the SAMs.[12] Due to the differences in the
chemical and physical behavior of molecules attached to surfaces
compared to their crystalline or liquid form, the composition of
these molecular layers is ideally characterized upon each
assembly and modification step. For such purposes, however,
only a few surface analytical methods exist that provide a high
enough sensitivity to determine the elemental composition (X-ray
photoelectron spectroscopy), the topology (scanning tunneling
microscopy, atomic force microscopy), the lattice structure (near
edge X-ray absorption fine structure spectroscopy), the surface
layer thickness (ellipsometry) or the surface coverage (thermal
gravimetric analysis and electrochemical methods).[13–16] While
these methods provide the above-mentioned information about a
concentrate incident electromagnetic radiation, Elocal
,
by
employing field-enhancing near-field methods, e.g. surface
plasmon polaritons. These methods are generally referred to as
surface-enhanced or tip-enhanced Raman spectroscopy (SERS
and TERS, respectively) with many variants. Their corresponding
Raman scattering intensities, IRaman, roughly scale with |Elocal|4 (at
least close to the near-field surface).[23–30] SERS and TERS
thereby enable ambient and label-free analytics of the molecule’s
unique vibrational fingerprint. While TERS relies primarily on
coating a scanning microscopy tip with an appropriate material
and bringing it in close proximity with the specimen,
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Figure 1. A) Schematic representation of a monocrystalline Si chip with Au nanohole arrays (0.5 nm Cr, 30 nm Au), nanohole dimensions: i = 180 nm, ii = 225 nm;
B) scanning electron micrographs of a Au nanohole array (scale bar = 1 μm, insert scale bar = 200 nm). C) Calculated enhancement factor (|E|4) in log scale for a
Au nanohole array on Si (x–y plane of the Au‒air interface). D) 100 SERS spectra (gray traces) and averaged spectrum (solid orange trace) of a 4-nitrothiophenol
(4-NTP) SAM on a Au nanohole array, in comparison to the Raman spectrum of 4-NTP on an unstructured, planar Au surface (dashed black trace). The vertical
gray lines indicate the peak positions of a reference spectrum.[35] E) Box plot of the peak position distribution for the three most intense Raman signals of 4-NTP on
a Au nanohole array as a deviation from the respective mean peak position. The gray boxes and the whiskers indicate the inner quartile range (IQR) and the range
within 1.5×IQR, respectively. F) Heat map of the peak height at 1327 cm–1 of 4-NTP on Au nanohole arrays, illustrating the quantitative uniformity of the signal
intensity over the entire map measurement.
various SERS methods and substrates have been developed
(usually containing plasmonically active materials such as Cu, Ag
or Au): On the one hand, SERS substrates, such as bottom-up
self-assembled Au nanospheres[31] and top-down fabricated
nano-gaps[23] or black Si-based nanoforests,[32] can deliver
exceptionally high field enhancements (FEs). But on the other
hand, it is difficult to control the nanoscale hotspots as they are
usually non-uniformly distributed on the surface and found to be
sensitive to irradiation, temperature or chemical environments.
Consequently, these substrates cannot be treated by single-spot
measurements alone but require time-consuming surface
mappings to locate the hotspots. Top-down substrates, which are
micro- and nanopatterned using lithography, are compatible with
mass-production manufacturing.[23,24,29,33,34] They usually provide
smaller field enhancements than the nano-gaps of bottom-up
assembled particles but are easier to control and more uniform.
Furthermore, they enable a deterministic and reliable integration
into more complex device architectures, e.g. in microfluidic
systems or gas and liquid sensing devices.[36] Over the last years,
SERS has evolved from an exploratory research topic to an
established analytical tool with fully characterized SERS
substrates and various applications, e.g. in microplate readers in
medicine and biology. In contrast, rather than tracking indirect
shifts in the plasmon resonance of the SERS substrate as is
commonly done, we employ the substrate’s field enhancements
for SERS to characterize SAMs that provide too small Raman
signals on unpatterned surfaces. Moreover, we use these
copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) “click”
reaction[37,38] and describe species-specific vibrational modes by
tracking their evolution for various molecular model systems upon
chemical modification.
