Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 229 (2020) 117994

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Spectrochimica Acta Part A: Molecular and Biomolecular

Spectroscopy
journal homepage: www.elsevier.com/locate/saa

SERS detection of sodium thiocyanate and benzoic acid preservatives in

liquid milk using cysteamine functionalized core-shelled nanoparticles
Abid Hussain a,b,c, Hongbin Pu a,b,c, Da-Wen Sun a,b,c,d,⁎
a
School of Food Science and Engineering, South China University of Technology, Guangzhou 510641, China
b
Academy of Contemporary Food Engineering, South China University of Technology, Guangzhou Higher Education Mega Center, Guangzhou 510006, China
c
Engineering and Technological Research Centre of Guangdong Province on Intelligent Sensing and Process Control of Cold Chain Foods, & Guangdong Province Engineering Laboratory for Intel-
ligent Cold Chain Logistics Equipment for Agricultural Products, Guangzhou Higher Education Mega Centre, Guangzhou 510006, China
d
Food Refrigeration and Computerized Food Technology (FRCFT), Agriculture and Food Science Centre, University College Dublin, National University of Ireland, Belfield, Dublin 4, Ireland

a r t i c l e i n f o a b s t r a c t

Article history: A cysteamine functionalized core shelled nanoparticles (Au@Ag-CysNPs) was presented for simultaneous and
Received 15 September 2019 rapid detection of sodium thiocyanate (STC) and benzoic acid (BA) preservatives in liquid milk using surface-
Received in revised form 25 December 2019 enhanced Raman spectroscopy (SERS) technique. A spectrum covering 350–2350 cm−1 region was selected to
Accepted 25 December 2019
detect STC with concentrations ranging from 0.5 to 10 mg/L and BA with concentrations ranging from 15 to
Available online 29 December 2019
240 mg/L in milk samples. Characterization of nanoparticles using high-resolution TEM confirmed that the suc-
Keywords:
cessful synthesis of Au@AgNPs with core (gold) size of 28 nm and shell (silver) thickness of about 5 nm was
SERS grafted with 120 μL of 0.1 nM cysteamine hydrochloride. Results showed that Au@Ag-CysNPs could be used to
Sodium thiocyanate detect STC up to 0.03 mg/L with a limit of quantification (LOQ) of 0.039 mg/L and a coefficient of determination
Benzoic acid (R2) of 0.9833 in the milk sample. For detecting BA, it could be screened up to 9.8 mg/L with LOQ of 10.2 mg/L and
Milk adulteration R2 of 0.9903. The proposed substrate was also highly sensitive and the employed method involved only minor
Nanoparticles sample pretreatment steps. It is thus hoped that the new substrate could be used in the screening of prohibited
chemicals in complex food matrices in future studies.
© 2018 Elsevier B.V. All rights reserved.

