Food Chemistry 315 (2020) 126276

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Cellulose nanofibers coated with silver nanoparticles as a flexible T

nanocomposite for measurement of flusilazole residues in Oolong tea by
surface-enhanced Raman spectroscopy
⁎ ⁎
Xi Chena,b, Hetong Lina,b, , Taotao Xuc, Keqiang Laic, Xue Hand, Mengshi Line,
a
College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China
b
Key Laboratory of Postharvest Biology of Subtropical Special Agricultural Products (Fujian Agriculture and Forestry University), Fujian Province University, Fuzhou,
Fujian 350002, China
c
College of Food Science and Technology, Shanghai Ocean University, No. 999 Hucheng Huan Road, Lingang New City, Shanghai 201306, China
d
School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin, Heilongjiang 150090, China
e
Food Science Program, Division of Food System & Bioengineering, University of Missouri, Columbia, MO 65211-5160, United States

A R T I C LE I N FO A B S T R A C T

Keywords: Cellulose nanofibers (CNF) coated with inorganic nanoparticles are novel hybrid nanocomposites that have great
Hybrid nanocomposites potential in various areas including agriculture and food science. The objectives of this study were to synthesize
Cellulose nanofiber nanocomposites consisted of CNF coated with silver nanoparticles (AgNPs), which can be used as a surface-
Silver nanoparticles enhanced Raman spectroscopy (SERS) platform for measuring pesticides in Oolong tea. CNF were coated with
Surface-enhanced Raman spectroscopy (SERS)
AgNPs to form uniform CNF-AgNP nanocomposites that were investigated by transmission electron microscopy.
Flusilazole
Three-dimensional and porous CNF structures were loaded with AgNPs with an average size of 41 nm. CNF-
Oolong tea
AgNP substrates were applied in characterization and measurement of flusilazole in Oolong tea samples by SERS.
A detection limit of 0.5 mg/kg for flusilazole was obtained based on partial least squares (PLS) regression
analysis. These results indicate that CNF-AgNP nanocomposites combined with SERS is an accurate, sensitive,
and efficient technique for identification and quantification of pesticide residues in Oolong tea.

1. Introduction et al., 2014). Oolong tea, a semi-oxidized tea, is widely consumed in

Nanocellulose is a nascent and promising material that has great
potential in various areas. It possesses large surface area, a high length-
to-width ratio, excellent optical properties, and high rigidity
(Kargarzadeh et al., 2017). Cellulose nanofibers (CNF) are nanoscale
cellulose structures composed of crystalline regions that are inter-
connected by non-crystalline regions. CNF can form structures with a
micron-level length and a width ranging from several nanometers to
several hundred nanometers, which is connected through the hydrogen
bond (Li, Mascheroni, & Piergiovanni, 2015). In recent years, CNF have
attracted much attention in material science, agriculture, food packa-
ging, and biomedical engineering. However, the application of CNF in
food analytical chemistry is still limited.
Flusilazole is an organosilicon fungicide that can control fungal
infections in fruit and vegetable crops (Eckert, Rossall, Selley, & Fitt,
2010). Flusilazole has also been used in the planting and cultivation
process of tea to prevent plant diseases (Zhang et al., 2010). Tea is the
most widely consumed beverage in the world (Ding et al., 2019; Zhao
East Asia because of its unique sensory qualities and health benefits
(Chen et al., 2020; da Silva Pinto, 2013; Ding et al., 2019; Liu, Bruins,
Ni, & Vincken, 2018; Zhao et al., 2014). However, pesticide residues in
tea have raised serious concerns due to growing use of pesticides in tea
cultivation and potential risks to consumers’ health (Abd El-Aty et al.,
2014; Chen et al., 2019, 2020; Ding et al., 2019; Sood, Jaggi, Kumar,
Ravindranath, & Shanker, 2004). In China, the maximum residue limit
(MRL) of flusilazole in cereals, fruits, and vegetables was set to be
0.2 mg/kg (GB, 2016). Currently, there are many testing methods to
detect flusilazole, including high-performance liquid chromatography
(HPLC) and liquid chromatography-mass spectrometry (LC-MS). How-
ever, the aforementioned methods have many drawbacks, such as being
laborious and time-consuming, and requiring complex sample pre-
treatment. Therefore, it is of critical importance to develop accurate,
and sensitive methods with easy sample pretreatment procedures for
detecting flusilazole in tea products.
Raman spectroscopy acquires the information on molecular vibra-
tions of detected analyte molecules based on inelastic scattering of the

