Biosensors and Bioelectronics 96 (2017) 167–172

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Biosensors and Bioelectronics

journal homepage: www.elsevier.com/locate/bios

Metal carbonyl-gold nanoparticle conjugates for highly sensitive SERS MARK

detection of organophosphorus pesticides
Mein Jin Tana,1, Zi-Yao Hongb,1, Mei-Hua Changc, Chih-Chen Liuc, Hwei-Fang Chengc,
⁎ ⁎
Xian Jun Loha,d, Ching-Hsiang Chene, Chia-Ding Liaoc, , Kien Voon Kongb,
a
Institute of Materials Research and Engineering, A*STAR Singapore, Singapore
b
Department of Chemistry, National Taiwan University, Taipei, Taiwan
c
Food and Drug Administration (FDA), Ministry of Health and Welfare, Taipei, Taiwan
d
Department of Materials Science and Engineering, National University of Singapore, Singapore
e
Sustainable Energy Development Center, National Taiwan University of Science and Technology, Taipei, Taiwan

A R T I C L E I N F O A BS T RAC T

Keywords: The binding of organometallic osmium carbonyl clusters onto the surface of gold nanoparticles (10OsCO-Au NPs)
SERS greatly enhanced the CO stretching vibration signal at ~2100 cm−1, which is relatively free from interference
Metal carbonyl due to the absorbance of biomolecules. By utilizing the acetylcholinesterase (AChE) mediated hydrolysis of
Glyphosate acetylthiocholine to thiocholine where the activity of AChE is inhibited by the presence of organophosphate
Acetylcholinesterase
pesticides (OPPs), the subsequent thiocholine-induced aggregation of 10OsCO-Au NPs can be monitored by the
Food safety
change in color of the NPs solution and the variation in intensity of the SERS CO signal. The change in color
offers a fast pre-screening method, whereas monitoring via SERS is used for greater accuracy and lower limit of
detection (0.1 ppb) for quantitative detection. Its potential as a quick and accurate method of OPPs monitoring
in consumer products was demonstrated in the detection of OPPs in real spiked samples such as beer.

1. Introduction Nonetheless, the authors report difficulty in getting uniform applica-

Organophosphorous pesticides (OPPs) compounds in particular
have drawn much attention due to its extensive use agriculture world-
wide and its suspected hazards (Fenik et al., 2011). The main
aggravating toxicity of OPPs is ascribed to their ability to inhibit the
activity of acetylcholinesterase (AChE), an essential enzyme required to
breakdown acetylcholine (a neuro-transmitter) at cholinergic synapses
(Kwiatkowska et al., 2014), which can severely affect the human
nervous system (Quinn, 1987). Thus it is highly desirable for the
development of reliable and sensitive methods for the detection of such
toxins in modern day's food supplies.
Surface-enhanced Raman scattering (SERS), which allows the
amplification of weak Raman signals due to the collective oscillation
of surface electrons on nanostructured metallic materials (Dieringer
et al., 2007; Jiang et al., 2003; Laurence et al., 2009; Moskovits, 1985),
offers very high sensitivity (Bell and Sirimuthu, 2006; Kneipp et al.,
1997). With respect to OPPs detection, efforts have been done to
develop an in-situ method for the direct probing of tea leaves and apple
skins (Chen et al., 2016; Hou et al., 2015; Li et al., 2013, 2014).
tion of the SERS probe onto the hydrophobic leaves and skin surfaces.
Furthermore, the detection window used (600–1800 cm−1) is crowded
with absorbance interference from biomolecules, making tracing of the
signature peaks of the respective target analytes tedious and challen-
ging (Salmain et al., 1999).
Hence considerable research effort was invested to develop SERS
probes that circumvent the problem of poor signal-to-noise ratio when
encountering complex biological samples. Organometallic compounds
thus exhibit such properties that can fulfill this function. A recent
example was demonstrated by Kong et al., using an air and moisture-
stable osmium carbonyl compound (Kong et al., 2007) that has
previously shown great efficacy as a SERS bio-sensor probe for glucose
detection (Kong et al., 2013) as well as SERS live-cell imaging (Kong
et al., 2012). The strong CO stretching vibration SERS peak at around
2030 cm−1 is in a relatively undisturbed IR region, which makes
detection and monitoring much simpler and direct. Thus in this work,
the osmium carbonyl SERS probe was further developed and extended
to detect OPPs in spiked and real samples, and demonstrate the
applicability of the newly developed assay for real-life applications.