1. Introduction bactericidal effect and is employed as a preservative in the dairy indus-

try [14], however adding STC in milk can hinder the iodine uptake, af-
Preservatives are chemicals, which are widely employed for the pre- fecting the production of thyroid hormone in human [11]. Therefore,
vention of agricultural commodities against bacteria, yeasts, and molds the admixing of BA and STC is prohibited in raw milk [10,15]. However,
spoilage. However, some preservatives are strictly prohibited by food their illegal adulteration is reported. Yong et al. [16] conducted an inves-
regulatory agencies in certain food products. Milk is highly nutritional tigation on thiocyanate ions (STC and percarbonate) in raw cow milk
and is consumed daily by many people, however, milk is also reported from China, New Zealand, and the Netherlands, and found that 65% of
to be adulterated with strictly prohibited chemical preservatives such the tested samples were contaminated with thiocyanate ions. Hong
as boric acid, hydrogen peroxide, formalin, salicylic acid, cyanuric acid et al. [11] evaluated the addition of BA in China and confirmed 76.8%
and others [1,2,3]. The objective of the admixing of such chemicals is tested samples containing the preservative, ranging from 0.51 to
to reduce the pasteurization cost, mask the poor quality of milk, adjust 111 mg/kg.
the pH of spoiled milk, extend the shelf life, etc. [4–9]. However, such Conventional approaches available for the assessment of BA and STC
chemicals can also cause health problems to the public. in different food matrices include spectrophotometry [13,17], high-
Benzoic (BA) is a widely used preservative in the food industry, performance liquid chromatography [18,19], gas chromatography
however, its addition in milk can lead to diarrhea, asthma, convulsion, [20,21] and ion chromatography [22]. However, these methods possess
and abdominal pain [10–13]. Likewise, sodium thiocyanate (STC) is a limitations such as the involvement of chemicals in experiments, the re-
chemical commonly used in different research areas due to its quirement of professionals to handle the expensive equipment, destruc-
tiveness in testing in nature, and lengthy data analyses [23–29].
Therefore, a versatile and sensitive tool is in need of non-invasively
⁎ Corresponding author at: School of Food Science and Engineering, South China
University of Technology, Guangzhou 510641, PR China.
monitoring these health-hazardous materials in foodstuffs.
E-mail addresses: [email protected], URL: http://www.ucd.ie/refrig, Surface-enhanced Raman spectroscopy (SERS) is among the most
URL: http://www.ucd.ie/sun (D.-W. Sun). reliable ultra-sensitive and specific spectroscopic approaches suitable

https://doi.org/10.1016/j.saa.2019.117994
1386-1425/© 2018 Elsevier B.V. All rights reserved.
2 A. Hussain et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 229 (2020) 117994

