Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 285 (2023) 121880

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Spectrochimica Acta Part A:

Molecular and Biomolecular Spectroscopy
journal homepage: www.journals.elsevier.com/spectrochimica-acta-part-a-
molecular-and-biomolecular-spectroscopy

Rapid and robust analysis of aristolochic acid I in Chinese medicinal herbal

preparations by surface-enhanced Raman spectroscopy
Xiao Meng a, Mengping Zhang a, Lingfei Liu b, Jie Du c, Nianlu Li a, e, Wei Zou a, Cuijuan Wang a,
Wenwen Chen a, Haiyan Wei a, Ranran Liu a, Qiang Jia a, Hua Shao a, *, Yongchao Lai d, *
a
Shandong Academy of Occupational Health and Occupational Medicine, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan 250062,
China
b
Diagnostic Imaging Department, Shandong Cancer Hospital and Institute, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan 250117,
China
c
Department of Pharmacy, The Third Affiliated Hospital of Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan 250013, China
d
Medical Science and Technology Innovation Center, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan 250117, China
e
Key Laboratory for Colloid & Interface Chemistry of Education Ministry, Department of Chemistry, Shandong University, Jinan 250100, China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• • SERS-based method for rapid detec­

tion of AAI in Chinese Medicinal herbal
preparations.
• • MA acted as adjusting the morphology
and mediating the adsorption of AAI.
• • Quantitative detection of AAI concen­
trations in the range from 0.2 to 120.0
μM.
• • Low detection limit of 0.06 μM.
• • Good recovery in Chinese Medicinal
herbal preparations.

A R T I C L E I N F O A B S T R A C T

Keywords: The use of Chinese herbs containing aristolochic acid can induce the exchange of adenine and thymine in gene
Surface-enhanced Raman spectroscopy mutations and even cause liver cancer. To eliminate the harm of aristolochic acids (AAs) to humans, a rapid and
Aristolochic acid I robust method of AAs screening is a prerequisite. In this work, a facile and robust Surface-enhanced Raman
Modification
spectroscopy (SERS) method was used for the qualitative and quantitative detection of AAs in Chinese medicinal
Mandelic acid
herbal preparations based on the mandelic acid modified Ag nanoparticles SERS substrate. Qualitative and
quantitative SERS detection of Aristolochic acid I (AAI) was achieved with a good linear relationship ranging
from 0.2 − 120.0 μM and a limit of detection (LOD) of 0.06 μM. The proposed method demonstrates a refined
strategy for sensitivity analysis of AAs with the advantages of easy operation, time-saving, high sensitivity, and
molecular specificity, making it a preferred platform for the screening of AAI in regular inspections of herbal
products and regulatory supervision of the supply chain.

* Corresponding authors.
E-mail address: yclai@sdfmu.edu.cn (Y. Lai).