⁎
Corresponding authors at: College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China (H. Lin).
E-mail addresses: hetonglin@163.com (H. Lin), linme@missouri.edu (M. Lin).

https://doi.org/10.1016/j.foodchem.2020.126276
Received 24 September 2019; Received in revised form 18 January 2020; Accepted 20 January 2020
Available online 23 January 2020
0308-8146/ © 2020 Elsevier Ltd. All rights reserved.
X. Chen, et al.
incident light. However, Raman scattering is an inefficient process and
signals of conventional Raman spectroscopy are very weak. This pro-
blem can be solved by surface-enhanced Raman spectroscopy (SERS).
SERS utilizes the excitation of the localized surface plasmon resonance
(LSPR) on a nanoparticle or nanostructured surface. When electro-
magnetic waves interact with a rough metal surface, the waves can
excite localized surface plasmon, leading to amplification of the elec-
tromagnetic fields near the surface (Stiles, Dieringer, Shah, & Duyne,
2008). Currently, two mechanisms about the SERS enhancement are
commonly accepted: electromagnetic enhancement and chemical en-
hancement. Both mechanisms have different mode of action but can
occur simultaneously. Particularly, electromagnetic enhancement plays
a major role in SERS in which LSPR occurs at “hot-spots” on a SERS-
active substrate (Sharma, Frontiera, Henry, Ringe, & Van Duyne, 2012).
Nanoscale surface features of a substrate affect the magnitude of the
SERS enhancement. SERS has been applied in various areas including
chemistry, medicine, environmental science, agriculture, and food
analysis (Alsammarraie et al., 2018; Chen et al., 2019, 2020; Ding et al.,
2019; Xiong, Lin, Lin, & Huang, 2018; Yaseen, Pu, & Sun, 2018).
The close interaction of nanomaterials with analyte molecules is a
key factor in SERS measurement, which requires a good platform for
loading nanomaterials. However, the loading nanoparticles on CNF can
be affected by various factors, which can lead to inhomogeneous dis-
tribution of nanoparticles on CNF and affect the sensitivity and stability
of SERS enhancement. To solve this problem, this study aimed to de-
velop a facile method to synthesize a nanocomposite with homo-
geneous distribution of silver nanoparticles (AgNPs) on CNF. Three
different sizes of AgNPs were synthesized and an optimal size of AgNPs
was selected to ensure uniform distribution of AgNPs on the surface of
the CNF. This novel CNF-AgNP nanocomposite possesses highly porous
three-dimensional (3D) structures that can serve as a platform in SERS
analysis. In addition, SERS was combined with CNF-AgNP substrates to
measure flusilazole in Oolong tea and the SERS spectral signals were
analyzed by multivariate statistical analysis.
2. Materials and methods
2.1. Materials and chemicals
Methanol, silver nitrate, acetonitrile, toluene, NaCl, Na2SO4 (an-
hydrous) were obtained from Macklin Biochemical Technology Inc.
(Shanghai, China). Flusilazole (99%) was purchased from Shanghai
Aladdin Biochemical Technology Inc. (Shanghai, China). Trisodium
citrate dihydrate (Na3C6H5O7·2H2O) was obtained from Innochem
Technology Inc. (Beijing, China) and used without any purification.
Silicon slides were obtained from Fisher Scientific Co Ltd. (Pittsburgh,
PA, USA) and CNF were purchased from the University of Maine
(Orono, ME, USA). Graphite carbon black column (GCBC, 250 mg,
3 mL, CNWBOND Carbon-GCB SPE Cartridge) as well as polyvinylidene
fluoride filter (PVDF, SYAA-114, 0.22 μm, 13 mm) were purchased
from ANPEL Scientific Instrument Inc. (Shanghai, China). Organic
Oolong tea samples (Tieguanyin) were provided by Fujian Guan He Tea
Industry Inc. (Anxi county, Fujian, China).
2.2. Preparation of AgNPs
AgNPs were synthesized according to a previously reported study
(Tashdjian, Sánchez Loredo, & González, 2013). First, different volumes
(0.7, 1.0, and 1.3 mL) of 1% w/w trisodium citrate solutions were added