for both food and non-food applications [30–33]. The high sensitivity of Brussels, Belgium). All glassware and magnetic stirring bars were
this method is due to the simultaneous chemical and physical phenom- treated with aqua regia (HCl: HNO3 = 3:1, v/v) for 24 h before
ena arising from nanoparticles employed [34]. Both chemical and phys- conducting experiments.
ical mechanisms can enhance the Raman signals for up to many folds,
enabling the detection of the target substance at up to single molecular 2.2. Sample preparation
levels [30,35,36]. In addition, rapidness, no interference to water, direct
or minor sample preparation procedure, and non-interruption with Stock solutions of STC (100 mg/L) and BA (1000 mg/L) were first
samples are among the advantages of SERS as compared with the con- prepared in distilled water and then milk samples were simultaneously
ventional methods [23,37,38]. spiked with STC at concentrations of 0.5, 1, 2, 5 and 10 mg/L and with BA
Gold (Au) and silver (Ag) are the nanoparticles most widely at concentrations of 15, 30, 60, 120 and 240 mg/L. The pH of the solu-
exploited as Raman substrates in different studies [39–41]. Feng et al. tions at each concentration was adjusted to the isoelectric point of pro-
[24] employed single AgNPs for the rapid detection of STC in liquid tein at 4.6 ± 0.1 using 0.1 M HCl, followed by centrifugation at
milk. However, the procedure involved 1 h boiling (at 120 °C) of 10,000 rpm for 5 min using a centrifuge machine (JW-3024HR, Anhui
chemicals for substrate preparation and the sample pretreatment in- Jiaven Equipment Industry Co., Ltd., Hefei, China). In addition to protein
volved the addition of aggregating agents and two times centrifugation removal, the sample pretreatment also helps to remove most of the fats
for 10 min, and then the sample had to stand for 6 min before detection. adhering to the casein, as good affinity exists between casein and fat
Moreover, in using AgNPs, their sizes are difficult to control and the ag- [48]. The centrifuged sample (supernatant) was then mixed with Au@
gregation process (due to the aggregating agent) is also uncontrollable, Ag-CysNPs (1:1 by volume) for 1 min and then suck (about 50 μL) in a
which affects the stability of nanoparticles [42]. Furthermore, Zhang capillary tube and placed on the Raman sample stage for Raman
et al. [14] designed an agarose microchip-based substrate for STC detec- analysis.
tion in milk using the SERS method. However, the microchip was appli-
cable only for the particular single analyte and the design of the chip 2.3. Synthesis of AuNPs
required much experience and expensive equipment. Likewise, Wu
et al. [43] employed DNA-assisted core-satellite nanoparticles to ascer- AuNPs were synthesized according to the procedure proposed by
tain STC and melamine in milk, however, the preparation of the sub- Wang et al. [33]. Briefly, a portion of 60 mL ultrapure water was
strate involved very complex steps and many chemicals. The added to a 150 mL conical flask, then 1000 μL of 5 g/L HAuCl4•4H2O
activation of the thiol group of DNA required 1 h and stabilization of was injected to the conical flask under continuous magnetic stirring
AuNPs for DNA conjugation needed standing of the solution for by a digital shaker (MS 3, IKA Inc., Staufen im Breisgau, Germany). The
30 min, followed by centrifugation at 10,000 rpm for 15 min. Further- solution was heated to 120 °C and kept for 1 min, after which 700 μL
more, the functionalization of AuNPs with DNA required 5 h at 55 °C of trisodium citrate (1%) was quickly poured into the solution under vig-
and then the solution was centrifuged for 15 min. The immobilization orous magnetic stirring at 1000 rpm and kept boiling for 6 min. Finally,
of AuNPs with streptavidin-coated polymers beads also required 12 h, the colorless solution turned into purplish-red color, indicating the suc-
and the removal of unbound AuNPs needed centrifugation for 3 min, cessful synthesis of AuNPs. In this procedure, HAuCl4 acted as the pre-
whereas grafting of Ag shell on AuNPs mixture required 6 h shaking, cursor, while trisodium citrate worked as both the reducing and
followed by centrifugation at 12,000 rpm for 2 min. Despite the above stabilizing agents. The size of the AuNPs was controlled by the amount
studies, no study has been reported for BA detection in milk using SERS. of trisodium citrate added to the solution. The solution was gradually
Recently, single nanoparticles are modified or functionalized to am- cooled and stored at 4 °C for further uses.
plify Raman signals for up to many times in various food safety experi-
ments [37,44–46]. On the other hand, bimetallic core shelled 2.4. Preparation of Au@AgNPs and functionalization with cysteamine
nanoparticles (Au@AgNPs) are among the most recently proposed sen-
sitive Raman active substrates, exhibiting simultaneous characteristics Core shelled nanoparticles were prepared by employing a seed-
of AuNPs and AgNPs [33,47]. Therefore, in the current study, Au@ mediated growth method. Briefly, a portion of 3 mL AuNPs colloid
AgNPs were functionalized with cysteamine hydrochloride for the first (28 nm) was added to a 10 mL Eppendorf tube and then sonicated for
time to achieve simultaneous detection of BA and STC in liquid milk 10 min using an ultrasonic cleaning machine (SB25-12D, Ningbo Xinzhi
using SERS. The thiol group-containing cysteamine generates more Ultrasound Equipment Co., Ltd., Ningbo, China). Then, 150 μL of 10 mM
hot spot regions for adsorption of target analytes, thus making Au@ ascorbic acid was added into the solution and agitated for 2 min using
AgNPs more sensitive for detection applications using SERS methods. the digital shaker. A portion of 100 μL of 10 mM AgNO3 solution was
The currently proposed method was simple and ecofriendly for the si- dropwise mixed into the solution at a rate of one drop per 30 s. The mix-
multaneous screening of both contaminates in liquid milk, it is thus ture was continuously shaken using the digital shaker at 1000 rpm for
hoped that the method could also be used for detecting other prohibited 20 min. Finally, the purplish-red color of the mixture changed into
substances in milk and other foods in the future. orange-yellow, depicting the successful deposition of the Ag shell on
the Au surface. The shell thickness depended on the amount of AgNO3
2. Materials and methods added to the solution. For the functionalization of bimetallic core shelled
nanoparticles, a portion of 120 μl of 0.1 nM cysteamine hydrochloride
2.1. Chemicals was added in 3 mL of Au@AgNPs and continuously shaken at 300 rpm
for 30 min using the digital shaker. In order to compare the Raman en-
Chloroauric acid (HAuCl4·4H2O) was obtained from Sinopharm hancement of Au@AgNPs and Au@Ag-CysNPs, freshly prepared nano-
Chemical Reagent Co., Ltd. (Beijing, China). Trisodium citrate, silver ni- particles were mixed with R6G (1:50) and the mixture was kept for
trate (AgNO3), sodium nitrate (NaNO3), ascorbic and Rhodamine 6G 40 min in dark and then Raman analysis was performed.
(R6G) acid were purchased from Aladdin Reagent (Shanghai) Co., Ltd.
(Shanghai, China). Sodium thiocyanate was supplied by Shanghai 2.5. Characterization of AuNPs, Au@AgNPs, and Au@Ag-CysNPs
Macklin Biochemical Co., Ltd. (Shanghai, China) and benzoic acid was
bought from Tianjin-Fuchen Chemical Reagents (Tianjin, China). The characterization of AuNPs, core shelled NPs and Au@AgNPs-Cys
Whole cow milk was purchased from a local market (Guangzhou, was performed by analyzing the UV–Vis absorption spectra ranging
China) and the ultrapure water used in experiments was produced from 300 to 700 nm, which were acquired by a UV-1800 spectrometer
from a Milli-Q® Integral Water Purification System (Millipore S.A., (Shimadzu Co., Ltd., Kyoto, Japan). Images were acquired by a JEM-
 A. Hussain et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 229 (2020) 117994 3

