Subscriber access provided by AUSTRALIAN NATIONAL UNIV
Article
A simple approach for rapid detection of alternariol in pear fruit by SERS with pyridine modified silver nanoparticles Tingtiao Pan, Da-Wen Sun, Hongbin Pu, and qing-yi wei J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05664 • Publication Date (Web): 14 Feb 2018 Downloaded from http://pubs.acs.org on February 19, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 28
Journal of Agricultural and Food Chemistry
1
A simple approach for rapid detection of alternariol in pear fruit by
2
SERS with pyridine modified silver nanoparticles
3
Ting-tiao Pan1,2,3, Da-Wen Sun1,2,3,4∗, Hongbin Pu1,2,3, Qingyi Wei1,2,3
4 5 6
1
School of Food Science and Engineering, South China University of Technology, Guangzhou 510641, China 2
7
Academy of Contemporary Food Engineering, South China University of Technology, Guangzhou Higher Education Mega Center, Guangzhou 510006, China
8 3
9
Engineering and Technological Research Centre of Guangdong Province on Intelligent Sensing and Process Control of Cold Chain Foods, Guangzhou Higher Education Mega Center, Guangzhou 510006, China
10 4
11
Food Refrigeration and Computerized Food Technology (FRCFT), Agriculture and Food Science Centre, University College Dublin, National University of Ireland, Belfield, Dublin 4, Ireland
12 13 14
Abstract: A simple method based on surface-enhanced Raman scattering (SERS) was developed for
15
rapid determination of alternariol (AOH) in pear fruit by using an easily prepared silver nanoparticles
16
(AgNPs) substrate. The AgNPs substrate was modified by pyridine to circumvent the weaker affinity
17
of AOH molecular on sliver surface and improve the sensitivity of detection. Quantitative analysis
18
was performed in AOH solutions at concentrations over a range of 3.16-316.0 µg/L, and the limit of
19
detection was 1.30 µg/L. The novel method was also applied to detect AOH residues in pear fruit
20
purchased from market and those were artificially inoculated with A. alternata. AOH was not found
21
in any of the fresh fruit, while it resided in the rotten and inoculated fruits. Finally, the SERS method
22
was cross validated against HPLC. It was revealed that SERS method have great potential utility in
23
rapid detection of AOH in pear fruit and other agricultural products.
24
Keywords: Mycotoxins, alternariol, pear, SERS, AgNPs ∗
Corresponding author. Tel: +353-1-7167342; Fax: +353-1-7167493.
E-mail address:
[email protected]. Website: www.ucd.ie/refrig; www.ucd.ie/sun.
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
25 26
1. Introduction
27
Alternaria is a cosmopolitan fungal genus that includes saprophytic, endophytic and pathogenic
28
species. Many Alternaria species are plant pathogens that can damage crops in the field. Moreover,
29
they are capable of causing significant postharvest decay of fruits, vegetables, and cereals, resulting
30
in quality degradation of the infected products.1 Even worse, some Alternaria species can generate
31
diverse toxic secondary metabolites, known as Alternaria mycotoxins, contaminating agricultural
32
products during storage.2,3 The occurrence of Alternaria mycotoxins has been reported in fruits,
33
vegetables, cereals, and beverages.4-12
34
More than 30 Alternaria mycotoxins belonging to different structural groups have been isolated
35
from Alternaria species, among which, one of the dibenzopyrone derivatives, alternariol (AOH,
36
3,7,9-trihydroxy-1-methyl-6H-dibenzo(b,d)pyran-6-one) is considered to be the most important
37
mycotoxins produced in alternata infected pear fruit and other products.13-15 From a toxicological
38
point of view, Alternaria mycotoxins are associated with a variety of adverse health effects, and
39
AOH is genotoxic, carcinogenic, mutagenic, and cytotoxic in microbial and mammalian cell
40
systems.16-19 To date, no in vivo toxicology researches in experimental animals for Alternaria
41
mycotoxins have been carried out, however, some symptoms of precancerous changes in esophageal
42
mucosa of mice have been presented.20 Although the acute toxicity of Alternaria toxins is low in