Results and Discussion
For SERS-based chemical structure determination and the
monitoring of chemical reactions that occur in surface-bound
molecules, one requirement is a surface patterning that yields
quantitively reproducible and homogeneously distributed field-
enhanced areas rather than a few high SERS-intensity hotspots
that are not representative of the entire SAM. To meet these
requirements, plasmonic nanohole arrays with a moderate FE
were chosen as a platform. In return, they probe a comparably
large number of molecules across the whole area of interest.[33]
A
poly(methyl methacrylate) (PMMA)/hydrogen silsesquioxane
(HSQ) two-layer electron-beam lithography (EBL) lift-off process
was developed (see SI) to fabricate such nanohole arrays
(10 × 10 μm2 in size each) with quantitative lift-off yield in a 30 nm
thick Au thin film on a monocrystalline Si substrate. By scanning
electron microscopy (SEM), the arrays were found to be defect-
free and reveal homogenous shapes and thicknesses (Figure 1A
and B). A variety of diameters and pitches were tested to optimize
the nanohole array for two excitation laser wavelengths (633 nm
and 785 nm) by empirically screening the geometrical parameter
space with a 4-nitrothiophenol (4-NTP) SAM as a reporter
substrates to monitor solid-phase reactions, specifically
a
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compound. Arene-thiols were chosen as they are known to form
stable SAMs on Au surfaces.[17] The nanohole array dimensions
that showed the highest plasmonic Au fluorescence and SERS
signals were a 180 nm hole diameter with a pitch of 225 nm in a
cartesian arrangement (elliptical holes were disregarded in the
consideration for simplified EBL). The SERS enhancement factor
(EF) of the nanohole arrays was determined experimentally (see
are compatible with monitoring chemical changes in SAMs prior
to and after exposing the entire substrate to wet chemical reaction
conditions.
As a test system for conducting chemical reactions on the SAMs
and to monitor them by SERS, we chose small molecules that
have got a low number of Raman active vibrational modes that
can be unambiguously assigned to specific chemical bonds. For
these compounds, the goal is to exemplarily show the
deprotection and a subsequent chemical transformation by SERS.
The synthesis and characterization of all compounds are
described in the SI. To allow a directed immobilization on Au
surfaces via Au–S linkage, thioacetate 1a carries two orthogonal
protecting groups; an acetyl protecting group on the S terminus
and trimethylsilyl (TMS) on the alkyne terminus. The acetyl moiety
prevents oxidative S–S bond formation, while TMS prevents
bonding of the alkyne to Au (Figure 2). This design allows an
asymmetric, controlled cleavage of the S-acetyl to form the Au–S
linkage, causing the alkyne to point upwards from the Au surface
because it is still TMS-protected. This configuration provides both
an immobilization on Au and a susceptibility to subsequent
chemical modifications. Thioacetate 1a was immobilized as a
SAM
Supporting Information (SI)) and reached a value of 86 ±29
[39,40]
(standard error).
This was confirmed by finite-difference
time-domain (FDTD) calculations (Figure 1C; see SI). To validate
the SERS performance and the uniformity of the substrate for the
4-NTP test compound, Raman spectra were recorded under
ambient conditions with a liquid N2 cooled Si-CCD and a
300 grooves mm–1 grating at 633 nm excitation (7.5 mW laser
power) with a 100× objective with 0.86 NA and by using variable
neutral density filters. A 633 nm excitation wavelength was
chosen even though the maxima of the plasmonic activity of Au
would be more in the near-infrared region of the electromagnetic
spectrum. However, measurements with 785 nm excitation do not
lead to stronger signals (see Figure S3 in the SI)). The confocal
Raman microscope has a spot size smaller than 1 µm and a
piezo-electric stage enables 3D scanning of entire nanohole
arrays with sub-nm spatial accuracy.