2100F high-resolution transmission electron microscopy (HRTEM) showed an Ag shell, while the inner dark region presented AuNPs core.
(JEM-2100F, JEOL Ltd., Tokyo, Japan), for which, the freshly prepared Fig. 1c shows the TEM images of Au@Ag-CysNPs, indicating that 120 μL
nanoparticles were first diluted in water for two times (1:1), followed of 0.1 nM cysteamine was enough to form 6 nm capping on the Au@
by ultrasonication and then dropped on an ultra-thin carbon film AgNPs without destruction of shell and core of silver-coated gold nano-
(T11032, Beijing Xinxing Braim Technology Co., Ltd., Beijing, China) particles. In addition, DLS tests were also conducted to confirm the aver-
and finally dried at room temperature of 25 °C. Dynamic light scattering age size of AuNPs, Au@AgNPs, and Au@Ag-CysNPs (Fig. 1d, e, and f),
(DLS) was performed with a Zetasizer system (Malvern Zetasizer Nano showing no aggregation in all the synthesized nanoparticles.
ZS, Malvern Instruments, Worcestershire, UK) to determine the diame-
ters of prepared samples. 3.1.1. Sensitivity of Au@Ag-CysNPs
SERS results depend on the affinity of substrate - analyte and the
2.6. Acquisition of SERS spectra sensitivity of proposed nanoparticles. Therefore, in order to estimate
the enhancement factor (EF) of Au@AgNPs and Au@Ag-CysNPs for BA
Raman spectra of all the samples were acquired using a confocal and STC, the following standard equation was employed:
Raman system (Horiba France SAS, Villeneuve d'Ascq, France) equipped
with a 633 nm laser as an excitation source with 50 mW laser power.
The Raman system was also furnished with a high-steady confocal mi- ISERS  C Raman
EF ¼ ð3Þ
croscope (BX41, Olympus Co., PA, USA), a grating of 600 grooves/mm, IRaman  C SERS
confocal coupling optics between the microscope and the spectrograph,
an 800 mm focal length achromatic flat field monochromator, and a
cooled charge-coupled device detector. All spectra were obtained with 10sb
where LOQ ¼ ISERS is the Raman intensity of STC and BA at
× 10 visible objectives from 350 to 2350 cm−1. The laser power was b
set at 100%, the exposure time was set at 20 s and the number of accu- 2112 cm and 996 cm−1 in the presence of Au@AgNPs and Au@Ag-
−1