⁎
Corresponding author.
E-mail addresses: cdliao@fda.gov.tw (C.-D. Liao), kvkong@ntu.edu.tw (K.V. Kong).
1
Author Contributions: These authors contributed equally.

http://dx.doi.org/10.1016/j.bios.2017.05.005
Received 11 February 2017; Received in revised form 19 April 2017; Accepted 3 May 2017
Available online 04 May 2017
0956-5663/ © 2017 Elsevier B.V. All rights reserved.
M.J. Tan et al.
Fig. 1. (A) Molecular structure and binding modes on nanoparticle, (B) FT-IR study, and (C) SERS spectra of (i) 10OsCO-Au, (ii) 3OsCO-Au and (iii) 1OsCO-Au NPs. (D) SERS spectra of
10Os
CO-Au NPs, Rhodamine 6G-Au-NPs and beverage (beer).
2. Materials and methods
2.1. Characterization Instruments & Methods
TEM images were recorded on a Philips CM300 FEG TEM. TEM
samples were prepared by placing a drop of the nanoparticles onto a
carbon coated Cu grid. Mean particle sizes were obtained by measuring
the sizes of the nanoparticles in a few randomly chosen areas of the
digitized image, each containing approximately 100–200 nanoparti-
cles. IR spectra were obtained using a Thermo Fisher Scientific
(OMNIC) Fourier Transform IR (FT-IR) spectrometer. The spectral
measurements were carried out using a Renishaw InVia Raman (UK)
microscope with a Peltier cooled CCD detector and an excitation
wavelength at 633 nm, where the laser beam is directed to the sample
through a 50× objective lens, which was used to excite the sample and
also to collect the return Raman signal. All Raman spectra were
processed with WiRE 4.2 software. The maximum laser power at the
sample was measured to be 6.2 mW and the exposure time was set at
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Biosensors and Bioelectronics 96 (2017) 167–172
10 s throughout the measurements. Prior to each measurement, the
instrument was calibrated with a silicon standard whose Raman peak is
centered at 520 cm−1. All the Raman results was also verified with
another fully automated Raman system (UniDRON model,
UniNanoTech Co. Ltd). The hydrodynamic sizes of the prepared NPs
were determined using a Brookhaven dynamic lightering scattering
(DLS) instrument at 90° (632.8 nm) using NNLS analysis.
2.2. General procedure
All manipulations for chemical synthesis were carried out using
standard Schlenk techniques under an argon or nitrogen atmosphere.
The CpOs(CO)2-I (Zhang et al., 2006), Os3(CO)10(μ-H)2 (Poe et al.,
1993), [Os10(μ6-C)(CO)24]-(Johnson and Powell, 2008), was prepared
according to reported procedures. Os3(CO)12 was purchased from
Oxkem; all other chemicals were purchased from other commercial
sources and used as supplied. Freshly prepared solutions of compounds
in ethanol (1 mg/mL) were mixed with 60 nm gold (Au) colloid pellets
M.J. Tan et al.
(2.6×1011 particles in 100 mL, BBInternational UK) in ethanol. After
incubation 24 h (except for CpOs(CO)2-I is for 36 h), 1 mL of DI water
was added and centrifuged (10,000 rpm, 2 min) to remove excess of
compounds. This process was repeated three times. The resultant pellet
of metal carbonyl NPs was suspended in DI water for storage. Standard
Ellman method (acetylcholinesterase assay kit; Abcam) and organo-
phosphorus compound ELISA kit (CD Creative Diagnostics) was
performed manually and according to the manufacturer's instructions.
2.3. SERS Detection of Glyphosate in Test and Spiked Samples
A 10 mM stock solution of acetylthiocholine (Sigma Aldrich) was
freshly prepared in DI water and not used for more than 3 h after
preparation to minimize possible hydrolysis. Organophosphorus com-
pound (glyphosate (Sigma Aldrich)) was initially dissolved in ethanol to
a relatively high concentration (1000 ppm), which was later diluted at
testing concentration. In the resulting solutions, such low levels of
organic solvent have been shown to have negligible effects on the
activity of acetylcholinesterase (AChE) (Sigma Aldrich). Phosphate-
buffered saline (PBS) was used to dissolve AChE (1 unit/μL), which
was diluted with PBS water and used immediately for the following
experiments. To aliquots of 1 μL of AChE (0.1 units/mL) solution were
added various concentrations of glyphosate/samples (10 μL) and
incubated for 10 min. To each mixture was added acetylthiocholine
and incubated for 5 min. 1 μL of resulting solution was added to
10Os
CO-Au NPs solution (200 μL) for 5 min, and SERS were taken. For
beer spiked samples, beer in cans were bought from a supermarket and
used without pretreatment. 10 μL of spiked beer sample was added to a