Figure 1D shows a 10 x 10 single-spot SERS map of a 4-NTP
SAM immobilized on a Au nanohole array (gray traces) mapped
with a step size of 0.96 µm, covering an area of approx. 75 µm2.
The solid black trace represents the averaged spectrum and the
gray dashed lines the peaks of a reference spectrum.[41] All
spectra show eight distinct peaks with the most prominent one
located at 1327 cm–1, corresponding to the symmetric stretch
frequency of NO2. The intensity varies from approximately 1600
to 3600 counts for the most prominent peak, which is a
reasonable range, showing that every single-spot measurement
delivers a suitable spectrum (Figure 1F). The noise in the spectra
is approximately 200 counts prior to signal processing. In contrast
to measurements on Au nanoholes, a SAM of 4-NTP grown on an
unpatterned Au surface under the same conditions does not yield
discernible Raman peaks (Figure 1D, dashed orange trace).
Hence, the FE of the nanohole arrays causes Raman signatures
of 4-NTP SAMs to appear. In addition to the good quantitative
uniformity of the SERS intensity over the entire nanohole array,
the corresponding Raman peak positions are also located
±1.0 cm–1 (fitted values) around the mean peak values, as shown
for the three strongest signals in the box plot in Figure 1E
(spectral resolution = 1.8 cm–1). This confirms that the molecular
intrinsic modes are only weakly, if at all, disturbed by the covalent
coupling of the Raman active moieties to the Au surfaces via thiols.
The fact that SERS spectra can be acquired everywhere on the
Au nanohole substrate with a high uniformity allows single-spot
measurements to be conducted instead of time-consuming
mappings to detect suitable hotspots. Consequently, the
nanohole arrays serve the purpose of chemically characterizing
SAMs by SERS with stable and uniform FEs. Notably, no
additional SiOX adhesion layer on top of the Au was used as a
mediating SAM-binding layer, for instance via silanes, or as a
stabilizing layer to prevent diffusion of Au surface atoms at
elevated temperatures (induced by the incident laser power).
Instead, the SAMs are directly grown on Au and immobilized via
a Au–S linkage. Finally, all the employed materials (Si, Au and Cr
as adhesion layer between the former two) are chemically inert
under the environments of interest. Therefore, these substrates
Figure 2. Immobilization of 1a on a Au nanohole array and subsequent
trimethylsilyl (TMS) deprotection: SERS spectra of a blank Au nanohole array
(black), TMS-protected alkyne (1b, red) and deprotected alkyne (2, blue) SAMs
on a Au nanohole array at 633 nm excitation. The Si signal of the substrate (2nd
order LO) is marked with a gray bar. The values above the signals denote the
respective Raman shift in cm–1. The spectra were separated for better visibility.
The vertical dashed gray lines indicate the peak positions of the Raman
spectrum of the corresponding solid. Reagents and conditions: a) Au nanohole
array chip, 1a, aq. NH3 (7 M in MeOH), EtOH, 23 °C, 24 h; b)
tetrabutylammonium fluoride (TBAF), tetrahydrofurane (THF), ultrasound, 40 °C,
1 h.
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Figure 3. A) SERS spectra taken after different reaction times (0 min, 1 min, 5 min, 10 min) during TMS deprotection of a 1a SAM (red) forming a 2 SAM (blue) with
TBAF (50 mM) in THF, followed by washing in DI-H2O and THF and drying (633 nm excitation; non-treated data in Figure S21A in the SI). Each spectrum is the
average of three spectra that were recorded at different, randomly chosen points on the nanohole array. The inset shows a zoom on the symmetric C≡C stretch
frequency located at 2155 cm–1 which vanished after 10 min. B) Illustration of the repeated sequence of immersing the sample in the reaction solution, washing,
drying followed by spectra acquisition. C) Development of the peak intensities of the three signals that are labelled with a square, a circle and a triangle in A) over
the course of 10 min total reaction time. The peak intensities in counts were determined by fitting the respective peaks (Gaussian fit) of three spectra recorded at
different, randomly chosen points on the nanohole array. The error bars indicate the standard deviation of the three measurements. None of the three spectra after
10 min displayed a signal at 2155 cm–1 above the measurements noise-level (see inset in A)) and the blue triangle was therefore set to zero and no error bar was
added.