https://doi.org/10.1016/j.saa.2022.121880
Received 8 July 2022; Received in revised form 22 August 2022; Accepted 10 September 2022
Available online 16 September 2022
1386-1425/© 2022 Elsevier B.V. All rights reserved.
X. Meng et al.
1. Introduction
AAs usually exists in Aristolochia and Asarum plants which have
been widely used in Chinese medicine for various diseases due to its
therapeutic effects such as anti-infective, anti-tumor, analgesic, anti­
fertility, and anti-inflammatory [1]. However, in recent years, more and
more toxic effects of AA have been reported including renal toxicity,
hepatotoxicity, and genetic toxicity. Previous studies have confirmed
that AA can induce the exchange of adenine and thymine in gene mu­
tations [2], resulting in errors in the replication process of genes and
gradually evolving into cancer. Researchers sequenced the genomes of
hepatocellular carcinomas in Taiwan and found that nearly 80 % of
cases demonstrated the unique mutational signature of AA exposure [3].
The kidney injury associated with AA exposure is known as aristolochic
acid nephropathy [4], caused either by ingestion of AA-containing drugs
as part of traditional therapies or by the environmental pollutants in the
food chain [5,6]. Considering the wide availability of AA-containing
plants, an urgent measure to reduce its exposure is to ensure the
safety of herbs through regular inspections of herbal products, and
regulatory supervision of the supply chain. Therefore, the prerequisite
issue is developing a rapid and robust method for AA screening.
Aristolochic acid I (AAI) is the major component in the Aristolochia
species and its internal exposure concentration is a critical indicator of
the nephrotoxicity and carcinogenicity of AA. Traditional analytical
techniques proposed to detect AAI in Aristolochia species and their
preparations include thin-layer chromatography [7], cyclic voltamme­
try [8], and capillary electrophoresis with electrochemical detection [9],
high-performance liquid chromatography with UV absorption detection
[10], high-performance liquid chromatography-mass spectrometry
[11], and so on. Some of these methods are accurate in determining AAI
concentrations and have been used as the gold standard. However, the
complicated and tedious operation processes of these methods hinder
them to be rapid and robust methods for the regulatory screening of AAs
[12]. A refined method that takes advantage of easy operation, time-
saving, high sensitivity, and molecular specificity is expected to cater
to these requirements. Surface-enhanced Raman spectroscopy (SERS) is
a preferred candidate, which exhibits unique advantages for in-suit or
on-site analysis [13–15]. SERS is a vibrational spectroscopic technique
that could amplify Raman signals when the Raman-active molecules are
close to the substrate surface [16] and provide rich chemical structure
fingerprinting information of most molecules at ultra-low concentra­
tions even at the single-molecule level [17]. SERS has been proven to be
a very effective tool in biosensors [18], food safety [19], the protection
of cultural heritage [20], forensic analysis [21], explosive screening
[22], environmental monitoring [23] and public security [24].
In this work, a facile and robust SERS method was used for the
qualitative and quantitative detection of AAI in Chinese medicinal
herbal preparations (Scheme 1). Silver nanocrystals on copper foil
which is fabricated by an AgNO3/ Mandelic acid (MA) system are used
as SERS substrates. MA played triple roles in the process: adjusting the
Scheme 1. The diagram of SERS detecting AAI process.
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 285 (2023) 121880
morphology of silver nanocrystals to optimize SERS activity, pre­
concentration of AAI close to the substrate surface through their
hydrogen bond interaction and acting as the internal spectral reference
in the quantitative detection. The thermal stability under continuous
laser radiation, SERS activity, and reproducibility were evaluated to
verify the feasibility of the MA-modified substrate. Qualitative and
quantitative SERS detection of AAI was achieved with a good linear
relationship ranging from 0.2 − 120.0 μM and a LOD of 0.06 μM. The
proposed SERS method shows satisfactory sensitivity and selectivity and
will pose a good prospect in practical application.
2. Experimental section
2.1. Chemical reagents
AAI was purchased from Sigma-Aldrich Inc. (St. Louis, USA). Silver
nitrate (99.8 %), 4-aminothiophenol (PATP, 98.0 %), salicylic acid (SA,
99.8 %), 4-hydroxybenzoic acid (HA, 99.0 %), and copper foils (Cu, 0.1
mm in thickness, 99.99 %) were purchased from Sinopharm Chemical
Reagent Co., ltd. (Shanghai, China). MA (99.0 %) was purchased from
Macklin Biochemical Co., ltd. (Shanghai, China). Citric acid (CA, 99.8
%) was obtained from Tianjin Guangcheng Chemical Reagent Co., ltd.
(Tianjin, China). Huoxiang Zhengqi Oral Liquid was purchased from Tai
Chi Group Chongqing Fuling Pharmaceutical Factory Co., ltd.
(Chongqing, China). All reagents were used without further purification.
Ultrapure water (18.25 MΩ/cm) used in all experiments was purified on
a Millipore Q system.
2.2. Apparatus
The morphology of the Ag nanoparticles was characterized by a cold
field emission scanning electron microscope (FE-SEM) (ZEISS Gemini
300, Japan) operating at an accelerating voltage of 3.0 kV. UV–vis ab­
sorption spectra were recorded on a UV2600 UV–vis spectrometer
(SHIMADZU, Japan). Raman spectra were recorded with a confocal
Raman spectrometer (Thermo DXR2, USA) equipped with a 10 ×
objective (NA = 0.5) and a semiconductor laser of 785 nm wavelength,
the excitation power was 10 mW and the accumulation time was 1 s.
2.3. Synthesis of SERS substrate
The copper foils were cut into 0.5 × 0.5 cm2 pieces and separately
rinsed with acetone, ethanol, and ultra-pure water under ultrasonic
irradiation for 10 min to degrease the surface and then washed with
water and dried for further use. Four chemical reagents (MA, CA, HA,
and SA) were selected to adjust the morphology of silver nanocrystals.
The copper foils were immersed in the aqueous solution of AgNO3 (1.0
mM) and MA/CA//HA/SA with different ratios (1:1–1:10) and different
times (1–25 min). Then the treated copper pieces were taken out and
rinsed with water and ethanol in turn before drying at room
X. Meng et al.
temperature.
2.4. SERS detection of AAI
2.4.1. Standard solutions and calibration curves
A 1.0 mM stock solution of AAI was prepared in absolute ethanol and
then the solutions of different concentrations (0.2 to 120.0 μM) were
prepared by diluting the stock solution with Ultra-pure water. The
prepared substrates were incubated for 80 min in the AAI solution of
different concentrations, and the SERS signal was recorded at various
times to obtain the optimal incubation time (Schematic 1). SERS spectra
of the substrates collected from different random points were recorded
to evaluate the reproducibility of the substrates.
2.4.2. Real sample detection
Huoxiang Zhengqi oral liquid (Dilute 1000 times with ultrapure
water) was chosen as the interference reagent to verify the selectivity of
the proposed method. Huoxiang Zhengqi oral liquid water diluent was
chosen to dilute AAI stock solution into different concentrations (0.2 to
120.0 μM). The prepared substrates were added to AAI solutions at
different concentrations and the signal intensities were recorded.
2.4.3. Determination of AAI by HPLC
HPLC was conducted to verify the reliability of the SERS method. The
chromatographic separation was performed on a C18 column (4.6 mm
× 250 mm, 5 μm) at the temperature of 30 ◦ C with gradient elution using
a mixed solution composed of methanol (A) and 0.2 % glacial acetic acid
(B). The gradient elution procedure was as follows: 0–15 min, 40–80 %
A; 15–20 min, 80–100 % A; 20–30 min, 100 % A; 30–35 min, 100–40 %
A. The flow rate was set to 1.0 mL/min and the injection volume was 10
μL. The signals were recorded at the wavelength of 254 nm with a DAD
detector.
Fig. 1. (a) FE-SEM images of silver nanoparticles on the SERS substrates of AgNO3:MA at a ratio of 1:1. (b) Raman contour plot of PATP adsorbed on the SERS
substrates. (c) The temporal stability and (d) the reproducibility of the substrate probed with 10− 4 M PATP.
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 285 (2023) 121880
2.5. Interaction between MA and AAI
UV–vis absorption spectroscopy was used to verify the interaction
between MA and AAI. 1.0 mL aqueous solutions of AAI (0, 0.2, 20.0, and
200.0 mM) were mixed with 1.0 mL aqueous solution of MA (200.0
mM), and the mixtures were incubated for 30 min at room temperature.
Then the mixtures were transferred to a quartz cuvette and their spectra
were recorded at 300–500 nm.
The interaction between MA and AAI was simulated by density
functional theory (DFT) at RM062X/def2tzvp level [25,26]. The ge­
ometry optimization and vibrational spectra calculation were performed
for the complex of MA and AAI by using the Gaussian 16 software
package [27].
3. Results and discussion
3.1. Fabrication and characterization of the SERS-active substrate
In this study, MA, CA, HA, and SA were used to regulate the silver
nanostructures during the galvanic replacement reaction between
AgNO3 and copper foils and galvanic replacement reaction [28–30] is a