1 μL of AChE (0.1 units/mL) solution and incubated for 10 min. To
each mixture was added acetylthiocholine (1 mM) and incubated for
5 min (Please note that 1 mM of acetylthiocholine will not cause
aggregation of Au colloid pellets). 1 μL of resulting solution was added
to 10OsCO-Au NPs solution (200 μL), and SERS were taken after 5 min.
3. Results and discussion
3.1. Formation Mechanism and Spectroscopic Properties
Several osmium metal cluster sizes were utilized in this work,
namely 1Os, 3Os and 10Os, and the mode of interaction of Os with gold
nanoparticles (Au NPs) is summarized in Fig. 1A. As shown by the FT-
IR results in Fig. 1B, a plausible mechanism to describe the formation
of 1OsCO-Au NPs involve the reduction of Cp(CO)2Os-I to the osmium
anion [Cp(CO)2Os]-, which could lead to the bonding onto the Au NPs
to form a Cp(CO)2Os-Au conjugate. A minor band formed as a result of
the attachment to the Au NPs surface after incubation, which became
more dominant after 36 h of incubation, indicating that Cp(CO)2Os-I
has fully bonded to Au NPs (Zhang et al., 2006). For 3Os, significant
differences in the IR spectra before and after incubation with Au NPs
were observed; two moderately intense νCO bands at 2031 and
1945 cm−1 respectively were formed after incubation. This is consistent
with the reported values for Os3(CO)10(μ-H)(μ-Au) of 2034 and
1952 cm−1, confirming that binding of 3Os to Au NPs (Li et al., 2009;
Li and Leong, 2008). 10Os clusters on the other hand are stable anionic
clusters that bind to surface of Au NPs via electrostatic interactions
(Fig. 1B). This is confirmed by FT-IR measurements where the IR
bands of 10Os incubated with Au NPs are similar to that of free 10Os
clusters. Furthermore, it is good to note that the functionalization of Au
NPs with 10OsCO did not cause any agglomeration as determined by
DLS (Fig. S1, Supporting information) as well as TEM (Fig. 2B).
All three non-aggregated OsCO-Au conjugates (1OsCO-Au, 3OsCO-Au
and 10OsCO-Au) exhibit varying intensity of CO signals, with the highest
being that for 10Os (10Os > 3Os > 1Os) (Fig. 1C). The difference in
intensity is due to the presence of more CO molecules per Os cluster as
the cluster size increases. Furthermore, it was shown via a control
experiment using Au NPs coated with PEG (20,000 kDa) that the
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Biosensors and Bioelectronics 96 (2017) 167–172
observed SERS peak at 2100 cm−1 was indeed caused by the binding of
10
Os clusters (PEG prevents the binding of Os-CO clusters onto the Au
NP surface) (Fig. S3, Supporting information). As shown in Fig. 1D,
10Os
CO-Au NPs has a clearly visible sharp CO peak in ~2000 cm−1,
indicating that 10OsCO-Au NPs have the potential to be a non-
interference SERS NPs (Kong et al., 2012, 2013). In contrast, the
SERS signals of rhodamine 6G-Au-NPs overlaps with those from other
molecules present in samples such as beer. Therefore the developed
10Os
CO-Au NPs were subsequently used as a SERS probe for OPPs
detection.
3.2. Mechanism of the Dual Mode Assay for Glyphosate
In the absence of glyphosate (one of the most common OPPs used),
AChE is active and will hydrolyze acetylthiocholine to thiocholine,
which can adsorb onto surface of Au NPs and cause strong aggregation
of Au NPs in solution via electrostatic interactions (Liu et al., 2013)
(Fig. 2A). The thiocholine-induced aggregation of 10OsCO-Au NPs
caused a distinct color change in solution from red to purple.
Aggregation of 10OsCO-Au NPs was also confirmed with transmission
electron microscopy (TEM) (Fig. 2B) and UV–vis measurements
(additional absorbance peaks was observed at region > 530 nm)
(Fig. 3B). This is in line with numerous observations about the red-
shifting of the absorbance peak of Au NPs due to aggregation.
Furthermore, the aggregation of the 10OsCO-Au NPs also resulted in
an increase in intensity of the SERS CO stretching vibration signal
(Fig. 3C) (an estimated factor of ~ 5.4×106).
The excitation of the surface Plasmon resonance (SPR) of metallic
nanostructures is a fundamental process of SERS (Moskovits, 2013;
Zhang et al., 2010). Our results showed that the SPR peak of non-
aggregated 10OsCO-Au NPs occurs at 530 nm, but shifted to longer
wavelengths (650 nm and 793 nm) upon aggregation (Fig. 3B). Thus, it
follows on that using a longer excitation wavelength could give rise to a
stronger SERS effect, as demonstrated by several other studies
(Bonifacio et al., 2014; Braun et al., 2009; Tian et al., 2014).