into boiling silver nitrate solutions (50 mL, 0.018% w/w), followed by
stirring for 30 min at 1200 rpm. The color of solutions during the
preparation process was changed from transparent to light brown, and
finally stayed in gray green. Three different diameters of AgNPs were
then obtained and cooled in an ice bath method. The as-prepared
AgNPs were kept at 4 °C for further uses.
2
Food Chemistry 315 (2020) 126276
2.3. Synthesis of CNF-AgNP nanocomposite
Two grams of NFC were added in 50 mL of Milli-Q water and stirred
for 1 h, then the NFC solution was vibrated for 30 min by sonication
(sonicator model No. KQ-500DB, Shumei Ultrasonic Instrument Inc.
Kunshan, Shanghai, China) to disperse the NFC homogeneously in the
solution. A volume (1 mL) of the prepared CNF was concentrated twice,
and then washed four times to eliminate other chemicals by cen-
trifugation (Eppendorf 5810R, Hamburg Germany) at 3824 G-force for
5 min. AgNP solution (1 mL) was centrifuged four times at 2655 G-force
for 5 min to collect AgNPs. AgNPs and CNF were mixed thoroughly in a
2-mL centrifuge tube with vortexing by a vortex oscillator (UVS-1,
YOUSHENG, China). The as-prepared CNF-AgNP nanocomposites were
placed on a gold slide and dried by a hot plate at 45 °C, which was then
ready for SERS measurement.
2.4. Sample preparation
Two grams of treated Oolong tea samples (N = 35) were mixed with
10 mL of acetonitrile in a 50-mL centrifuge tube, vortexed for 10 s, and
vibrated by the sonicator for 5 min. Next, the mixture solution was
centrifuged at 2600 G-force for 5 min, followed by extracting the su-
pernatants of the centrifuged samples. Then, the precipitates were
mixed with acetonitrile solution (10 mL). The supernatant was ex-
tracted after repeating the aforementioned extraction procedure. The
combined supernatants were dried by the nitrogen blowing method and
5 mL of acetonitrile was used to dissolve the residues. An aliquot of the
mixture (1 mL) was used for purification. Then, the GCBC containing
Na2SO4 (anhydrous) and 5 mL of acetonitrile/toluene solution (3:1, v/
v) was activated, followed by the addition of 1 mL of the mixture. A
volume of 25 mL of acetonitrile/toluene solution (3:1, v/v) was used for
elution. The filtrates of the samples were collected and dried by rotary
evaporation. A volume of 1 mL of acetonitrile/water (1:1, v/v) was used
to dissolve the residues, and the mixture was filtered through a 0.22 μm
PVDF filter before the SERS or GC–MS/MS measurement.
2.5. Characterization of CNF-AgNP nanocomposites
An ultraviolet–visible (UV–vis) absorption spectroscopy
(UV3000PC, Mapada Instruments Co. Ltd., Shanghai, China) was used
to measure the optical properties of CNF-AgNP nanocomposite. The
size, shape, and other physical properties of AgNPs were characterized
by a transmission electron microscopy (TEM) (JEM-1200, JEOL Inc.,
Tokyo, Japan). The morphology and size distribution of AgNPs were
measured based on the TEM images of AgNPs.
2.6. Acquisition of SERS spectra
The collection of SERS spectral data was based on using a Raman
DXR system (Thermo Fisher Scientific, Waltham, MA, USA) that con-
sisted of a microscope and a He-Ne 632.8-nm laser source. During
measurement, a sample was placed on a microscope stage and mea-
sured by the light from the laser (~5 mW) through a 20× objective.
The Raman spectra were acquired with three scans for each measure-
ment and 5-second exposure time for each scan. The range of Raman
shift was from 200 to 2000 cm−1 in the extended mode. Raman scat-
tering spectra acquired from a serial concentration of flusilazole
(0–10 mg/L) were collected from ten random spots of the substrate. A
volume (5 µL) of the as-prepared CNF-AgNP nanocomposite was placed
onto a slide and then air-dried, forming a rough film in round shape,
which had a diameter of 4 mm. A volume (5 µL) of the extract solution
was deposited onto the film, then dried by a hot plate prior to the SERS