43
animals, limited evidences are available for long term toxicity effects of them and their synergistic
44
effect with other toxins or contaminants.1,20 In addition, many researchers believe that the
45
incremental incidence of human esophageal cancer in China was related to the contamination of
46
Alternaria toxins, and A. alternata might be one of the etiological factors.17,21
47
The traditional methods reported in the literatures for AOH and other Alternaria mycotoxins
48
analysis include thin-layer chromatography,22 gas chromatography (GC),23 liquid chromatography
49
(LC),13,24,25 and high-performance liquid chromatography (HPLC).11,14,26-34 Among them, HPLC is
ACS Paragon Plus Environment
Page 2 of 28
Page 3 of 28
Journal of Agricultural and Food Chemistry
50
the most commonly used method. Even though these chromatographic methods are specific, accurate,
51
they are laborious, time-consuming, and need skilled staff due to a series of complex clean-up and
52
pre-concentration steps are required prior to analysis. Besides, these methods are insufficiently
53
sensitive to detect nanogram amounts of Alternaria mycotoxins.
54
Recently, some emerging analytical techniques, such as stable isotope dilution assays (SIDAs),5-8
55
polymerase chain reaction (PCR),27,33,35 enzyme-linked immunosorbent assay (ELISA),26 and
56
molecularly imprinted polymer,36 offer alternative tools for the detection of Alternaria species in
57
agricultural products, and could be used as an indirect marker of the presence of toxins. These novel
58
techniques usually have the advantage of a high sensitivity, low limit of detection (LOD), and high
59
selectivity. However, they also require complex preparation steps prior to analysis. For example,
60
PCR method based on the internal transcribed spacer (ITS) genetic marker needs a complex DNA
61
extraction and PCR amplification process before analysis.33 In using SIDAs for the determination of
62
alternata mycotoxins, the deuterated mycotoxins are first synthesized by palladium catalyzed
63
protium-deuterium exchange from the unlabeled toxins.6,7 While in ELISA, polyclonal or (and)
64
monoclonal antibodies need to be prepared in advance, which is a complex process that takes a long
65
time.26 In the process of molecularly imprinted polymer analysis, template selection and preparation
66
is a necessary step, but also an extremely complex step.36
67
Surface-enhanced Raman spectroscopy (SERS) is a powerful analytical technique that provides
68
molecular-structural information of the target molecule adsorbed on the nanostructured metals. Due
69
to the surface plasmon resonance in the visible electromagnetic range, metal nanoparticles, such as
70
gold and silver, provide a considerable enhancement of the SERS signal from a molecule located in
71
the close vicinity of nanoparticles surface.37 Recent studies showed that SERS based on novel
72
nanoparticles substrates have increased in popularity as important tools in toxin detection.38-43
73
Therefore, it is our intention to develop a SERS method for the detection of Alternaria mycotoxins,
74
considering the advantages such as rapidness, high sensitivity and low cost that the technique can
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
75
offer.
76
To the best of our knowledge, this is the first report dealing with the determination of AOH by
77
using SERS, and the aim of this study was thus to develop a simple and rapid SERS approach to
78
detect AOH in pear fruit. An important issue in the development of the SERS approach is the low
79
affinity of target molecules for metallic surfaces. Therefore, a new strategy, which could modify the
80
silver nanoparticles (AgNps) by pyridine, was developed to avoid above limitation and improve the
81
detection sensitivity in the current study. Finally, the proposed method was tested on the detection of
82
AOH. In addition, the occurrence of AOH in pear fruit was analyzed by HPLC method to validate
83
the feasibility of the SERS method to identify the samples contaminated with AOH.