Moreover, Wustholz, K. L. and his members also suggested that the
SERS efficiency at the original (shorter) excitation wavelength is
affected by the shifting of the SPR peak due to aggregation (Braun
et al., 2009; Wustholz et al., 2010). This is clearly evident when the
solution of aggregated 10OsCO-Au NPs exhibited a significant reduction
in intensity when irradiated with a shorter wavelength laser (532 nm)
as compared to irradiation with a 633 nm laser (Fig. 2C). This
observation is consistent with other reported observations where a
significant boost to the SERS signal was observed when using a 633 nm
laser as an excitation source due to the aggregation-induced red-
shifting of the SPR peak (Tian et al., 2014). It is also important to note
that whilst a change in intensity was observed, the vibrational
frequencies of the CO band were not affected (i.e. no change in the
spectra feature). This indicates that the structure/geometry of 10OsCO-
Au NPs remained unchanged, which is markedly different as compared
to other probes such as rhodamine where molecular orientation
variations during aggregation resulted in shifting of the SERS peak,
making detection of target analytes tricky and complex.
3.3. Sensitivity of dual mode assay for Glyphosate
We next tested the sensitivity of this assay for different concentra-
tions of glyphosate in aqueous solutions (0, 0.0001, 0.0005, 0.001,
0.005, 0.01, 0.05, 0.1, 0.5, 1, 5 and 10 ppm). Color changes can be
observed within the first minute, and we found that the color of the
solution changed from red to purple with decreasing concentration of
glyphosate (Fig. 3A). This is particularly useful for a fast, initial visual
colorimetric screening to confirm the presence glyphosate when deal-
ing with a large number of samples. To demonstrate the developed
assay's specificity to OPPs, chlorphyrifos was tested as well while
bifenthrin, a non-organophophorus compound was used as a negative
M.J. Tan et al.
Fig. 2. (A) Schematic showing the design strategy of the glyphosate sensor using 10OsCO-Au NP as SERS probe. (B) TEM images of Au nanoparticles,
10Os
CO-Au NPs. and (C) SERS spectra of 10OsCO-Au NPs scanned at 633 nm (top) and 532 nm (bottom).
control. Only bifenthrin induced a color change (Fig S3, Supporting
information) due to thiocholine-induced aggregation, whereas chlor-
phyrifos inhibited the activity of AChE and prevented the production of
thiocholine. A simple comparison with existing bioassay methods such
as Ellman method was done (Fig. 3A, bottom row) too. The Ellman
method is known to suffer from background interference, where
proteins and thiol amino acids are capable of producing false positives.
In contrast, the developed 10OsCO-Au assay worked under high dilution
(10OsCO-Au NPs mixed with only 1 μL of solution from the mixture of
sample, AChE and acetylthiocholine) which minimizes various non-
specific interactions (including biothiols interferences). Under the
same condition, a false positive glyphosate result was obtained for
the Ellman method (Fig. S4, Supporting information).
Next, the SERS method was used for detection of glyphosate and
the variation in the CO stretching vibration signal intensity vs
glyphosate concentration was plotted in Fig. 3C-D, with the linear
correlation (plotted inset) determined to be r2=0.991. The detection
limit of glyphosate using SERS was determined to be 0.1 ppb, which is
much lower than the maximum residue limits (MRL) as reported in the
United of Kingdom and European database (MRL for grapes wine is
0.5 ppm for glyphosate). For Taiwan, the permitted residues level in
food for glyphosate is 0.1 ppm, whereas the maximum level of
glyphosate residue permitted in the United States is 30.0 ppm. In
comparison with a commercial Elisa glyphosate assay which has a limit
of detection (LOD) of 0.05 ppb, the developed 10OsCO-Au assay was
found to have similar LOD. The recovery is good on the whole in the
detection of glyphosate in the presence of other components (such as
beer) (Table S1, Supporting information). Nonetheless, the developed
assay required a shorter preparation time and lower sample volume
(1 μL from the mixture of 10 μL sample, AChE (1 mL) and acetylthio-
choline (10 μL) as compared to 50 μL of sample for Elisa assay). Thus,
we have demonstrated that our assay can achieve good efficacy in
colorimetric mode (with better sensitivity and accuracy as compared to
Ellman method) and SERS mode (with comparable detection limit as
