Detection and identification of estrogen based on surface-enhanced resonance raman scattering (SERRS)

Article abstract of DOI:10.3390/molecules23061330

Many studies have shown that it is important to consider the harmful effects of phenolic hormones on the human body. Traditional UV detection has many limitations, so there is a need to develop new detection methods. We demonstrated a simple and rapid surface-enhanced resonance Raman scattering (SERRS) based detection method of trace amounts of phenolic estrogen. As a result of the coupling reaction, there is the formation of strong SERRS activity of azo compound. Therefore, the detection limits are as low as 0.2 × 10^?4 for estrone (E1), estriol (E3), and bisphenol A (BPA). This method is universal because each SERRS fingerprint of the azo dyes a specific hormone. The use of this method is applicable for the testing of phenolic hormones through coupling reactions, and the investigation of other phenolic molecules. Therefore, this new method can be used for efficient detection.

Full text of DOI:10.3390/molecules23061330

molecules
Article
Detection and Identification of Estrogen Based on
Surface-Enhanced Resonance Raman
Scattering (SERRS)
Yang Liu 1 , Yue Chen 1,2, Yuanyuan Zhang , Qiangwei Kou 1,2, Yongjun Zhang 1,2
,2
1,2
,
Yaxin Wang 1 , Lei Chen 1,2,*, Yantao Sun , Honglin Zhang 3 and Young Mee Jung *
,2
1,2
4,
1
College of Physics, Jilin Normal University, Siping 136000, China; liuyang@jlnu.edu.cn (Y.L.);
1
7649973053@163.com (Y.C.); 13944139606@163.com (Yu.Z.); 13944949603@163.com (Q.K.);
zhangyongjunwyx@126.com (Yo.Z.); wangyaxin1010@126.com (Y.W.); syt@jlnu.edu.cn (Y.S.)
Key Laboratory of Functional Materials Physics and Chemistry of the Ministry of Education,
Jilin Normal University, Changchun 130103, China
School of Electronic and Information Engineering, South China University of Technology,
Guangzhou 510640, China; eezhl@scut.edu.cn
2
3
4
Department of Chemistry, Institute for Molecular Science and Fusion Technology,
Kangwon National University, Chunchon 24341, Korea
*
Correspondence: chenlei@jlnu.edu.cn (L.C.); ymjung@kangwon.ac.kr (Y.M.J.);
Tel./Fax: +86-434-329-4566 (L.C.); +82-33-250-8495 (Y.M.J.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 25 April 2018; Accepted: 31 May 2018; Published: 1 June 2018
Abstract: Many studies have shown that it is important to consider the harmful effects of phenolic
hormones on the human body. Traditional UV detection has many limitations, so there is a need to
develop new detection methods. We demonstrated a simple and rapid surface-enhanced resonance
Raman scattering (SERRS) based detection method of trace amounts of phenolic estrogen. As a result
of the coupling reaction, there is the formation of strong SERRS activity of azo compound. Therefore,
−
4
the detection limits are as low as 0.2
×
10 for estrone (E1), estriol (E3), and bisphenol A (BPA).
This method is universal because each SERRS fingerprint of the azo dyes a specific hormone. The use
of this method is applicable for the testing of phenolic hormones through coupling reactions,
and the investigation of other phenolic molecules. Therefore, this new method can be used for
efficient detection.
Keywords: phenolic hormones; SERRS; detection
1
. Introduction
Estrogen, which is also known as a “female hormone”, is a natural hormone and steroid that has
very important physiological roles [
estrogen or estrogen-like compounds is related to the development of certain diseases, such as
breast cancer, which has attracted considerable interest [ ]. This type of hormone can increase
1,2]. However, researchers have found that the use of synthetic
3,4
the incidence of breast cancer, heart disease, stroke and thrombosis. Thus, estrogen can be considered
a carcinogen [5–7]. Therefore, it is very important to maintain the hormone concentration within safety
standards and to develop rapid and highly sensitive detection methods of estrogens.
At present, there are two types of estrogen detection methods: direct and indirect detection. The direct
detection method of estrogen includes liquid chromatography–mass spectrometry (LC–MS) [
performance liquid chromatography–tandem quadrupole mass spectrometry (HPLC–MS/MS) [10,11]
The objectives of Bogg’s study was to develop a liquid chromatography–tandem mass spectrometry