reliable way to produce a planar substrate with various nanostructured
materials for SERS. The process is simple and easy to scale up. Those
dense silver nanostructures, covering the substrates, are free from
oxidation with the help of metallic copper and the substrates show
strong surface plasmon resonance in visible light region. Under contin­
uous laser radiation, the substrates can still give stable SERS signals.
During the reaction process, silver nanostructures are formed by the
diffusion-controlled crystal growth process [31]. MA is used as a com­
plex reagent to adjust the morphology of silver nanocrystals. As shown
in Fig. 1a, aggregated silver nanoparticles were observed on copper foils
in the four systems. The SERS substrate prepared by AgNO3/MA has
more dense silver nanoparticles indicating better SERS performance.
X. Meng et al.
The SERS performance of these substrates was evaluated with PATP, in
which the SERS spectra of PATP were recorded with a confocal Raman
system under the same experimental conditions. As shown in Fig. S1, the
substrate prepared by AgNO3/MA has the best SERS performance among
the four substrates. Therefore, the MA-Ag substrate was chosen in the
following study.
The ratios of AgNO3/MA and reaction time were investigated to
obtain the optimal reaction conditions. As shown in Fig. S2 and Fig. S3,
the surface morphology of SERS substrates is highly related to the ratios
of AgNO3/MA and reaction time. PATP, a well-known probe molecule,
was used to evaluate the SERS performance of the SERS substrates
prepared with AgNO3 and MA at different ratios and at different times.
As shown in Fig. S4, the best SERS performance can be obtained when
the ratio of AgNO3/MA is 1:2 and the reaction time is 15 min. The spatial
uniformity of the SERS substrate was also evaluated with PATP. It was
found the SERS substrate had a relatively homogeneous SERS perfor­
mance with some small undulations which may be caused by the un­
evenness of the commercial copper foil (Fig. 1b). The reproducibility of
the SERS substrate was assessed by examining SERS spectra at 20
random points in the same substrate, as shown in Fig. 1d and Fig. S5b. A
relative standard deviation (RSD) of 6.4 % indicated the substrate had a
good reproducibility.
As far as the practical application of SERS analysis is concerned,
thermal stability is an important parameter that affects the good practice
of SERS substrates [32,33]. The thermal effect generated by the laser
power may decompose the sample molecules or destroy the prepared
metal nanostructures on the metal surface, thereby weakening the SERS
performance. We determined the stability of the SERS substrate by
recording the SERS spectra of PATP at the concentration of 10− 4 M
under continuous Raman laser irradiation. The laser excitation wave­
length was 785 nm and the power was 10 mW. The acquisition time for
all spectra was 1 s for each Raman spectrum and an interval time of 5 s
between two SERS spectra. As shown in Fig. 1c, no significant change
was found in 300 s, which confirmed the good thermal stability of the as-
prepared SERS substrate. The RSD of the Raman intensity at 1436 cm − 1
and 1076 cm − 1 were 4.4 % and 6.1 %, respectively, as shown in
Fig. S5a. The good thermal stability can be attributed to the high thermal
conductivity of silver and copper. The tightly integrated structure of the
SERS substrate can effectively transfer and reduce the heat on the laser
spot, thereby limiting the temperature rise and preventing the negative
thermal effect for SERS measurement.
In short, a stable SERS substrate was prepared with the improved
galvanic replacement on a commercial copper foil. The as-prepared
substrate not only has good SERS activity, thermal stability, and
reproducibility, but is also robust in manufacture, cost, and practicality.
The above advantages provide a preferred candidate for the following
study.
3.2. The basis of AAI detection
It is well-known that sufficient SERS signals could be only obtained
when the analytes are close to or adsorb on the substrate surface.
However, many analytes, such as AAI, show weak affinity to the SERS-
activated metal substrates and scarcely adsorb on the substrates,
which makes their direct detection by SERS difficult. Therefore, a sur­
face modification strategy should be executed for facilitating AAI
adsorbed on the surface of the substrate. Since the as-prepared substrate
is obtained by a galvanic replacement reaction of AgNO3 and MA on
copper foil, MA is inevitably adsorbed on the substrate surface
(Fig. S6b), which might act as a surface modifier and mediate the
adsorption of AAI on the SERS substrate.
As shown in Fig. S6a, the SERS spectrum of AAI (120.0 μM) on MA-
Ag substrate mainly corresponded with the Raman spectrum of pure AAI
powder, indicating AAI can be adsorbed on the surface of MA-Ag sub­
strate with the assistance of MA. To further confirm the role of MA, the
MA-Ag substrate was treated with NaBH4 and KI to remove the surface
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 285 (2023) 121880
MA. NaBH4 and KI were known as competitive adsorption agents to
remove impurities on the substrate surface [34–37]. Compared with the
SERS response on untreated MA-Ag substrate, AAI on NaBH4 and KI-
treated MA-Ag substrates showed a much weaker signal response,
proving the presence of MA enhances the adsorption of AAI on the SERS
substrate.
To clarify the role of MA in SERS detection of AAI, UV − vis ab­
sorption spectroscopy was used to study the interaction between MA and
AAI conducted. As shown in Fig. 2a, the UV absorption intensity of AAI
changed as the concentration of MA increased, which confirm the
interaction between MA and AAI. Theoretical calculation was used to
clarify the type of interaction between MA and AAI. Specifically, the
complex of MA and AAI was optimized with DFT at RM062X/def2tzvp
level [25,26], then interaction energy and vibration analysis were per­
formed with the optimal configuration. As shown in Fig. 2c, the inter­
action between MA and AAI was realized through two hydrogen bonds
with band distances of 2.117 and 1.801 Å. The calculated Raman
spectrum of AAI-MA is illustrated in Fig. 2b (curve C) which accord with
the experimental data (curve B). The peaks at 1325, 1362, and 1543 cm−
1
can be assigned to the –COOH and –NO2 groups in AAI [38,39]. The
stretching mode of carbon–carbon bonds in the benzene ring of MA
(Fig. 2c) was located at 1002 cm − 1. The tiny shifts of some peaks can
attribute to the interaction between AAI and the substrate surface. Based
on these results, the adsorption of AAI onto the MA of the substrate was
attributed to the interaction between –COOH and –NO2 groups in AAI
and –COOH and –OH groups in MA. For the quantitative evaluation of
the stabilities of AAI-MA complexes, interaction energy (ΔE) between
them were obtained using the following equation:
ΔE = (EAAI + EMA ) − EAAI− (1)
MA
where EAAI , EMA , and EAAI-MA are the energies of AAI, MA, and AAI-
MA, respectively. The calculated ΔE values were − 9.10 kcal/mol and
− 7.99 kcal/mol after basis set superposition error (BSSE) correction
[40], verifying the existence of interactions between AAI and MA. The
formation of hydrogen bonds between AAI and MA can capture AAI
molecules close to the metal surface, which is the basis of AAI detection
in this study. As far as we know, this is the first example of using SERS
technology to detect AAI through MA for substrate modification, which
allows AAI to be concentrated near the substrate surface, which makes
their SERS detection possible.
3.3. Qualitative and quantitative detection of AAI
The time-dependent adsorption behavior of AAI on MA-Ag substrate
was investigated by continuously recording SERS spectra for 80 min
(Fig.S7a). As shown in Fig. 3a, The SERS intensity at 1338 cm− 1 rose as
the reaction time increases in the first 60 mins and then remained steady
in the next 10 mins, which indicated that the adsorption was saturated
after 60 mins and could keep a relatively long signal stabilization time
for spectral acquisition. To explore the reproducibility of the as-
prepared SERS substrate for AAI, the MA-Ag substrate was immersed
in 5.0 μM AAI solution for 60 mins, and the SERS spectra were recorded
on 30 random points on the same substrate (Fig. S7b). The corre­
sponding relative intensity variations at the characteristic Raman shift
of 1338 cm− 1 were listed in Fig. 3b. An RSD of 3.3 % indicated as-
prepared SERS substrate had a good reproducibility for AAI detection.
The quantitative performance of the MA-Ag substrate was evaluated
under optimal experimental conditions. A series concentration of AAI
(0.2–120.0 μM) were prepared and detected with MA-Ag substrates. As
shown in Fig. 3c, the Raman intensity of AAI increases gradually with
the increase of AAI concentration. As the amount of MA was constant on
the surface of the MA-Ag substrate, the SERS intensity of MA could be
considered as an internal standard, which could provide a more reliable
method for studying the correlation between molecular concentration
and its SERS intensity. The characteristic Raman band of MA at 1002
cm− 1 was selected as the reference to normalize the variance of the
X. Meng et al. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 285 (2023) 121880