analysis. The uniformity of CNF-AgNP nanocomposite was measured
from 10 random spots using 10 mg/L of flusilazole in the methanol–-
water solution. Flusilazole was added in the methanol/H2O organic
solvent solution (methanol: H2O = 1: 1, v/v) to prepare a stock
X. Chen, et al.
flusilazole solution (100 mg/L). A series of flusilazole solution samples
(0.1, 0.5, 1, 2, 5, and 10 mg/L) were obtained by serial dilutions from
the 100 mg/L of stock solution. The solvent solution without spiked
pesticide was used as a control.
2.7. Analysis by GC–MS/MS
The tea samples were measured by GC–MS/MS method using gas
chromatograph (Shimadzu, GC-2010 plus, Japan) equipped with an
auto-sampler and a mass spectrometer (Shimadzu, TQ8040, Japan)
equipped with EI ion source. Chromatographic columns
(30.0 m × 0.25 mm × 0.25 µm, SH-Rxi-5Sil MS capillary column) were
obtained from Shimadzu Co., Ltd. (Shimadzu, Japan) and a Rotary
evaporator (N-1000/NVC-2100, Tokyo Rikakikai, Tokyo, Japan) was
used for the study.
2.8. Data analysis
The spectral data were analyzed by the software Delight version
3.2.1 (D-Squared Development Inc., LaGrande, OR, USA). Multivariate
statistical tools were used to analyze the acquired SERS spectral data
(Alsammarraie & Lin, 2017). Briefly, the background interferences from
the Raman spectrometer were reduced by Gaussian smoothing at
6 cm−1. Moreover, the second-derivative analytical tool at 12 cm−1
intervals was applied prior to the PLS regression analysis to reduce the
overlapping problem of spectra. PLS was used to predict pesticide
concentrations in tested samples. Leave-one-out cross-validation
(LOOCV) was utilized to build the PLS models based on the information
from the entire spectral samples in a calibration model. In LOOCV, all
but one sample was used to build a calibration model. This calibration
model was then utilized to predict the remaining sample. Next, a second
sample was set aside from the group, all other samples were used to
build a new calibration model that was used to predict the second
sample. The LOOCV procedure was repeated until every sample was left
out and predicted by a calibration model. To avoid over-fitting of
spectral data, the lowest root-mean-square error of prediction (RMSEP)
values were calculated (Eq. (1)) to optimize the number of PLS latent
variables.
n
∑ ( ci ̂ − ci ) 2
RMSEP = i The as-prepared CNF-AgNP substrates were utilized to detect flusi-
n (1)
In this formula, n is the number of samples, ĉi is the predicted
analyte concentration (mg/L), and ci is the actual analyte concentration
(mg/L). To validate the PLS models, RMSEP and the correlation coef-
ficient (r) were calculated and evaluated. Principally, a higher r value
or a lower RMSEP value indicates a better prediction model. In addi-
tion, calibration curves were established and the Eq. (2) was used to
calculate the detection limit (DL) with 99.86% of confidence interval
(Strickland & Batt, 2009):
DL = 3σ / m (2)
In which σ is the standard error of predicted concentration and m is
the slope of the calibration curve. In a PLS model, σ equals to RMSEP.
Furthermore, the quality of the PLS model was evaluated based on the
ratio of performance to deviation (RPD) of the PLS models.
3. Results and discussion
3.1. Synthesis and analysis of CNF-AgNP nanocomposites
Fig. 1a–c shows the TEM micrographs of AgNPs, demonstrating that
the size of AgNPs were 85 ± 30 nm, 41 ± 18 nm, and 37 ± 10 nm
(n = 50), which were corresponding to the added volume of 0.7, 1.0,
and 1.3 mL of trisodium citrate solution, respectively. Fig. 1d shows the
UV–vis absorption spectra of AgNPs, revealing surface plasmon
3
Food Chemistry 315 (2020) 126276
resonance (SPR) peaks at 430, 420, and 410 nm, which corresponded to
the added volume of 0.7, 1.0, and 1.3 mL of trisodium citrate, respec-
tively. Fig. 1e shows an optical image of CNF-AgNP nanocomposites