84 85
2. Materials and methods
86
2.1. Chemicals and reagents
87
Silver nitrate (99.9%), trisodium citrate (dihydrate, 98%), pyridine, sodium nitrate, sodium
88
chloride, anhydrous sodium sulfate, acetonitrile, methanol, dichloromethane, ethyl acetate,
89
methanoic acid, and acetic acid were purchased from Shanghai Aladdin Bio-Chem Technology Co.
90
Ltd. (Shanghai, China). Both acetonitrile and methanol are of HPLC grade and the other chemicals
91
are of analytical grade. AOH (>98.5%) was purchased from Sigma-Aldrich (Shanghai, China).
92
Ultrapure water was prepared by a Milli-Q system (EMD Millipore Co., Billerica, MA, USA). The
93
pear fruit were collected from local retail shops in Guangzhou, China.
94 95
2.2. Silver nanoparticles synthesis and characterization
96
AgNPs were synthesized with slight modification based on the method available.44 Briefly, 90.0
97
mg of silver nitrate was dissolved in 500.0 mL of milli-Q water in a clean flask, the solution was
98
stirred and heated to boiling point in a constant temperature heating magnetic whisk (DF-101S,
99
Yuhua Instrument Co. Ltd., Yiwu, China). The solution was kept boiling and stirring, 5.0 mL solution
ACS Paragon Plus Environment
Page 4 of 28
Page 5 of 28
Journal of Agricultural and Food Chemistry
100
of trisodium citrate (1%, m/v) was quickly added to the boiling solution. The resulting solution was
101
continued to boil and stir for 1 h, and then cooled to 25oC.
102
A UV-1800 spectrophotometer (Shimadzu Co., Kyoto, Japan) was applied to measure the visible
103
adsorption spectra from 300 to 700 nm with a 1 nm interval. The newly synthesized AgNps were
104
diluted five times and detected. The transmission electron microscope (TEM) images were obtained
105
on a field-emission high-resolution JEM-1400Plus (JEOL Ltd., Tokyo, Japan) at an acceleration
106
voltage of 120 kV. The samples for TEM analysis were prepared by dropping the diluted solution of
107
freshly synthesized AgNPs on carbon film (T11023, Beijing Xinxing Braim Technology Co. Ltd,
108
Beijing, China) and air dried at 60 oC. Dynamic light scattering (DLS) measurements were
109
performed using a two angle particle and molecular size analyzer (Zetasizer Nano ZS, Malvern
110
Instruments Ltd., Worcestershire, UK) at 25 °C under a scattering angle of 173° at λ = 633 nm.
111 112
2.3. Standard preparation for SERS
113
The AOH stock solution of 1.0×106 µg/L was prepared by diluting AOH standard with methanol
114
and kept in darkness at -20 oC. A working solution (1.0×105 µg/L) was prepared by diluting the AOH
115
stock solution with methanol. Standards for SERS detection were obtained by diluting working
116
solutions with milli-Q water. A series of standards with the concentrations of 3.16, 10.0, 31.6, 100.0,
117
and 316.0 µg/L were prepared.
118 119
2.4. Detection of AOH in standard solution by SERS
120
In order to provide a reference for the SERS spectra of AOH, 1.0 mL of 1.0×105 µg/L AOH
121
standard solution was loaded into a cuvette, and then scanned by a Raman microscope (LabRAM HR,
122
HORIBA Scientific, Longjumeau, France) with a 633 nm laser as the excitation source. The SERS
123
measurement was carried out within the wavelength range of 300-1800 cm-1. Two accumulations
124
were used, and the acquisition time was set to 30 s.