Fig. 2. (a) UV − vis absorption spectra of 200.0 mM AAI incubated with different concentrations of MA. (b) AAI powder(A), the experimental SERS spectrum (B), and
the calculated Raman spectrum (C) of AAI-MA, (c) Optimized molecular configuration of AAI-MA from DFT.

intensity of AAI at 1338 cm− 1. As displayed in Fig. 3d, the normalized ( )

Raman signal intensity of AAI at 1338 cm− 1 increases with the
[MA • AAI n ]
log = logK + log[MA] + nlog[AAI] (4)
increasing concentrations of AAI. The log − log plot of the normalized [MA]
Raman intensity of AAI at 1338 cm− 1 versus its concentration exhibits a where [MA] is the concentration of MA adsorbed on the substrate
good correlation (the inset of Fig. 3d). The linear regression equation is surface, [AAI] and [MA • AAIn ] are the concentrations of AAI in the
y = − 0.55 + 0.15x (where × is the logarithm of the AAI concentration liquid matrix and on the MA-modified substrate. In this study, the
and y is the logarithm of the normalized SERS intensity of the Raman molecules adsorbed on the surface are limited due to the low surface
peak at 1338 cm− 1) with the correlation coefficient of 0.99. The error areas of MA-Ag substrates. So, the concentration of AAI is approximately
bars were estimated from three times repeating measurements. The LOD equal to the initial concentration c0. Therefore, the equation (4) could be
was calculated to be 0.06 μM at S/N = 3. The enhancement factor (EF) written as.
for AAI was 3.6 × 104 (detailed EF calculations are provided in Fig. S8). ( )
The enhancement factor of AAI is obtained by calculation. The above [MA • AAI n ]
log = logK + log[MA] + nlogc0 (5)
results indicate that the aggregates of silver nanoparticles on the copper [MA]
foil can be used as an effective material for qualitative and quantitative as.
detection of AAI. ISERS ∝ N(6) [47] where ISERS is the SERS signal, N is the number of
The proposed method has a comparable or superior linear range and molecules involved in the SERS process. The following equation can be
limit of detection compared with other methods for AAI detection, obtained.
which are summarized in Table 1. The SERS detection of AAI using MA-
SERS substrate has similar quantitative results (a good linear relation­ ISERS,MA•AAI n NMA•AAI n [MA • AAI n ]
= = (7)
ship of the log − log plot) as SERS detection of PAHs using alkanethiols ISERS,MA NMA [MA]
modified substrate [45,46]. The adsorption between the substrate and Therefor the equation (5) can be expressed as according to equation
the analyte solution at equilibrium could use adsorption isotherm (7).
equations. Assuming n is the number of AAI molecules interacting with ( )
MA on the substrate surface, the equilibrium could be considered as log
ISERS,MA•AAI n
= logK + log[MA] + nlogc0 (8)
follows: ISERS,MA