Elisa assays). A comparison with various other methods used in the
detection of OPPs is summarized in Table S2, Supporting information.
Although method 1 has the lowest LOD, it requires 2 h of detection
time. As compared with method 2, 3 and 4, our method exhibited
broader detection range. Our assay also possess similar LOD with
methods 4 and 5, but requiring a shorter detection time than method 4.
As compared with other SERS techniques (Method 6 and 7), our
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Biosensors and Bioelectronics 96 (2017) 167–172
10Os
CO-Au NPs and aggregated
method exhibited greater sensitivity.
To demonstrate the efficacy of the 10OsCO-Au assay in real samples,
beer spiked with glyphosate was used. There was a recent food and
healthy safety issue regarding the integrity of the beer produced in
Germany, where reports of glyphosate-laden beer (with levels almost 5
times higher than the legal limit in drinking water) have been found (A
similar issue has also just been reported for wines in California). Beer is
a complex solution and contains multiple bio-based components
(which will produce significant interference between 100 and
1800 cm−1) that make detection of glyphosate in conventional SERS
measurements challenging. Thus, this issue can be circumvented with
the use of the 10OsCO-Au NPs with a clear detection peak at 2123 cm−1
that is relatively free from any biomolecule interference.
Beer was spiked with varying concentrations of glyphosate, namely
0, 0.005, 0.01, 0.05, 0.1, and 0.25 ppm and the variation in solution
color with respect to glyphosate concentration was similar to that
observed for prepared glyphosate samples (i.e. color of the solution
changed from red to purple with decreasing concentration of glypho-
sate). We also note that there was no color interference from the beer
itself due to the extremely low volume (1 μL) used for the testing.
Nevertheless, significant color interference caused by beer was ob-
served when larger volumes ( > 20 μL) were used instead. Correlation
of the intensity of CO stretching frequency vs various concentrations of
glyphosate in beer is plotted in Fig. 3E, obtaining r2=0.957 and a
detection limit of 0.01 ppm. Therefore, this demonstrated the devel-
oped assay's capability to detect OPPs in real-life samples. The SERS
assay in this work does not require any complicated sample prepara-
tion and/or pre-treatment before testing, and only require a minute
amount of sample for testing.
4. Conclusions
We have demonstrated an osmium carbonyl-based SERS probe for
the highly sensitive detection of OPPs (glyphosate) via SERS technique.
The SERS-enhanced CO stretching vibration signal of 10OsCO-Au NPs
at the mid-IR region (1800 – 2200 cm−1) is free from biomolecule
interference, making detection simple and direct. Utilizing the inhibi-
tory properties of glyphosate on AChE and its subsequent effect on
thiocholine production, quantification of glyphosate concentration in
solution can be correlated with the change in color and SERS signal
intensity due to thiocholine-induced agglomeration of the 10OsCO-Au
M.J. Tan et al. Biosensors and Bioelectronics 96 (2017) 167–172

Fig. 3. (A) Color changes of 10OsCO-Au NPs with respect to varying concentrations of glyphosate (top row). The same procedures were compared with standard Ellman method (bottom
row). (B) Absorption of non-aggregated and aggregated 10OsCO-NPs. (C) SERS spectra of 10OsCO-Au NPs (2123 cm−1) with vary concentration of glyphosate. (D) Plot of intensity of CO
stretching frequency vs various concentrations of glyphosate. Inset: Linearity of the expression CO signal against the concentration of glyphosate. (Relative standard deviations =8.3%).
(E) Plot of the intensity of CO stretching frequency vs various concentrations of glyphosate in beer. Standard deviations of the samples were calculated for a sample size of 10. (Relative
standard deviations =12.4%).

NPs. A detection limit of 0.1 ppb and dynamic range of 5 orders of Appendix A. Supporting information
magnitude was achieved using the 10OsCO-Au NPs, and the efficacy
was further demonstrated in real-life samples (spiked beer) with a Supplementary data associated with this article can be found in the
detection limit up to 0.01 ppm glyphosate content. Future work is online version at doi:10.1016/j.bios.2017.05.005.
underway to improve detection beyond the 10 ppm range.
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