and the black part of the optical image indicates the AgNPs on the
substrate. Fig. 1f shows the corresponding SERS intensity map of 1 mg/
L of flusilazole. In general, the darker the color in Fig. 1e, the high
(redder) the SERS intensity in the map (Fig. 1f). These figures indicate
that AgNPs are uniform and highly monodisperse, making the CNF-
AgNP nanocomposite a potential SERS substrate.
TEM image in Fig. 2a shows that most synthesized AgNPs were in
spherical shape with an average size of 41 nm. The width of the CNF
was from a few nanometers to several hundred nanometers (Fig. 2b).
TEM images (Fig. 2c and d) show that CNF were coated with AgNPs.
Fig. 3a shows the particle size distribution and most AgNPs were in the
size range of 30–40 nm, which was based on the measurement of 150
AgNPs. The effects of AgNP size of CNF-AgNP nanocomposite on the
detection of flusilazole is shown in Fig. 3b, indicating that the particle
size is crucial to the SERS measurement. AgNPs were able to achieve
satisfactory results in SERS measurement due to their excellent optical
and electrical properties, which could be used for chemical and biolo-
gical sensing applications (Stamplecoskie, Scaiano, Tiwari, & Anis,
2011; Tashdjian et al., 2013). The LSPR can occur in the vicinity of
nanoparticles, creating strong electromagnetic field enhancement (Luo,
Huang, Lai, Rasco, & Fan, 2016).
3.2. SERS activities of CNF-AgNP nanocomposite
After the synthesis of CNF-AgNP nanocomposite, AgNPs were uni-
formly distributed on the CNF via mechanical mixing (Fig. 2c and d) in
which AgNPs were tightly coated on the CNF. Most AgNPs were ad-
sorbed on the surface of CNF. Numerous SERS hot-spots were generated
at the gaps between adjacent nanoparticles and their attached surface.
CNF are composed of nanoscale cellulose fibers loaded with AgNPs,
providing a platform for the LSPR that occurs when the collective os-
cillation of valence electrons in AgNPs in resonance with the frequency
of incident light, which contributed to enhanced Raman scattering
signals from analyte molecules (Stiles et al., 2008).
3.3. Detection of flusilazole in the methanol–water solution
lazole in the methanol–water solution. The average (N = 10) SERS
spectra of various concentrations of flusilazole solutions are shown in
Fig. 4a. “Fingerprint-like” Raman peaks are located at 632, 807, 828,
1102, 1167, 1356, and 1588 cm−1. The Raman signal intensity of those
peaks grew as the flusilazole concentration grew from 0 to 10 mg/L. A
characteristic peak at 632 cm−1 arises from the out-of-plane bending
vibration of CeH, a Raman peak at 807 cm−1 is attributed to stretching
vibration of C]C and two Raman peaks at 828 and 1356 cm−1 arise
from the stretching vibration of CeN. A peak at 1102 cm−1 is due to
stretching vibration of C-F, and a Raman peak at 1167 cm−1 is attrib-
uted to the scissor bending vibration of CeH. A peak at 1588 cm−1
arises from the in-plane bending vibration of C]C group (Pan et al.,
2012; Sun et al., 2009; Tang et al., 2011). The band assignments of
flusilazole are summarized in Supplementary Table S1.
Fig. 4b displays the 2nd derivative transformation of SERS spectra
of flusilazole in the methanol–water solution within the Raman shift of
1070–1140 cm−1. Poor reproducibility of the measurement has been a
major issue for SERS methods using various substrates. Adopting var-
ious types of substrates to get a good reproducibility is the major point
of SERS measurement. The CNF/AgNP nanocomposite can overcome
this issue. Fig. 3c shows the uniformity of the CNF-AgNP substrate
which was measured from 10 random spots using 10 mg/L of flusilazole
in the methanol–water solution, the relative standard deviation (RSD)
of the peak intensity at 828 cm−1 was calculated to be 3.5% from spot
to spot on substrates and Fig. 3d confirms that CNF-AgNP substrates are
X. Chen, et al. Food Chemistry 315 (2020) 126276