LC–MS/MS) method for multi-class steroid hormone detection using biologically relevant
8,9] and high
.
(
Molecules 2018, 23, 1330; doi:10.3390/molecules23061330
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1330
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concentrations, before testing limits of detection (LOD) in a high-background matrix by
ˇ
spiking charcoal-stripped fetal bovine serum (FBS) extract [12]. Celi c´ et al. used an online
ultra-high-performance-liquid chromatography–triple quadrupole tandem mass spectrometry
(UHPLC–MS/MS) method for detection and quantification of natural and synthetic estrogens and
their conjugates in aqueous matrices [13]. However, these methods have some limitations as the
sample pretreatment process is relatively complex, the testing time is relatively long and the results
from these methods cannot be widely applied. On the other hand, the indirect detection method,
such as the high pressure liquid chromatography–radioimmunoassay (HPLC–RIA) [14], enzyme-linked
immunosorbentassay [15] and plasma biosensor [16], has high sensitivity. Nevertheless, these methods
require complex antibody preparation and blurring in specific and non-specific recognition boundaries.
Therefore, the development of a simple, rapid, highly sensitive and selective detection method of
estrogen has become the top priority of researchers.
Surface-enhanced Raman spectroscopy (SERS) is a highly sensitive analytical technique that
enables the detection of single molecules [17
–21]. As an ultrasensitive method, SERS has been widely
used in the determination of chemicals and biological molecules [22
–
24]. The SERS study for estrogen
is focused on improving the adhesion of these estrogens to Ag (or Au) surfaces, thereby enhancing
the SERS. For the detection of estrogen based on SERS, one of the most important issues is the
direct connection of phenolic estrogens and Ag (or Au) with specific difficulties [25,26]. Therefore,
the interaction between Ag (or Au) and estrogen has been intensively studied. The selectivity and
sensitivity of this method can be further improved by combining the surface enhancement with the
molecular resonance Raman (RR) effect (i.e., the excitation line in resonance with both the SPR and
electronic transition of the close molecules). This approach has been called the surface-enhanced
resonance Raman scattering (SERRS) [27,28].
There is a coupling reaction between the phenol and diazonium ions, which produces products
known as azo dyes. Thus, Pauly’s reagent is often used for tyrosine detection based on the optical
absorption of the products, azo compounds. These azo compounds have been proven to have strong
Raman activity and a propensity toward binding to Ag NPs [29]. Coupled with surface-enhanced
resonant Raman scattering (SERRS), the SERRS “fingerprint” spectra of different azo dyes correspond
to the detection and identification of different phenolic estrogens.
In this study, we investigated a sensitive SERS-based detection method of phenolic estrogens.
To greatly enhance the adhesion of analytes to the surface of metal nanoparticles as well as the SERS
signal, we used the coupling reaction between phenolic estrogens and Pauly’s reagents. This proposed
method has the following advantages. First, it is very simple as it only involves the mixing of estrogen,
Pauly’s reagent and silver colloid without the need for a separation or purification procedure. Second,
it takes about 2 min to complete the coupling reaction and SERS measurement, which allows for rapid
analysis. Third, the ultra-sensitive detection was realized using SERS. These results indicate that
this detection method shows significant potential in being highly sensitive for most molecules with
phenolic groups due to the phenolic coupling reaction.
2
. Experimental Section
2
.1. Materials
Estrone (E9750, E1), estriol (E1253, E3) and bisphenol A (239658, BPA) were the three estrogens
used in this present study, with their structures shown in Figure 1. Silver nitrate was purchased
from Sigma-Aldrich Co., Ltd., (Shanghai, China), at the highest purity available and used as received
without further purification. The estrogens were dissolved in ethanol or dioxane. Infant formula
−
1
was purchased from Meiji Co., Ltd., (Shanghai, China). Ultrapure water (18.0 M
throughout the present study.
Ω cm ) was used