[MA] + n[AAI] ↔ [MA•AAIn ] (2) As [MA] is fixed in this study, logK +log[MA] can be considered as a
constant. The equation (8) could be written as follows.
The equilibrium constant (K) could be written as. ( )
ISERS,MA•AAI n
[MA • AAI n ] log = A + nlogc0 (9)
K= (3) ISERS,MA
[MA][AAI]n
where A is a constant that are not related to AAI (MA) concentration
After taking the log at both ends of the equation, we can get. and includes all Raman and SERS related parameters. From equation (9),

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X. Meng et al. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 285 (2023) 121880

Fig. 3. (a) The SERS intensity at 1338 cm− 1 of 100.0 μM AAI with different adsorption time. (b) the reproducibility of the MA-Ag substrate from 30 random points at
peak of 1338 cm − 1. (c) SERS spectra of AAI on MA-modified substrate with concentrations of (A) 120.0, (B) 60.0, (C) 40.0, (D) 20.0, (E) 10.0, (F) 5.0 and (G) 2.0, (H)
1.0, (I) 0.5 and (J) 0.2 μM, respectively. (d) Calibration curves and the inset log–log plot for AAI based on MA-modified substrate.

developed SERS method based on MA-modified Ag NPs is a reliable,

Table 1
sensitive, and rapid AAI detection technique for daily exposure.
Comparison of different sensors and methods for the detection of AAs.
Method LOD (μM) Linear range (μM) reference

Ag@BSA NPsa AAI:0.28 AAI: 0.5–50 [41] 3.4. Real samples detection
HPLC-DADb AAI: 5.86 × 10− 4 AAI: 0.58–1.76 [42]
OMC/GCEc Total AA: 0.186 Total AA: 0.6–50 [43] As a commonly used over-the-counter (OTC) drug, the Huoxiang
CE-MSd AAI: 1.46 × 10− 4 AAI: 8.79 × 10− 4 − 4.83 × 10-2 [44]
Zhengqi Oral Liquid use Magnolia officinalis and Magnolia officinalis as
Ag@MA NPs AAI: 0.12 AAI: 0.2–120 This study
raw materials, which may lead to the adulteration of aristolochic acid.
a
Surface-enhanced Raman scattering, bhigh-performance liquid chromatog­ Therefore, the Huoxiang Zhengqi Oral Liquid was selected as the
raphy–diode array detector, cpyrolytic graphite electrode, dcapillary electro­ interference matrix to investigate the anti-interference ability of the
phoresis–mass spectrography, eenzyme-linked immune sorbent assay.
proposed SERS method in this study. Different concentrations of AAI
were spiked into Huoxiang Zhengqi oral liquid and detected by the
it could be interpreted that the good linear relationship of the log − log proposed SERS method. As shown in Fig. 4a, the SERS intensity of AAI
plot is rational. increases gradually with the increase of AAI concentration. The char­
The performance of this method was compared with that of other acteristic peak of MA at 1002 cm− 1 was selected as the reference and the
detection methods (Table 1). The proposed method not only provides a peak of AAI at 1338 cm− 1 was normalized in the same way. As shown in
relatively wider detection range and lower detection limit compared Fig. 4b, the normalized Raman signal intensity of AAI at 1338 cm− 1
with other methods but is also superior to those methods in simple increases with the increasing concentrations of AAI. The log − log plot of
operation processes, rapid detection, and fewer environmentally unfa­ the normalized Raman intensity of AAI at 1338 cm− 1 versus its con­
vorable procedures. Comparison with the HPLC results and the analysis centration exhibits a good linear correlation with R2 = 0.98 (the inset of
performance were summarized in Table S1. The SERS method based on Fig. 4b). The LOD was calculated to be 0.11 μM at S/N = 3 and the re­
Ag-SERS substrate has satisfactory recovery rates from 81.9 % to 120.0 covery of the spiked samples were within the range of 76.5–131.5 %.
%, and RSD from 3.8 % to 7.3 %. Obviously, the results obtained by the These results have also been cross-compared with HPLC and the analysis
SERS method are in good agreement with the results of the HPLC performance is summarized in Table S2. Although the detection limit of
method, indicating that the method is highly reliable. Compared with HPLC is lower than that of SERS, the process of SERS is simple and ef­
the HPLC and other methods, our method has the advantage of fast ficiency. To evaluate the repeatability of this method, three concentra­
detection time, using only several seconds to acquire one spectrum [48], tions of AAI (1.0 μM, 20.0 μM, and 120.0 μM) were analyzed in 10
performing well in the mixed sample without the pre-separation [49] parallel experiments (Figs. S9a–c) and the corresponding RSDs were 2.2
due to its fingerprint characteristics. The above results indicate that the %, 1.8 % and 8.2 %, respectively (Fig. 4c). These results indicate that the