(a) (b) (c)

(d) (e) (f)

Fig. 1. TEM micrographs of silver nanoparticles prepared with 50 mL of silver nitrate and different volumes of trisodium citrate added: 0.7 mL (a); 1.0 mL (b); 1.3 mL
(c); and their UV–vis absorption spectra (d) showing surface plasmon resonance peaks at 430, 420, and 410 nm corresponding to AgNPs (a, b, and c); the optical
image of NFC-AgNP nanocomposite (e); the corresponding SERS intensity maps of 1 mg/L of flusilazole measured by NFC-AgNP substrate in methanol–water solution
(f).

(a) (b)

(c) (d)

Fig. 2. TEM images of silver nanoparticle prepared with 1.0 mL trisodium citrate (a); unmodified CNF (b); modified CNF coated with AgNPs (c & d).

reliable substrates for measurement of flusilazole in the methanol–-
water solution.
3.4. Detection and quantification of flusilazole in Oolong tea samples
Measurement by SERS was conducted to directly measure the liquid
samples extracted from the tea samples containing flusilazole. Fig. 4c
displays the averaged SERS spectra of the extract samples from the tea
samples containing flusilazole. Three strong peaks are located at 828,
1102, and 1167 cm−1. The peak intensity at those Raman shift wave-
numbers grew as the concentration of flusilazole in the tea samples
grew from 0 to 10 mg/kg. Fig. 4d displays that the 2nd derivative
transformation of SERS spectra of the samples within the Raman shift of
615–645 cm−1. The results demonstrate that flusilazole in Oolong tea
samples can be accurately detected and quantified using SERS in
combination with CNF-AgNP substrates.

4
X. Chen, et al. Food Chemistry 315 (2020) 126276

(a) (b)

Raman shift (cm -1 )

Particle size (nm)

(c) (d)

Fig. 3. The nanoparticle size distribution with 1.0 mL trisodium citrate added (a); the SERS measurement effect of NFC-AgNP substrate with different particle size on
the detection of 5 mg/L flusilazole in methanol–water solution (b); the SERS spectra of flusilazole (10 mg/L) measured from 10 random sites on NFC-AgNP substrate
(c); the corresponding bar chart for the peak intensity at 828 cm−1 of flusilazole (10 mg/L) from 10 random sites on NFC-AgNP substrate (d).

(a) (b)
828

1102

10 mg/L
Second derivative of intensity

5 mg/L
(counts)
intensity (counts)

2 mg/L
1 mg/L
0.5 mg/L
0.1 mg/L
1167
Raman intensity

807

0 mg/L 0 mg/L
1356

0.1 mg/L
0.5 mg/L
632

1 mg/L
Raman

1588

2 mg/L
5 mg/L
10 mg/L

Raman shift (cm -1 ) Raman shift (cm-1)

(c) (d)
10 mg/kg
5 mg/kg
2 mg/kg
Second derivative of intensity
Raman intensity (counts)

828

1 mg/kg
1102

0.5 mg/kg
0 mg/kg
1167

0 mg/kg
1356

1588

0.5 mg/kg
807

1 mg/kg
2 mg/kg
632

5 mg/kg
10 mg/kg

-1 ) Raman shift (cm -1 )

Raman shift (cm

Fig. 4. Averaged SERS spectra of 0–10 mg/L of flusilazole in methanol–water solution (a); second derivative transformation of Raman spectra within the range of
1070–1140 cm−1 of flusilazole in methanol–water solution (b); averaged SERS spectra of 0–10 mg/kg of flusilazole in tea samples (c); second derivative trans-
formation of Raman spectra within the range of 615–645 cm−1 of flusilazole extracted from tea samples (d).

5
X. Chen, et al. Food Chemistry 315 (2020) 126276

Fig. 5. Partial least squares (PLS) analysis results of 0–10 mg/L flusilazole in the methanol–water solution: prediction results of the PLS model (a); latent variables of
the PLS model (b); partial least squares (PLS) analysis results of 0–10 mg/kg flusilazole extracted from tea samples: prediction results of the PLS model (c); the
loading plot of the PLS model (d).