on the Au nanohole arrays by immersing a sample in a 5 mM
ethanolic solution of 1a with an excess of NH3 (to deprotect the
thiol) for 24 h (step (a) in Figure 2). After functionalization, the chip
was immersed in H2O and EtOH baths accompanied by
ultrasonication to remove physisorbed 1a as well as byproducts
from the acetyl deprotection and impurities. Optical inspection
under the microscope was performed to rule out the presence of
visible aggregates and particles. Subsequently, single spot SERS
spectra were recorded under ambient conditions and using a
633 nm excitation source. A SERS spectrum of the TMS-
protected monolayer (1b) is depicted in Figure 2 (red). The most
prominent Raman signals (full range spectra in Figure S19 in the
SI) include the signals at 1072 cm–1, 1176 cm–1 and 1581 cm–1
that can be assigned to vibrational modes of the substituted
benzene. The peak at 2155 cm–1 corresponds to the symmetric
C≡C stretch frequency of the TMS-protected alkyne.[22,42] The
spectrum of the SAM of 1b is closely comparable to the Raman
spectrum of its bulk analogue (1a) in crystalline form (gray dashed
lines; see Figure S17 in the SI for the full spectra). The small
spectral shifts observable between the SERS spectrum of the
immobilized molecule and to the one of its crystalline state can be
attributed to the change in the substitution at the aromatic ring (–
SAc vs. –S@Au) and to the different intermolecular forces the
molecule experiences in a solid compared to a SAM.
it is still highly sensitive for the detection of changes in the
chemical structure. After the SAM formation, the alkyne was
deprotected by releasing the TMS protecting group to enable a
CuAAC reaction on the terminal alkyne. TMS deprotection with
K2CO3 was found to be unsuccessful, as no change in the Raman
signals occurred. However, deprotection with TBAF in an
ultrasonic bath led to the disappearance of the signal at 2155 cm–1
as shown in Figure 2 (blue). A notable difference was observed
between the expected and the measured changes in the Raman
spectrum after TMS deprotection; the symmetric C≡C signal is
expected to have a vibrational frequency between 2130–
2100 cm–1,[22,42] which is a slightly lower frequency range than the
one for the TMS-protected alkyne (2155 cm–1). Surprisingly, no
signal for the alkyne was detected after the deprotection. These
results underline the importance of evaluating the unique
characteristics of Raman-active vibrations of immobilized
molecules on plasmonic surfaces.
After detecting a change in the spectra before and after the TMS
deprotection for an immersion time of 1 h, it was particularly
interesting to investigate whether the decay of the signal at
2155 cm–1 during the conversion from 1b to 2 could be monitored
over time by measuring several spectra after short reaction
intervals. Under homogeneous conditions, this reaction would be
expected to complete within just a few seconds. This was
confirmed by deprotecting the precursor 1a in a homogeneous
solution with TBAF. NMR analysis showed the deprotection to be
finished within less than 1 min (see Figure S21B in the SI).