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Figure 1. Molecular structures of two natural estrogens (estrone (E1), and estriol (E3)) and a synthetic
estrogen (bisphenol A (BPA)).
2
.1.1. Preparation of Silver Nanoparticles
Colloidal silver nanoparticles were prepared by the aqueous reduction of silver nitrate
10 M, 200 mL) with trisodium citrate (1%, 4 mL) using the method described by Lee and Meisel [30].
−
3
(
The colloidal silver nanoparticles showed a maximum absorption at 420 nm.
2
.1.2. Pauly’s Reagent and Coupling Reaction
We prepared three reagents. Reagent A was prepared by dissolving 4.5 g of p-aminobenzenesulfonic
◦
acid in 500 mL of deionized water, before adding 5 mL of 12 M HCl (the solution was stored at 4 C).
◦
Reagent B was a 5% sodium nitrite solution (the solution was stored at 4 C) and reagent C was a 10%
sodium carbonate solution. The coupling reagent configuration was reagent A, reagent B, reagent C
and estrogen in a volume ratio of 1:1:1:2.
2
.1.3. SERS Measurement
After the coupling reaction, 25
µL of each sample and an equal amount of colloidal silver
nanoparticles were added drop wise to the aluminum pan (No. 0219-0041, Perkin-Elmer, Waltham,
MA, USA before the mixture was irradiated with a laser beam for 30 s before each SERS measurement.
The SERS spectra were measured using a Renishaw Raman microsystem 2000 equipped with
a charge-coupled device (CCD) detector and a holographic notch filter. A wavelength of 514.5 nm
+
Ar ion laser (Spectra Physics, Harwell, UK) was used as an excitation source for SERS measurement.
The typical exposure time used during this study was 10 s with 15-mW power directed at each sample.
2
.1.4. Characterization Methods
A detailed structure of the obtained sample was obtained by a JEOL 2100 transmission electron
microscope (TEM) operating at 200 kV. The light scattering experiments (both SLS and DLS) were
performed using the Zetasizer Nano instrument (Malvern, UK). Ultraviolet–visible spectroscopy
(UV–Vis) absorbance spectra were recorded on a Shimadzu UV 3600 spectrophotometer. The SERS
spectra were measured using a Renishaw Raman microsystem 2000 equipped with a charge-coupled
+
device (CCD) detector and a holographic notch filter. A wavelength of 514.5 nm Ar ion laser
(Spectra Physics) was used as an excitation source for SERS measurement.
3
. Results and Discussion
Two natural steroid estrogens (estrone (E1) and estriol (E3)) and a nonsteroidal estrogen
(bisphenol A (BPA)) as a typical hormone were used in this study. The molecular structures of these

Molecules 2018, 23, 1330
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estrogens are shown in Figure 1. As shown in Figure 1, the molecular structures of two natural
estrogens are quite similar. The fundamental difference between these two natural steroid estrogens is
the number of hydroxyl groups: E1 has a hydroxyl group and E3 has two hydroxyl groups. In contrast,
the nonsteroidal estrogen BPA has a symmetrical structure. As mentioned earlier, the affinity of
these phenolic estrogens to the silver nanoparticles is weak, which leads to a great reduction in the
SERS sensitivity.
In fact, the pure silver colloids still have good dispersibility and demonstrate a narrow particle
size distribution, which is shown in Figure 2. The corresponding average particle size in average
is 41 nm. The particle size of the nanocrystals can also be determined by measuring the random
changes in the intensity of light scattered from a suspension or solution. The dynamic light scattering
(
DLS) is used to analyze the particle size using the associated distribution of the as-prepared Ag NPs,
which is depicted in the inset of Figure 2. From the size distribution histograms obtained via DLS
measurement, the average size of Ag NPs abnormally reaches up to ~70 nm. The abnormal increase in
the average diameter as demonstrated by DLS measurements is mainly attributed to the aggregation
of Ag NPs in aqueous solution [31]. Although we utilized the dried form of the synthesized samples
in TEM, the size of the samples was estimated in an aqueous dispersion in DLS, which provides the
hydrodynamic diameter of the samples. Furthermore, DLS gives an overall size distribution, but the
size of the aggregation is usually measured instead of the individual nanoparticles. Therefore, the size
obtained from the DLS measurement tends to be larger than that of TEM as DLS takes the aggregates
and surfactants into account.
Figure 2. TEM images of the pure Ag NPs with the distribution of hydrodynamic sizes of Ag NPs (inset).
To improve the affinity of estrogen and silver nanoparticles, we used the azo reaction, which is
shown in Figure 3. This azo reaction can further enhance SERS. A coupled reaction is a process in
which two organic chemical units (hydrocarbon fragments) are coupled with the aid of a metal catalyst