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
125
Prior to the SERS measurement, 80.0 µL of aqueous pyridine solution (0.1 M) was added to 400.0
126
µL of AgNps in the centrifuge tube and the mixed solution was stirred for 5 s, and then 400.0 µL of
127
standard solution was dropped into the tube and the solution in the tube was mixed for 5 s. Next step,
128
80.0 µL of sodium nitrate solution (1 M) was also added to the tube and mixed for 5 s to facilitate
129
AgNps aggregation. Finally, the mixed solution was analyzed after these preparations. For SERS
130
detection, the SERS spectrum was collected within the range of 300-1800 cm-1 using the Raman
131
microscope with a 532 nm laser source at 50 mW laser power for excitation. Two accumulations
132
were used, and the acquisition time was set to 30 s. Baseline correction and denoising were fulfilled
133
for all measurements. SERS measurement was repeated 3 times for each concentration. The intensity
134
value of the peak at 1252 cm-1 was plotted against the log concentration of AOH.
135 136
2.5. Spiked samples determination by SERS
137
The applicability and reliability of the proposed SERS method were evaluated by performing a
138
recovery test using blank pear fruit. The fruit purchased were confirmed to be negative for AOH by
139
China Entry-Exit Inspection and Quarantine Bureau (Guangzhou, China). AOH standard solution
140
was injected into the blank pear fruit and the spiked fruit were stored at room temperature for 2 h
141
before initiating the extraction process. The fortification levels in the recovery test were 20.0, 50.0,
142
and 100.0 µg/kg, and five replicates were performed for each level. AOH extraction procedure was
143
adapted based on previously published methods.26,32 Briefly, 5.0 g of sample was transferred into a
144
50-mL centrifuge tube, and then 4.0 g of sodium chloride, 10.0 mL of water and 15.0 mL of
145
acetonitrile (with 1% acetic acid) were added. The tube was first shaken in an oscillator for 10 min,
146
then was centrifuged for 5 min at 5000 rpm. The upper organic solvent was transferred into a flask,
147
while the underlying substance was re-extracted two times with the same solvent composition. All of
148
the upper organic solvent portions were pooled and evaporated (vacuum-rotary evaporation, 40 oC)
149
to nearly dry, then was concentrated to dryness using a nitrogen flow, and the residue was dissolved
ACS Paragon Plus Environment
Page 6 of 28
Page 7 of 28
Journal of Agricultural and Food Chemistry
150
in 1.0 mL of methanol, and this solution was diluted by 9.0 mL of milli-Q water, the diluted solution
151
was detected directly by SERS method as described above, without further purification. The recovery
152
(%) was calculated as measured concentration divided by fortification level.
153 154
2.6. SERS determination of AOH in real samples
155
The validated SERS method was then used for determination the presence of the AOH and
156
quantification its amount in the pear fruit. In total, 10 pear fruit (5 fresh fruit and 5 rotten fruit) were
157
analyzed. All the samples were stored at 4 oC prior to analysis. Moreover, AOH production
158
measurement was performed on the 10 pear fruit artificially inoculated with A. alternata at the
159
concentration of 1.0×106 cfu/mL, which were kept at room temperature for 2 and 8 days.45 The
160
extraction of AOH from the fruit samples was performed as per procedure described previously.
161 162
2.7. Comparative analysis by HPLC
163
2.7.1 Extraction and cleanup
164
The extraction of AOH from the fruits was performed following the same procedure described for
165
SERS analysis. The cleanup was conducted by using the extract solution by gravity onto an Oasis
166
HLB 3 cc (60 mg) extraction cartridge (Waters Co., Milford, MA, USA), and the cartridge was first
167
conditioned and equilibrated by 5.0 mL of methanol and milli-Q water, respectively, then washed
168
with 2.0 mL of methanol:water (1:4, v/v). 2.0 g of sodium sulfate was injected into a 3 mL/500 mg
169
Supelclean LC-NH2 SPE Tubes (Supelco Inc., Bellefonte, PA, USA). After that, the tubes were
170
conditioned with 5.0 mL of dichloromethane and the lower end of the HLB cartridge was connected
171
to the tubes, this series was then washed with 5.0 mL of dichloromethane. Finally, AOH was eluted
172
with 7.0 mL of 1% methanoic acid in methanol:ethyl acetate (1:1, v/v). The eluent was transferred
173
into a flask and evaporated (vacuum-rotary evaporation, 40 oC) to nearly dry, which was then
174
concentrated to dryness using a nitrogen flow, and the residue was reconstituted in 5.0 mL of
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
175
acetonitrile:water (2:3, v/v). The reconstituted solution was filtrated through 0.22 µm PTFE filters
176
(EMD Millipore Co., Bedford, MA, USA) and moved to the autosampler vials for the instrumental
177
analysis by an Acquity® ArcTM HPLC system (Waters Co., Milford, MA, USA).