Contrary to the reaction in solution, the TMS deprotection of SAM
1b was expected to proceed at a lower rate (potentially within
minutes instead of seconds) because of the steric hindrance the
As indicator for chemical modifications of the SAM of 1b,
alterations in the corresponding SERS spectrum, ranging from
shifts in the peak positions to disappearances or appearances of
peaks, were used as discriminating features after each step. This
monitoring only takes a few minutes and can be done non-
destructively, non-invasively and contact-free. At the same time,
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TMS group experiences due to the neighboring immobilized
molecules of the SAM, as well as reduced mass-flow exchange at
the surface. The deprotection of the TMS group of 1b was
therefore studied by repetitively removing the chip from the TBAF
solution in THF at specific intervals to wash and dry it and
subsequently measure the SERS of the SAM. Figure 3A shows
the SERS signal of the pristine (as grown) 1b SAM on the Au
nanohole array right after its formation (0 min; red trace), after
1 min (dark red trace), 5 min (violet trace) as well as 10 min (blue
trace) of immersion in the TBAF solution in THF. Each spectrum
is the average of three individual point spectra that were recorded
at three randomly chosen points on the nanohole array. The
signal strength of the disubstituted alkyne peak (symmetric C≡C
stretch frequency) at approximately 2155 cm–1 decreases after
1 min as well as after 5 min, but it is still visible (Figure 3A, inset).
The measurement after 10 min of immersion, however, does not
reveal a signal that can be distinguished from the baseline
anymore. This suggests that the TMS deprotection of a 1b SAM
is completed after 10 min while a partial conversion of the 1b SAM
towards a full 2 SAM could be monitored in time. The spectra in
Figure 3A (like all herein presented spectra) have been smoothed
and baseline-subtracted. The non-treated data show the same
result and can be consulted in the SI (Figure S21A). The
development of the intensity of the three signals in Figure 3A that
are marked with a gray, dashed line and have a square, circle or
triangle as a label is plotted over time in Figure 3C. The error bars
indicate the standard deviation from three randomly chosen point
measurements on the surface, in agreement with the fluctuation
in Figure 2 and typical for SERS. Despite these fluctuations, it can
be concluded from Figure 3C that the signal at 2155 cm–1
(triangle) gradually declines while the other signals remain at a
relatively high level. Although quantitative evidence about peak
intensities for these measurements is difficult to establish due to
the high intensity fluctuations between SERS measurements, the
stability of the SAMs 1b and 2 in ultrasonic conditions were
studied to learn whether large quantities of the SAMs dissociate
under these conditions by following the signal intensities over time.
Figure S22 in the SI shows that prolonged ultrasonic exposure
does not cause the signal intensities to fall below the detection
limit. To summarize, the above-mentioned time-resolved
experiments show that the presented method not only can be
used to characterize the SAMs prior to and after a reaction, but
also to follow the partial and gradual conversion of molecules
inside a SAM in time, at least for slow reactions such as the TMS
deprotection on the surface. The stepwise monitoring provides
clear evidence that the reaction is taking place at the SAM
interface.
in Figure 4A. When comparing it to the spectrum before
deprotection and CuAAC reaction, the alkyne peak at 2155 cm–1
has vanished and three new peaks appeared: Next to the peak at
1581 cm–1 of 1b, a peak at 1601 cm–1 appears with a comparable
intensity; the two signals are not baseline-separated. The new
peak can be attributed to a vibrational mode of the added benzene
ring with a slightly different energy caused by the different
substitution of the two rings. At 1404 cm–1, a weaker peak evolves
from the baseline that originates from the C‒N stretching mode of
the triazole.[43] Further, a third new peak located at 967 cm–1 can
be discerned. It superimposes the broad 2nd order longitudinal
optical (LO) band from the Si substrate (Figure 4A, inset). Around
1325 cm–1, however, no peaks appear in the spectra of 4b. Such
signals representing the symmetric stretch frequency of the
terminal NO2 of 4b would be expected after the CuAAC reaction
clicking 3 to 2. The NO2 moiety is known to be Raman active and
was also clearly observed in the 4-NTP spectra acquired on Au
nanohole arrays, even as the most prominent peak at 1327 cm–1
presented in Figure 1D. Figure 4B illustrates the appearance, the
evolution and the disappearance of the individual SERS peaks
over the course of all steps of the experiment. While this evolution
was deemed reasonable, the disappearance instead of a shift of
the peak at 2155 cm–1 upon TMS deprotection remains
anomalous as it clearly corresponds to the symmetric C≡C stretch
frequency of the alkyne. Also, the missing NO2 peak, as
discussed above, is distinct from existing literature data. In order
to confirm that 4b has indeed been formed by the solid-phase
reactions on the SAM, a control experiment was executed through
synthesizing 4a with classic batch synthesis methods (see SI).