6
X. Meng et al.
Fig. 4. (a) SERS spectra of AAI on MA-Ag substrate in real samples with concentrations of (A) 120.0, (B) 60.0, (C) 40.0, (D) 20.0, (E) 10.0, (G)5.0 and (G) 2.0, (H)
1.0, (I) 0.5 and (J) 0.2 μM, respectively. (b) Calibration curves and the inset log–log plot for AAI based on MA-Ag substrate. (c) The repeatability of MA-modified Ag
substrate at different concentrations. (d) The results of SERS versus HPLC method.
proposed method exhibits high repeatability for the quantitative anal­
ysis of AAI. Furthermore, the results of the SERS versus HPLC method
with an R2 of 0.989, indicating the results are in very good agreement
(Fig. 4d). In all, the SERS-based method used in this study shows a
satisfactory sensitivity and selectivity; and will pose a good prospect in
practical application.
Zhisou Huatan Granules (Nanjing Tongrentang, China), another
Chinese patent medicine containing aristolochia, were selected as the
matrix to verify the feasibility of the proposed method. Zhisou Huatan
Granules were diluted with ultrapure water by 250 times as the matrix,
and then the AAI was spiked into this diluent with a finally concentra­
tion of 100.0 μM. The SERS spectra of the spiked (curve A) and unspiked
(curve B) samples on MA-Ag substrate were illustrated in Fig. S10, in
which characteristic Raman peaks of AAI could be found in spiked
Zhisou Huatan Granules, indicating that the proposed method can be
applied to other samples.
4. Conclusions
In this work, silver nanoparticle aggregates on copper foil by AgNO3/
MA system were successfully synthesized and used to perform rapid
detection of AAI with a confocal Raman spectrometer. The effect of re­
action time and the ratio of AgNO3 and MA were investigated. Under the
optimal conditions, the Ag-SERS substrate has high temporal stability
under continuous laser radiation, good uniformity, and reproducibility,
which indicated that the substrate could provide reliable detection
performance. The surface MA can be acted as an intermediary to adsorb
AAI on the substrate surface. UV–vis and DFT calculations revealed the
interaction of MA and AAI originated from hydrogen bonds. The log −
log plot of the normalized Raman intensity of AAI to its concentration
7
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 285 (2023) 121880
exhibits a good linear relationship in the concentration ranges of
0.2–120.0 μM. The LOD was estimated to be 0.06 μM (at S/N = 3).
Moreover, the proposed method shows a good linear range and low
detection limit in the interference matrix of Chinese patent medicines,
making it a potential platform for the detection of AAI.
CRediT authorship contribution statement
Xiao Meng: Investigation, Conceptualization, Methodology, Writing
– original draft. Mengping Zhang: Formal analysis, Funding acquisi­
tion. Lingfei Liu: Data curation. Jie Du: Software. Nianlu Li: Software,
Visualization. Wei Zou: Supervision. Cuijuan Wang: Software, Data
curation. Wenwen Chen: Investigation. Haiyan Wei: Methodology,
Software. Ranran Liu: Validation. Qiang Jia: . Hua Shao: Funding
acquisition, Project administration. Yongchao Lai: Supervision,
Conceptualization, Resources, Funding acquisition, Writing – review &
editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
X. Meng et al.
Acknowledgments
This work is supported by the National Natural Science Foundation
of China (NSFC) (Grant No. 82003503), the Natural Science Foundation
of Shandong Province (ZR2020QH285 and ZR2019PH112), the Medical
and health science and technology development plan of Shandong
Province (202112070425), and the Traditional Chinese Medicine Proj­
ect of Shandong Province (2021M152).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.saa.2022.121880.
References
[1] N. Anandagoda, G.M. Lord, Preventing aristolochic acid nephropathy, Clin J Am
Soc Nephrol 10 (2015) 167–168.
[2] R. Li, L. Di, J. Li, W. Fan, Y. Liu, W. Guo, W. Liu, L. Liu, Q. Li, L. Chen, Y. Chen,
C. Miao, H. Liu, Y. Wang, Y. Ma, D. Xu, D. Lin, Y. Huang, J. Wang, F. Bai, C. Wu,
A body map of somatic mutagenesis in morphologically normal human tissues,
Nature 597 (2021) 398–403.
[3] A.W.T. Ng, S.L. Poon, M.N. Huang, J.Q. Lim, A. Boot, W. Yu, Y. Suzuki,
S. Thangaraju, C.C.Y. Ng, P. Tan, S.-T. Pang, H.-Y. Huang, M.-C. Yu, P.-H. Lee, S.-
Y. Hsieh, A.Y. Chang, B.T. Teh, S.G. Rozen, Aristolochic acids and their derivatives
are widely implicated in liver cancers in Taiwan and throughout Asia, Sci Transl
Med 9 (412) (2017).
[4] D. Krell, J. Stebbing, Aristolochia: the malignant truth, The Lancet Oncology 14
(2013) 25–26.
[5] M. Stiborová, V.M. Arlt, H.H. Schmeiser, Balkan endemic nephropathy: an update
on its aetiology, Arch Toxicol 90 (11) (2016) 2595–2615.
[6] M. Stiborova, E. Frei, H.H. Schmeiser, Biotransformation enzymes in development
of renal injury and urothelial cancer caused by aristolochic acid, Kidney Int 73
(2008) 1209–1211.
[7] S.C. Hsieh, M.F. Huang, B.S. Lin, H.T. Chang, Determination of aristolochic acid in