Table 1 Our pretreatment method can efficiently exclude the interferences of

Partial least squares analysis results of the flusilazole samples measured by food matrices and concentrate analyte molecules maximally. As shown
SERS. in Fig. 4c, the extraction process significantly removed complex food
Flusilazole samples R RMSEP RPD Recovery components and the Raman characteristic peaks of flusilazole are ob-
vious with no discernible interfering peaks. The enhancement factor of
Methanol-water solution 0.957 0.962 2.70 93% the CNF-AgNP substrates was calculated to be 106. GC–MS/MS tech-
Oolong tea 0.970 0.859 2.45 87%
nique was used to verify the accuracy and reliability of the SERS
method. Supplementary Fig. S1 shows the GC–MS/MS analysis of or-
ganic Oolong tea samples that indicated the absence of flusilazole and
3.5. Spectral analysis by PLS
other pesticides. Supplementary Fig. S2 demonstrates a chromatogram
of GC–MS/MS analysis using flusilazole standard solution. The reten-
SERS spectra were acquired from the methanol–water solution or
tion time of flusilazole was 15.66 min. The chromatogram was used for
Oolong tea samples containing flusilazole, which were analyzed by PLS
qualitative analysis of flusilazole in Oolong tea samples. Supplementary
(Fig. 5). Based on the PLS regression model (Fig. 5a), a clear linear
Fig. S3 is the standard curve of flusilazole standard solution, showing a
relationship between the predicted and spiked flusilazole content in the
linear equation (y = 14407.29x + 15399.80, R2 = 0.9925). Accep-
methanol–water solution was established (r = 0.957; slope = 0.921;
table recovery values were acquired for both GC–MS/MS analysis
RMSEP = 0.962 mg/L). Fig. 5b shows that the optimum number of the
(Supplementary Table S2) and SERS method (Table 1). Furthermore, a
latent variables of the PLS model is five, which was indicated by the
comparison was conducted between GC–MS/MS and SERS methods
lowest value of RMSEP. These results demonstrate that SERS method
about the measurement of flusilazole in Oolong tea, which is shown in
coupled with CNF-AgNP substrates is a sensitive and efficient approach
Supplementary Table S3. The results show that the relative errors were
to characterize and quantify flusilazole in a model system in the me-
within a range of ± 10%, indicating that the SERS method can be used
thanol–water solution. Fig. 5c shows the PLS analysis results based on
in detection of flusilazole in Oolong tea. In addition, compared with
the Raman spectral data of the Oolong tea contaminated with flusila-
traditional analytical methods such as LC-MS or HPLC, a major ad-
zole. In general, the optimal number of the latent variables is selected
vantage of SERS is its rapid acquisition of Raman scattering signals,
based upon the lowest RMSEP value. The loading plot of the PLS model
which takes only a few minutes to measure a sample that was prepared
was shown in Fig. 5d. An excellent relationship between the predicted
and deposited on a substrate. These results demonstrate that the CNF-
and spiked flusilazole content in Oolong tea samples was established as
AgNP nanocomposites coupled with the sample extraction process is a
shown in Fig. 5c (r = 0.970; slope = 0.939; RMSEP = 0.859 mg/kg).
practical and efficient method for detecting pesticides in Oolong tea.
Table 1 shows that the RPD value was 2.70 and 2.45 for the flusilazole
in the methanol–water solution and in Oolong tea, respectively, in-
dicting robust PLS models established in the data analysis.
4. Conclusion
Food ingredients can interfere with the acquisition of Raman scat-
tering signals. In this study, tea is a complex matrix because it contains
In conclusion, this study established a facile method to synthesize
various components, such as pigments, chlorophyll, polyphenols, etc.
SERS substrates using NFC coated with AgNPs. NFC have porous 3D
(Cabrera, Giménez, & López, 2003). Thus, adopting appropriate pre-
structures that possess large surface areas, which are suitable for
treatment method is crucial to exclude those interfering components.
loading large amount of AgNPs. Flusilazole in Oolong tea samples was

6
X. Chen, et al.
measured and quantified by SERS measurement that generated strong
electromagnetic enhancement of Raman scattering signals. The PLS
analyses show that the SERS method combined with NFC-AgNP nano-
composite can detect flusilazole with a DL of 0.5 mg/kg in Oolong tea
samples. These results reveal that the NFC-AgNP nanocomposites are
suitable substrates that can be utilized in SERS for analysis of pesticide
in tea products. Future research is needed to develop different sub-
strates, optimize the extraction protocol, and test different food pro-
ducts.
Author contributions section
Hetong Lin and Mengshi Lin designed the research program; Xi
Chen, Taotao Xu, Keqiang Lai, Xue Han performed the experiments; Xi
Chen analyzed the data and wrote the manuscript; Hetong Lin and
Mengshi Lin revised the manuscript; Mengshi Lin edited English lan-
guage of the manuscript. All authors have approved the submission and
publication of the manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
This study was financially supported by the Science and Technology
Innovation Foundation at Fujian Agriculture and Forestry University of
China (Grant Nos. KF2015051, CXZX2016086, and CXZX2016256) and
the National Natural Science Foundation of China (Grant
No. 31728016).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodchem.2020.126276.
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