Batch synthesis derived 4a was immobilized on Au and its SERS
spectrum measured (Scheme 1 and Figure 4A, dashed green
curve). When comparing it with 4b produced by solid-phase
synthesis (solid green curve), the two spectra are qualitatively
almost identical,
Next, we performed a CuAAC reaction to attach 1-azido-4-
nitrobenzene (3) to the liberated alkyne of 2 (Scheme 1). Working
with
a SAM-coated surface on Au as reactant requires
homogeneous reaction mixtures rather than suspensions, as the
presence of precipitates may cause aggregation of particles
forming thin films on the surface. These thin films can be difficult
to remove without affecting the nanostructures underneath and
the SAM itself. Hence, a series of reaction conditions was
screened to evaluate a suitable catalyst, as well as other reagents,
solvents and concentrations (see SI). CuSO4·5 H2O with sodium
ascorbate as reducing agent in THF/H2O with ultrasound at 40 °C
was found to be a suitable combination to provide the required
homogeneous mixture. The SERS spectra prior to and after the
coupling of azide 3 onto alkyne 2, yielding triazole 4b, is depicted
Scheme 1. Deprotection of (a) the TMS-protected alkyne of 1b with (b) the
immediately following CuAAC reaction on the liberated alkyne of 2 on Au
nanohole arrays, yielding triazole 4b. c) Immobilization of 4a on Au nanohole
arrays. Reagents and conditions: a) TBAF, THF, ultrasound, 40 °C, 1 h; b) 1-
Azido-4-nitrobenzene (3), CuSO4·5 H2O, sodium ascorbate, THF/H2O,
ultrasound, 40 °C, 1 h, then 23 °C, 16 h; c) Au nanohole array chip, NH3 (7 M in
MeOH), CHCl3, 23 °C, 5.5 h.
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Figure 4. A) SERS spectra of TMS-protected alkyne at (1b, red), as well as the triazole originating from the solid-phase synthesis on the SAM (4b, solid green) and
from the batch synthesis (4b, dashed green) at 633 nm excitation. The broad 2nd order LO band of the Si substrate is marked with a gray bar. The values above the
signals denote the respective Raman shift in cm–1. The vertical dashed gray lines indicate the peak positions of the Raman spectra of the corresponding solids. B)
The appearance, evolution and disappearance of the individual SERS signals before and after functionalization, upon TMS deprotection and CuAAC reaction.
with some small quantitative deviations in the relative peak
intensities of the two benzene vibrations at 1580 cm–1 and
1601 cm–1. The peaks at 1077 cm–1 and 1180 cm–1 are almost
equally intense in the solid green curve, but the peak at 1073 cm–1
is much more pronounced than the peak at 1176 cm–1 in the
dashed green curve. More importantly, however, also in the
batch-synthesis derived 4b, the peaks at 2155 cm–1 as well as
1327 cm–1 are clearly below the detection limit. When comparing
this with bulk Raman spectra of the microcrystalline solids (see
Figure S17 of the SI) excited at 785 nm, however, the NO2 signal
is present, both in 3 and 4a. In contrast, bulk measurements at
633 nm provide less informative spectra as they are dominated
by a fluorescent background (see Figure S18 in the SI).We
therefore tentatively rationalize the absence of the usually Raman
active symmetric C≡C stretch frequency of the alkyne moiety of
the TMS-deprotected 1b and the NO2 in the Au-bound 4b by the
very low Raman cross-section due to specific optical scattering
geometries on surfaces or quenching of the Raman-scattered
phonon. The overall agreement of the two SERS spectra of the
differently derived compounds 4b, however, leads to the
conclusion that the deprotection and CuAAC reaction on the SAM
immobilized on Au nanohole substrates can be unequivocally
monitored by SERS. Further control experiments were performed
to rule out that the reagents of the CuAAC reaction adhere to the
Au surface and could be responsible for the change in the
spectrum (see Scheme S2 and Figure S20 in the SI). Given the
double-checked and confirmed differences in the Raman spectra
of immobilized compounds to their bulk counterparts, it becomes
apparent that SERS is distinctly suitable to detect minor features
in the chemical structure of SAMs, but its empirical determination
is currently still mandatory for SERS-based surface
characterizations.