178 179
2.7.2. Standard preparation
180
A stock solution of 1.0×106 µg/L was prepared by diluting AOH standard with methanol and kept
181
in darkness at -20 oC. A working solution (1.0×104 µg/L) was prepared by diluting the AOH stock
182
solution with methanol. AOH standard solutions for HPLC calibration and recovery test were
183
prepared by diluting the working solution with methanol. A series of standard solutions with the
184
concentrations of 10.0, 50.0, 100.0, 200.0, and 400.0 µg/L were prepared.
185 186
2.7.3 HPLC conditions
187
All analyses were conducted using the HPLC system equipped with a 2475 FLR detector (Waters
188
Co., Milford, MA, USA), which excites at wavelength of 339 nm and emits at wavelength of 404 nm.
189
ChromQuest software (Version 4.2, ThermoQuest Italia S.p.A., Milano, Italy) was used to manage
190
the HPLC data acquisition and processing. An XBridge® BEH-C18 (Waters Co., Milford, MA, USA)
191
column (100 × 2.1 mm, 2.5 µm) was used as chromatographic column. The mobile phase consisted
192
of two eluents (eluent A: water and eluent B: acetonitrile). A gradient program with a flow rate of
193
0.204 mL/min was used, starting with 85% A and 15% B, reaching 70% B after 8.82 min and then
194
maintained for 1.47 min. Afterward, the gradient was returned to 15% B in 0.3 min and allowed to
195
equilibrate for 4.41 min. The column temperature was set to 35 oC and the injection volume was set
196
to 10 µL. For quantitative analysis, an external calibration curve was used. AOH standard solutions
197
with the concentrations of 10.0, 50.0, 100.0, 200.0, and 400.0 µg/L were used for construction of
198
five-point calibration curves, and the peak areas versus concentrations were plotted.
199
ACS Paragon Plus Environment
Page 8 of 28
Page 9 of 28
200
Journal of Agricultural and Food Chemistry
2.7.4 Recovery test
201
Blank pear fruits, previously analyzed with negative result for the presence of AOH, were injected
202
with working solutions to reach 20.0, 50.0, and 200.0 µg/kg of AOH. Spiked pear fruit were prepared
203
and analyzed using the same procedure described for the pear fruit, i.e., extraction, cleanup and
204
HPLC analysis. Recovery tests were based on quintuplicate spiking and triplicate analysis. The fruit
205
samples detected by SERS previously were also analyzed by HPLC.