Chinese herbal medicine by capillary electrophoresis with laser-induced
fluorescence detection, J Chromatogr A 1105 (2006) 127–134.
[8] Z. Sun, L. Liu, X. Zheng, C. Fan, Q. Wang, G. Li, An easy and rapid method to
determine aristolochic acids I and II with high sensitivity, Anal Bioanal Chem 378
(2004) 388–390.
[9] C.H. Kuo, C.W. Lee, S.C. Lin, I.L. Tsai, S.S. Lee, Y.J. Tseng, J.J. Kang, F.C. Peng,
L. Wei-Chu, Rapid determination of aristolochic acids I and II in herbal products
and biological samples by ultra-high-pressure liquid chromatography-tandem mass
spectrometry, Talanta 80 (2010) 1672–1680.
[10] A.P. Grollman, S. Shibutani, M. Moriya, F. Miller, L. Wu, U. Moll, N. Suzuki,
A. Fernandes, T. Rosenquist, Z. Medverec, K. Jakovina, B. Brdar, N. Slade, R.
J. Turesky, A.K. Goodenough, R. Rieger, M. Vukelic, B. Jelakovic, Aristolochic acid
and the etiology of endemic (Balkan) nephropathy, Proc Natl Acad Sci U S A 104
(2007) 12129–12134.
[11] H. Liu, Q.P. Lei, M. Washabaugh, Characterization of IgG2 Disulfide Bonds with
LC/MS/MS and Postcolumn Online Reduction, Anal Chem 88 (2016) 5080–5087.
[12] P. Niemela, Possibilities for using Raman spectroscopy to determine the amount of
analyte directly from a solid-phase extraction cartridge in water-based
applications, Appl Spectrosc 62 (2008) 1378–1383.
[13] N. Yang, T.T. You, Y.K. Gao, C.M. Zhang, P.G. Yin, Fabrication of a Flexible Gold
Nanorod Polymer Metafilm via a Phase Transfer Method as a SERS Substrate for
Detecting Food Contaminants, J Agric Food Chem 66 (2018) 6889–6896.
[14] K. Wu, T. Li, M.S. Schmidt, T. Rindzevicius, A. Boisen, S. Ndoni, Gold Nanoparticles
Sliding on Recyclable Nanohoodoos-Engineered for Surface-Enhanced Raman
Spectroscopy, Advanced Functional Materials 28 (2018) 1704818.
[15] K. Xu, R. Zhou, K. Takei, M. Hong, Toward Flexible Surface-Enhanced Raman
Scattering (SERS) Sensors for Point-of-Care Diagnostics, Adv Sci (Weinh) 6 (2019)
1900925.
[16] X.M. Qian, S.M. Nie, Single-molecule and single-nanoparticle SERS: from
fundamental mechanisms to biomedical applications, Chem Soc Rev 37 (2008)
912–920.
[17] S.Y. Ding, E.M. You, Z.Q. Tian, M. Moskovits, Electromagnetic theories of surface-
enhanced Raman spectroscopy, Chem Soc Rev 46 (2017) 4042–4076.
[18] L.A. Lane, X. Qian, S. Nie, SERS Nanoparticles in Medicine: From Label-Free
Detection to Spectroscopic Tagging, Chem Rev 115 (2015) 10489–10529.
[19] Y. Hu, S. Feng, F. Gao, E.C. Li-Chan, E. Grant, X. Lu, Detection of melamine in milk
using molecularly imprinted polymers-surface enhanced Raman spectroscopy,
Food Chem 176 (2015) 123–129.
[20] C.L. Brosseau, K.S. Rayner, F. Casadio, C.M. Grzywacz, R.P. Van Duyne, Surface-
enhanced Raman spectroscopy: a direct method to identify colorants in various
artist media, Anal Chem 81 (2009) 7443–7447.
[21] A. Raza, B. Saha, Silver nanoparticles doped agarose disk: highly sensitive surface-
enhanced Raman scattering substrate for in situ analysis of ink dyes, Forensic Sci
Int 233 (2013) 21–27.
[22] C. Muehlethaler, M. Leona, J.R. Lombardi, Review of Surface Enhanced Raman
Scattering Applications in Forensic Science, Anal Chem 88 (2016) 152–169.
8
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 285 (2023) 121880
[23] L.L. Qu, Y.T. Li, D.W. Li, J.Q. Xue, J.S. Fossey, Y.T. Long, Humic acids-based one-
step fabrication of SERS substrates for detection of polycyclic aromatic
hydrocarbons, Analyst 138 (2013) 1523–1528.
[24] H. Liu, D. Lin, Y. Sun, L. Yang, J. Liu, Cetylpyridinium Chloride Activated
Trinitrotoluene Explosive Lights Up Robust and Ultrahigh Surface-Enhanced
Resonance Raman Scattering in a Silver Sol, Chemistry - A European Journal 19
(2013) 8789–8796.
[25] F. Weigend, R. Ahlrichs, Balanced basis sets of split valence, triple zeta valence and
quadruple zeta valence quality for H to Rn: Design and assessment of accuracy,
Phys Chem Chem Phys 7 (2005) 3297–3305.
[26] Y. Zhao, D.G. Truhlar, The M06 suite of density functionals for main group
thermochemistry, thermochemical kinetics, noncovalent interactions, excited
states, and transition elements: two new functionals and systematic testing of four
M06-class functionals and 12 other functionals, Theoretical Chemistry Accounts
120 (2007) 215–241.
[27] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman,
G. Scalmani, V. Barone, G.A. Petersson, H. Nakatsuji, X. Li, M. Caricato, A.V.
Marenich, J. Bloino, B.G. Janesko, R. Gomperts, B. Mennucci, H.P. Hratchian, J.V.
Ortiz, A.F. Izmaylov, J.L. Sonnenberg, Williams, F. Ding, F. Lipparini, F. Egidi, J.
Goings, B. Peng, A. Petrone, T. Henderson, D. Ranasinghe, V.G. Zakrzewski, J. Gao,
N. Rega, G. Zheng, W. Liang, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J.
Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, K.
Throssell, J.A. Montgomery Jr., J.E. Peralta, F. Ogliaro, M.J. Bearpark, J.J. Heyd,
E.N. Brothers, K.N. Kudin, V.N. Staroverov, T.A. Keith, R. Kobayashi, J. Normand,