Conclusion
We have shown the possibility to characterize different
intermediates during the chemical modification of a SAM on a
structured Au nanohole array. The Au nanohole array thereby
acts as a plasmonic surface that provides a uniform FE over the
whole patterned area, allowing single-spot SERS measurements.
The starting material and the product of the studied solid-phase
CuAAC reaction produce distinguishable Raman spectra that
match with the spectra of the corresponding batch synthesized
compounds. While the CuAAC reaction serves as a suitable
model reaction, a broad range of short-chained SAMs is
considered as suitable substrates and analytes for functional
surface modification. While most peaks in the SERS spectra can
be unambiguously attributed to chemical bond vibrations from
data bases, SERS shows unique effects as peaks may disappear
on surfaces as shown in this work, motivating the further
investigation of this interesting feature. Therefore, it is important
to compare the reactions on the surface with their batch-synthesis
analogues. Notably, the presented SERS platform enables
detailed monitoring of chemical reactions for solid-phase
synthesis by providing very fast, contact-free, non-destructive and
6
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10.1002/anie.202102319
Angewandte Chemie International Edition
RESEARCH ARTICLE
non-invasive information about the surface functionalizations and
the modifications thereof.
acknowledged. M.M. acknowledges support from the 111 project
(Grant No. 90002-18011002).
As the nanohole arrays can be created with a variety of
plasmonically active materials and only a tiny area needs to be
nano-patterned, it is anticipated that such monitoring tags might
become useful for process monitoring in a variety of industry-
relevant applications. As an example, we foresee this method to
be used to miniaturize currently existing systems for reaction
screenings in which the chemical substrates are immobilized on
solid supports (e.g. on resins). Reaction screenings that aim to
find pharmaceutical leads require a vast number of possible
candidate substances. Such systems include the combinatorial
synthesis of peptides and other biologically active molecules on
solid supports.[44] Miniaturization of these systems would mean
using a single molecular monolayer on a Au surface instead of the
substrate being immobilized on resin beads. The need for less
resources with possibly lower required reaction volumes could be
an advantage of such a miniaturized system. If plasmonic
surfaces are integrated in a microfluidic device and if the
synthesized molecules can be released in a controlled manner, it
could be linked to other lab-on-a-chip modules such as biological
assays to assess the binding to a protein target in an integrated
system, for instance. The herein reported method would greatly
benefit from further developments to be viable for such an
application. Firstly, the FE produced by the presented
nanostructures is only strong enough in a certain energy range of
the spectrum and does not cover the parts of the spectrum above
Keywords: Click chemistry • Field enhancement • Raman
spectroscopy • Self-assembled monolayers • Solid-phase
synthesis
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Entry for the Table of Contents
A highly reproducible, homogeneous plasmonic substrate was fabricated by electron-beam lithography to detect the Raman
fingerprints of molecular self-assembled monolayers (SAMs) by surface-enhanced Raman spectroscopy (SERS) at all points of the
substrate with high uniformity. This substrate was employed for characterizing the intermediates and the product of a solid-phase
synthesis in a SAM by SERS.
9
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