Molecules 2018, 23, 1330
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to obtain an organic molecule. In coupling reactions, the acidity medium is very important. In Figure 3,
Azo-N = N- is linked to a hydroxyl group. The R-N = N-R’ azo compounds have cis and trans isomers,
with the trans isomer being more stable than the cis isomer. The two isomers can be converted to each
other under light or heating conditions. Azo compounds are a class of colored compounds, some of
which can be directly used as a dye or indicator. In organic analysis, the use of coupling reactions to
produce colors can identify phenolic or aromatic amine structures.
Figure 3. Coupling reaction between estrone (E1) and diazonium ions.
As a result, when estrogen is added to Pauly’s reagent, we observed a significant change in color
(the inset of Figure 4). When observing with the naked eye, E1 and E3 turn orange, while BPA and
other substances turn yellow after the reaction with Pauly’s reagent. The color changes were more
clearly distinguished by UV–Vis spectroscopy (Figure 4). We noted that two of the azo dyes had
a similar absorption at
When E1, E3 and BPA reacted with the azo dye with the subsequent addition of silver, we found that
the absorption was still around 500 nm, because the absorption peak of Ag was coupled with the
∼
500 nm, which differed from that of the azo product from BPA ( 450 nm) [26].
∼
∼
absorption peak of estrogens (see Figure S1 in the Supplementary Materials). Therefore, 514.5-nm laser
excitation was selected for all estrogens, which was chosen according to the resonance effect of the
samples and the plasmon resonance of silver nanoparticles with the laser. The UV–Vis detection of
hormones has the disadvantage of a relatively low sensitivity and, thus, we chose the SERRS method
for testing. This method that is based on the azo coupling reaction and SERRS technique has the
following advantages of being: (1) simple and fast as the azo-Ag mixture for SERRS test is very easy
to prepare, which only requires direct mixing and can be completed in 2 min; and (2) practical and
reliable in the detection of phenolic endocrine disruptors.
Compared with the UV–Vis spectra, the Raman spectra have a significant advantage
as the high-resolution structure exhibited excellent quantitative and qualitative applications.
This characteristic property encouraged us to select SERS as the basic research method. Compared
with the traditional UV–Vis detection, the sensitivity is greatly improved. The estrogen fingerprint
information can be observed by using the SERS technology (Figure 5). Moreover, the normal Raman
and SERS spectra of diazonium ions before reacting with phenolic estrogens are very weak, which
almost have no interference with SERS spectra of the estrogen-derived azo dyes (see Figure S2 in the
−
1
Supplementary Materials). The concentration-dependent SERRS intensity of the bands at 1072 cm
for E1 and E3, respectively, adsorbed on Ag substrate is shown in Figure 5. At all frequencies, the SERS
intensity varies monotonously with a change in concentrations.

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Figure 4. Visual color change before (a) and after (b) coupling reactions between the estrogens E1, E3
and BPA; and the UV–Vis spectra of the corresponding azo dyes.
−
4
Figure 5. Raman test results after azo reaction of E1 (
a
) and E3 (
c
) at concentrations of 1.0
×
10 M,
−
4
−4
−4
−4
0.8
×
10 M, 0.6
×
10 M, 0.4
×
10 M, 0.2
×
10 M and 0 M, respectively. Concentration-dependent
−
1
SERRS intensities of the Raman bands at 1072 cm for E1 (b) and E3 (d).
From the concentration-dependent SERS spectra of BPA shown in Figure 6a, we found that,
−
4
−1
when the concentration of BPA was 0.2
×
10 M, there was one vibrating band at 1420 cm ,
which was mainly attributed to the –N=N– stretching vibration of the trans isomer. Compared with the
other SERS spectra of the estrogen-derived azo dyes investigated in this study, the SERS spectra of the