mulations was set at 2 for Raman analysis. The capillary tube with an CysNPs, IRaman is the intensity of both preservatives in the absence of
inner diameter of 1 mm was used to suck the sample. All the acquired nanoparticles. Likewise, CSERS is the concentration of STC (1 mg/L) and
Raman spectra were analyzed using the LabSpec 6 spectroscopy soft- BA (15 mg/L) for SERS, and CRaman is the concentration of STC
ware suite (Horiba France SAS, Villeneuve d'Ascq, France). Three spec- (200 mg/L) and BA (200 mg/L) for normal Raman measurements, re-
tral preprocessing steps including despiking, denoising, and baseline spectively. EF calculations for STC showed improvement in the en-
correction were performed to improve the signal to noise ratio and to hancement from 3.3 × 103 with Au@AgNPs to 1.4 × 104 with Au@Ag-
minimize the interference of fluorescence. The details of the preprocess- CysNPs. Likewise, for BA, the EF was estimated to be 1.1 × 103 and
ing procedure can be found elsewhere [47,49–51]. 5.5 × 103 for Au@AgNPs and Au@Ag-CysNPs, respectively. The better
enhancement factor with Au@Ag-CysNPs for both preservatives might
2.7. Data analysis be due to the stronger adsorption of the analyte with the substrate
and the generation of more hot spot regions.
The limit of detection (LOD) for STC and BA was evaluated on blank
samples (n = 5) and calculated by using the following equation [52]: 3.2. STC and BA in standard solution

3Sb Fig. 2a depicts Raman spectra of solid STC, BA and simultaneous

LOD ¼ ð1Þ
m spectra of both STC and BA in standard solutions at various concentra-
tions. The levels of STC and BA spiked in standard solutions were 0.5,
where Sb is the standard deviation of the SERS intensity of the blank at
1, 2, 5 and 10 mg/L, and 15, 30, 60, 120 and 240 mg/L, respectively.
Raman shifts of 2112 (for STC) and 996 (for BA) while m represents
The characteristic peak at 2070 cm−1 in solid STC is due to S-C≡N anti-
the slope of plotted calibration curves. The limit of quantitation (LOQ),
symmetric stretching, shifted to 2112 cm−1 in standard solution,
was estimated with the following equation:
while a weak peak at 755 cm−1 assigned to C\\S symmetric stretching
10sb [53], which also shifted to 772 cm−1. The peaks shifting were confirmed
LOQ ¼ ð2Þ by analyzing Raman spectra of individual spectra (Fig. 2b) of each pre-
b
servative in standard solution and such a shifting trend of STC was
In order to assure the feasibility of the current SERS method, the re- also reported by Molinou and Tsierkerzos [53] and Zhang et al. [14].
covery (%) was also estimated. The calibration curves of STC and BA ob- Furthermore, a characteristic peak at 999 cm−1 and a band at
tained from milk were used to calculate the concentrations of the 1025 cm−1 were due to ring deformations in solid BA. The band at
samples and to determine the recovery percentage. 1025 cm−1 in solid BA shifted to 1027 cm−1 in the standard solution.
During the individual spectral analysis of analytes (Fig. 2b), three new
3. Results and discussion peaks positioned at 1310 cm−1, 1361 cm−1 and 1570 cm−1 were also
determined, which were due to C\\H in-plane bending, COO– in-
3.1. Characterization of AuNPs, Au@AgNPs, and Au@Ag-CysNPs plane scissoring and C\\C stretching of the benzene ring in BA, respec-
tively [54,55].
Fig. 1a depicts the UV–Vis spectra of AuNPs, Au@AgNPs, and Au@Ag- Moreover, Fig. 2c and d present the calibration curves of STC and BA
CysNPs. A band centered at 524 nm was observed for AuNPs, while two in standard solution at five different concentrations. The error bars are
bands centered at 401 and 481 nm were recorded for Au@AgNPs, as- the standard deviation of the average Raman intensity obtained with
cribed to overlapping and coupling plasmon resonance frequencies of five sets of SERS estimations, indicating high R2 values of 0.9951 for
AuNPs as seeds and AgNPs as shell, respectively. For Au@Ag-CysNPs STC (calculated from the 2112 cm−1 peak) and 0.991 for BA (calculated
the bands at 401 and 481 (for Au@AgNPs) shifted to 406 and 488 nm, from the 999 cm−1 peak). Limit of detection (LOD) and limit of quanti-
showing the increment in Au@AgNPs size, which depicted the success- fication (LOQ) for STC were calculated as 0.002 mg/L and 0.003 mg/L, re-
ful grafting of cysteamine on Au@AgNPs. AuNPs were purplish, while spectively, while for BA the LOD and LOQ were calculated as 3.38 mg/L
Au@AgNPs and Au@Ag-CysNPs were yellowish in appearance. and as 4 mg/L, respectively. The percent recoveries were calculated to
Fig. 1b shows the high-resolution TEM images of Au@AgNPs, having range from 99 to 109% for STC and from 90.8 to 113.1% for BA in stan-
a core size of 28 nm and a shell thickness of 5 nm. The outer bright layer dard solutions (Table 1).
4 A. Hussain et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 229 (2020) 117994