K. Raghavachari, A.P. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi, J.M.
Millam, M. Klene, C. Adamo, R. Cammi, J.W. Ochterski, R.L. Martin, K. Morokuma,
O. Farkas, J.B. Foresman, D.J. Fox, Gaussian 16 Rev. A.03, in, Wallingford, CT,
2016.
[28] A. Gutes, C. Carraro, R. Maboudian, Silver dendrites from galvanic displacement on
commercial aluminum foil as an effective SERS substrate, J Am Chem Soc 132
(2010) 1476–1477.
[29] C. Zuo, P.W. Jagodzinski, Surface-enhanced Raman scattering of pyridine using
different metals: differences and explanation based on the selective formation of
alpha-pyridyl on metal surfaces, J Phys Chem B 109 (2005) 1788–1793.
[30] W. Ye, Y. Chen, F. Zhou, C. Wang, Y. Li, Fluoride-assisted galvanic replacement
synthesis of Ag and Au dendrites on aluminum foil with enhanced SERS and
catalytic activities, Journal of Materials Chemistry 22 (2012) 18327.
[31] J. Fang, B. Ding, X. Song, Self-assembly ability of building units in mesocrystal
structural and morphological transitions in Ag nanostructures growth, CRYSTAL
GROWTH & DESIGN 8 (10) (2008) 3616–3622.
[32] T. Kang, S. Hong, Y. Choi, L.P. Lee, The effect of thermal gradients in SERS
spectroscopy, Small 6 (23) (2010) 2649–2652.
[33] K.W. Kho, Z.X. Shen, Z. Lei, F. Watt, K.C. Soo, M. Olivo, Investigation into a surface
plasmon related heating effect in surface enhanced Raman spectroscopy, Anal
Chem 79 (23) (2007) 8870–8882.
[34] S.M. Ansar, F.S. Ameer, W. Hu, S. Zou, C.U. Pittman, D. Zhang, Removal of
molecular adsorbates on gold nanoparticles using sodium borohydride in water,
Nano Lett 13 (3) (2013) 1226–1229.
[35] R. Hu, K. Zhang, W. Wang, L. Wei, Y. Lai, Quantitative and sensitive analysis of
polystyrene nanoplastics down to 50 nm by surface-enhanced Raman spectroscopy
in water, J Hazard Mater 429 (2022), 128388.
[36] L.J. Xu, Z.C. Lei, J. Li, C. Zong, C.J. Yang, B. Ren, Label-free surface-enhanced
Raman spectroscopy detection of DNA with single-base sensitivity, J Am Chem Soc
137 (2015) 5149–5154.
[37] D. Zhang, H. You, L. Zhang, J. Fang, Facile Surface Modification of Mesoporous Au
Nanoparticles for Highly Sensitive SERS Detection, Anal Chem 92 (2020)
15379–15387.
[38] L. Cao, H. Liu, W. Xie, S. Jiao, X. Wu, K. Yuan, X. Zhou, M. Yang, Y. Guan, H. Cai,
Z. Lai, J. Chen, H. Zhou, Real-time monitoring of aristolochic acid I reduction
process using surface-enhanced Raman Spectroscopy with DFT simulation, Biosens
Bioelectron 179 (2021), 113061.
[39] L. Ouyang, Q. Zhang, G. Ma, L. Zhu, Y. Wang, Z. Chen, Y. Wang, L. Zhao, New
Dual-Spectroscopic Strategy for the Direct Detection of Aristolochic Acids in Blood
and Tissue, Analytical Chemistry 91 (2019) 8154–8161.
[40] S.F. Boys, F. Bernardi, The calculation of small molecular interactions by the
differences of separate total energies, Some procedures with reduced errors,
Molecular Physics 100 (2002) 65–73.
[41] Y. Gao, T. Xuan, F. Chen, Y. Wu, X. Guo, Y. Wen, H. Yang, Protein-docking strategy
boosting Raman detection sensitivity for aristolochic acid I, Sensors and Actuators
B: Chemical 304 (2020), 127223.
[42] M. Araya, S. Garcia, M. Gonzalez-Teuber, Rapid Identification and Simultaneous
Quantification of Aristolochic Acids by HPLC-DAD and Confirmations by MS in
Aristolochia chilensis Using a Limited Biomass, J Anal Methods Chem 2018 (2018)
5036542.
[43] Y. Wang, M. Qiao, Y. Baikeli, X. Mamat, L. Li, X. Hu, Y. Dong, F. Chang, H. Zhang,
G. Hu, Soft-templated mesoporous carbon-modified glassy carbon electrode for
sensitive and selective detection of aristolochic acids, J Hazard Mater 385 (2020),
121550.
[44] X. Fu, Y. Liu, W. Li, Y. Bai, Y. Liao, H. Liu, Determination of dissociation constants
of aristolochic acid I and II by capillary electrophoresis with carboxymethyl
chitosan-coated capillary, Talanta 85 (2011) 813–815.
[45] X. Jiang, Y. Lai, M. Yang, H. Yang, W. Jiang, J. Zhan, Silver nanoparticle
aggregates on copper foil for reliable quantitative SERS analysis of polycyclic
aromatic hydrocarbons with a portable Raman spectrometer, Analyst 137 (2012)
3995–4000.
X. Meng et al.
[46] L. Guerrini, J.V. Garcia-Ramos, C. Domingo, S. Sanchez-Cortes, Sensing polycyclic
aromatic hydrocarbons with dithiocarbamate-functionalized ag nanoparticles by
surface-enhanced Raman scattering, Anal Chem 81 (2009) 953–960.
[47] J. Kneipp, H. Kneipp, K. Kneipp, SERS–a single-molecule and nanoscale tool for
bioanalytics, Chem Soc Rev 37 (2008) 1052–1060.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 285 (2023) 121880
[48] K.E. Shafer-Peltier, C.L. Haynes, M.R. Glucksberg, R.P. Van Duyne, Toward a
glucose biosensor based on surface-enhanced Raman scattering, J Am Chem Soc
125 (2003) 588–593.
[49] O. Guselnikova, P. Postnikov, M. Erzina, Y. Kalachyova, V. Švorčík, O. Lyutakov,
Pretreatment-free selective and reproducible SERS-based detection of heavy metal
ions on DTPA functionalized plasmonic platform, Sensors and Actuators B:
Chemical 253 (2017) 830–838.