Molecules 2018, 23, 1330
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−
1
BPA-derived azo dye is unique, due to the presence of a weaker band at 1449 cm and a new band at
1
−
1
420 cm , which probably originated from the stereo structure of BPA and its steric effects on –N=N–
stretching. Moreover, with an increase in the BPA concentration, the intensity of these two bands
−
−
1
gradually increases. The bands at 1598, 1385 and 1124 cm may originate from the –C–C– within
1
phenol and/or phenyl rings. Furthermore, a band at 1385 cm also comes from the phenol groups in
−
1
the –CH– and –OH vibration. A band at 1198 cm is the result of the effects of –CH, –OH and –C–C
in the phenol group in –CN and phenyl-N in –C–N–N [32 35]. The concentration dependent SERRS
–
−
1
intensity at the 1385 cm band for BPA adsorbed on Ag substrate is shown in Figure 6b. The SERS
intensity of all bands varies monotonically with a change in concentrations.
−4
−4
10 M,
Figure 6.
(
a
) Raman spectra after azo reaction of BPA at the concentrations of 1.0
×
10 M, 0.8
−4
10 M and 0 M, respectively; and (b) the plot of the SERS intensity of
×
−
4
−4
0.6
×
10 M, 0.4
×
10 M, 0.2
−1
×
the band at 1385 cm for BPA as a function of the concentration.
To investigate the reusability of the proposed method, we studied the spectrum of SERRS azo
dyes in estrogen mixtures. Figure 7a shows the SERRS spectra of the three hormones E1, E3 and BPA
separately, while Figure 7b shows the SERRS spectra of the three hormone cocktails. Due to their
azo structure, the affinity of the azo dyes for their silver nanoparticles allows the parallel adsorption
−
1
of overlapping SERRS bands. The 1168, 1485 and 1385 cm bands in Figure 7b correspond to the
characteristic peaks of E1, E3 and BPA in Figure 7a, respectively. As shown in Figure 7b, composite
SERRS bands were clearly observed in the azo dyes of the estrogen mixture, confirming the application
potential of the present method for the detection of an estrogen mixture.
Figure 7. SERRS spectra of the estrogen-derived azo dyes from estrogen mixtures (E1, E3 and BPA) (
and their individual spectra (each estrogen was at a concentration of 1.0 × 10−4 M) (a).
b);

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4
. Conclusions
In summary, this SERRS-based detection method significantly improves the sensitivity of the
detection of phenolic hormones. The use of coupling reactions to form a “bridge” between Ag and
phenolic hormones is an indirect means, which provides the advantages of being simple and fast
with universal applications and high selectivity. This can be used for the sensitive and rapid detection
of other phenolic hormones. Our approach provides a simple, fast and ultra-sensitive platform for
the detection of phenolic estrogen in practical applications. It can be extended to numerous other
applications in the food safety and environmental safety fields.
Supplementary Materials: The following are available online. Figure S1: The UV–Vis spectrum of silver colloids
added after the coupling reaction of phenol and diazo ion. Figure S2: Raman spectra of pure E1 dissolved and its
SERRS spectra in silver colloid; the concentration of E1 was 100 ppm.
Author Contributions: Y.L. wrote the original draft; Y.C., Y.Z. (Yuanyuan Zhang) and Q.K. carried out the
experiments and tested the data; Y.Z. (Yongjun Zhang) and Y.W. reviewed and enhanced the revised manuscript;
L.C. conceived the whole paper and provided the funding supports; Y.S. verified the experimental results; H.Z.
provided software and verified the experimental results; and Y.M.J. supervised the whole work and helped
perform the analyses with constructive theoretical discussions.
Funding: This research was funded by the National Natural Science Foundation of China (Grant Numbers
2
1676115, 51609100, 61705020, 61675090 and 61575080); Program for the development of Science and Technology of
Jilin province (Grant Numbers 20160101287JC and 20150519024JH); Guangdong Province Science and Technology
Projects (Grant Numbers 2015A030310342 and 2015A010103004); and Technology of Education Department of
Jilin Province (Grant Number JJKH20170374KJ).
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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