Fig. 1. (a) UV–Vis absorption spectra of AuNPs, Au@AgNPs and Au@Ag-CysNPs, high-resolution TEM images of (b) Au@AgNPs and (c) Au@Ag-CysNPs, and DLS results of (d) AuNPs,
(e) Au@AgNPs and (f) Au@Ag-CysNPs.

Fig. 2. (a) Raman spectra of solid STC and BA, and of STC and BA in standard solutions at different concentrations, (b) Raman peaks of the spectra and calibration curves for (c) STC and
(d) BA in standard solutions, (e) Raman peaks of STC and BA in liquid milk, and calibration curves of (f) STC and (g) BA in liquid milk.
 A. Hussain et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 229 (2020) 117994 5

Table 1
LOD, LOQ, R2 values, recoveries (%) and RSD (%) for STC and BA in standard solutions and in liquid milk samples.

Chemicals R2 LOD (mg/L) LOQ (mg/L) Spiked value (mg/L) Recovered value (mg/L) Recovery (%) RSD (%)

Standard solution STC 0.9951 0.002 0.003 1 1.09 109 7.08

5 5.05 101.02 10.8
10 9.9 99 8.7
BA 0.991 3.38 4 30 34.2 113.1 11.06
120 108.9 90.8 6.66
240 243.8 101.6 5.9
Liquid milk STC 0.9833 0.03 0.039 1 0.93 93 7.7
5 5.48 109.6 8.09
10 9.713 97.13 6.1
BA 0.9903 9.8 10.2 30 33.6 112 10.2
120 126.36 105.3 9.3
240 235.2 98 7.4

Note: STC = sodium thiocynate, BA = benzoic acid, R2 = coefficient of determination, LOD = limit of detection, LOQ = limit of quantification, RSD = relative standard deviation.

3.3. STC and BA in liquid milk 4. Conclusions

Fig. 2e compares the Raman spectra for STC at concentrations of 0.5, In the current work, SERS based on cysteamine functionalized core
1, 2, 5 and 10 mg/L and BA at concentrations of 15, 30, 60, 120 and shelled nanoparticles (Au@Ag-CysNPs) was employed for simultaneous
240 mg/L in milk samples with the Raman spectra of solid STC and BA. screening of STC and BA liquid milk. STC and BA at five concentrations of
A strong peak at 2112 cm−1 assigned to S-C≡N antisymmetric stretching 0.5, 1, 2, 5 and 10 mg/L and of 15, 30, 60, 120 and 240 mg/L, respectively,
was selected as the main peak for STC, in addition, a weak peak at were determined. The synthesized Au@AgNPs had a core of 28 nm in di-
772 cm−1 ascribed to C\\S symmetric stretching, which was associated ameter and shell thickness of 5 nm was successfully grafted with 120 μL
with STC in liquid milk [24,42,53,54]. cysteamine hydrochloride (0.1 nM), as confirmed by high-resolution
Furthermore, a peak at 996 cm−1 was selected as the characteristic TEM images. pH adjustment followed by centrifugation was the only
peak for BA in the liquid sample. Both peaks at 1030 cm−1 and sample pretreatment step in the study. Results indicated that the pro-
996 cm−1 were assigned to ring deformations of BA. Moreover, the posed procedure could achieve the detection liquid milk samples of
peaks at 1310 cm−1, 1362 cm−1, and 1570 cm−1 were also detected STC with LOD of 0.03 mg/L, LOQ of 0.039 mg/L and R2 of 0.9833, and
in liquid milk, which was due to C\\H in-plane bending, COO– in- BA with LOD of 9.8 mg/L, LOQ of 10.2 mg/L and R2 of 0.9903 mg/L. The
plane scissoring and C\\C stretching of the benzene ring, respectively current results revealed that the proposed Au@Ag-CysNPs was a sensi-
(Fig. 2b). tive SERS substrate, possessing the potential to screen other prohibited
Minor peak shifts were also observed for both STC and BA in stan- chemicals in complex food matrices such as milk and milk products.
dard solutions and liquid milk samples. For example, the main peak at
999 cm−1 for BA in standard solution shifted to 996 cm−1 in liquid Author contribution
milk, likewise peak positions at 1027 cm−1, 1361 cm−1 and
1570 cm−1 shifted to 1030 cm−1, 1362 cm−1 and 1572 cm−1, respec- In current manuscript, all the authors contributed equally to complete the
tively. Shifting of peaks from one position to another might be due to research work. The contribution details are illustrated in the table below.
different attachment sites of analytes with hot spot regions of nanopar-
ticles [40,54,55]. Abid Designed the research plan, performed all experiments and wrote the
Fig. 2g and h depict the calibration curves of STC and BA in liquid Hussain manuscript with input from all authors
milk samples at five different concentrations. The error bars are the Hongbin Verified the analytical methods and results, and revised the
Pu manuscript
standard deviation of the average Raman intensity obtained with five
Da-Wen Supervised the research work, revised and finalized the manuscript
sets of SERS estimations, showing R2 of 0.9833 for STC, which was calcu- Sun
lated from 2112 cm−1 peak position, and R2 of 0.9903 for BA, which was
determined from 996 cm−1 Raman band.
LOD and LOQ for STC were calculated as 0.03 mg/L and 0.039 mg/L Declaration of competing interest
respectively, with percent recoveries ranging from 93 to 109.6% in
milk samples, while for BA the LOD and LOQ were 9.8 mg/L and The authors declare that they have no known competing financial
10.2 mg/L, respectively, and percent recoveries ranged from 98 to interests or personal relationships that could have appeared to influ-
105% as shown in Table 1. ence the work reported in this paper.
However, in addition to substrates sensitivity, SERS results also de-
pend on the structure of analytes under study [56]. For example, in Acknowledgment
our study, Raman sensitivity for STC (NaSCN) was higher in both stan-
dard and milk samples as compared with BA (C7H6O2), which might The authors are grateful to the National Key R&D Program of China
be due to the presence of more functional groups, which significantly (2018YFC1603400) for its support. This research was also supported
enhanced the affinity of STC with nanoparticles. by the Contemporary International Collaborative Research Centre of
Furthermore, sharp Raman peaks and better LOD and LOQ were Guangdong Province on Food Innovative Processing and Intelligent
observed in standard solutions as compared with milk samples, Control (2019A050519001), the Common Technical Innovation Team
which might be due to the presence of Raman active constituents of Guangdong Province on Preservation and Logistics of Agricultural
in milk, as such substances competed for adsorption on active re- Products (2019KJ145, 2019KJ101), the Innovation Centre of Guangdong
gions of nanoparticles and hindered the attachment of target mate- Province for Modern Agricultural Science and Technology on Intelligent
rials on active sites of the proposed substrates. The introduction of Sensing and Precision Control of Agricultural Product Qualities and the
nanoparticles with high stability and more hot spots can further en- Fundamental Research Funds for the Central Universities
hance the detection ability of the SERS technique in complex food (2018MS056, 2017MS075). In addition, Abid Hussain is in receipt of a
matrices [33,45,47,57]. PhD scholarship from the China Scholarship Council.
6 A. Hussain et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 229 (2020) 117994

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