206 207
3. Results and discussion
208
3.1. Characterization of AgNPs
209
AgNPs are widely used as SERS substrate because they are easier to synthesize, in addition,
210
different batches of AgNPs are basically the same in shape, size, and size dispersion. As illustrated in
211
the UV-visible spectra of AgNPs (Fig. 1a), the wavenumber of maximum absorption peak was
212
414.6±0.9 nm and the full width at half-maximum was 116.1±0.1 nm, which suggesting that the
213
shape and size of AgNPs were similar from batch to batch. Besides, the UV-visible spectra were
214
overlapped, which indicated an excellent repeatability of AgNPs synthesis in various batches. DLS
215
analysis was performed to measure the diameter of AgNPs and the result is shown in Fig. 1b,
216
indicating that the average hydrodynamic diameter of the bare AgNPs was 48.9 nm, and the diameter
217
increased to 49.7 nm after pyridine modification (Fig. 1c). The TEM images of the bare AgNPs and
218
the modified AgNPs are shown in Fig. 1d, e and Fig. 1f. As shown in Fig. 1e, the diameters of bare
219
AgNPs range from 26.8 nm to 63.3 nm. Compared with the bare AgNPs (Fig. 1e), the modified
220
AgNPs had a silver core with a diameter of about 49 nm and about 2 nm thick shell (Fig. 1f) due to
221
that the multilayer pyridine molecules were tightly adsorbed on nanostructured surfaces of sliver. The
222
adsorption is favored by the heteroatom of nitrogen in the molecular structure.46,47 Pyridine is a
223
functional Raman reporter, its molecular structure contains nitrogen atom, which cannot only interact
224
with the AgNPs but can also conjugate with the AOH molecule, leading to the tight adsorption on the
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
225
surface of AgNPs and as many as possible number of the AOH molecules adsorbed on AgNPs.40,48
226
Therefore, the surface of AgNPs with a thin pyridine shell could circumvent the weak affinity of
227
AOH on metallic surfaces and to improve the sensitivity of detection. Moreover, due to that the shell
228
was thin enough, AOH molecules tightly adsorbed on the surface of AgNPs.
229 230
3.2. SERS activity of AOH on pyridine modified AgNPs
231
In order to confirm the SERS activity of pyridine modified AgNPs for the sensitive detection of
232
AOH, the SERS spectra of various solutions were given and compared. As shown in Fig. 2, the
233
SERS spectra of AOH added to the bare AgNPs (a) and to the modified AgNPs (b) without the
234
addition of sodium nitrate did not elicit distinctive SERS bands, and their intensities were extremely
235
low. Comparing Fig. 2b with Fig. 2c (the spectrum of the modified AgNPs), it can be seen that the
236
addition of AOH did not introduce a new Raman peak, that is to say, the modified AgNPs without the
237
addition of sodium nitrate cannot be used directly for the detection. The spectra of AOH added to the
238
bare and modified AgNPs with adding sodium nitrate were also collected (Fig. 2d, e). Sodium nitrate
239
was added to promote the AgNps close together, which could increase the signal of target.48
240
Although an apparent spectral change could not be discerned after the AgNPs was modified by
241
pyridine, some new peaks appeared, especially the ones at 1002 and 1033 cm-1 (in black dotted line),
242
these two peaks also appeared in the spectrum of pyridine solution (Fig. 2f). Thus, they are likely
243
assigned to pyridine ring breathing vibrational mode and in-plane deformation vibrational mode,
244
respectively.40 These two peaks showed different intensities in Fig. 2e and Fig. 2g (the spectrum of
245
the modified AgNPs with the addition of sodium nitrate), probably due to the different fluorescence
246
backgrounds of these two solutions, which have different effects on the spectra. As shown in Fig. 2d,
247
low SERS signal of AOH was obtained when detecting AOH with the bare AgNPs, while the
248
modified AgNPs exhibited very strong enhancement effect (Fig. 2e). The reasons may be attributed
249
to the weak affinity of AOH molecules on the surface of AgNPs. Compared to the Fig. 2g, the
ACS Paragon Plus Environment
Page 10 of 28
Page 11 of 28
Journal of Agricultural and Food Chemistry
250
spectra of pyridine modified AgNPs showed very strong characteristic bands about 1173, 1252, 1298,
251
1367, and 1615 cm-1 after the addition of AOH solution, which could be attributed to the vibration
252
modes of AOH molecules by comparison with the spectrum of AOH solution (Fig. 2h). The band at
253
1173 cm-1 is assigned to the β (C-H) ring.49 The intense peaks at 1298 cm-1 corresponds to the
254
stretching C-H vibration of benzene ring, while the band at 1252 cm-1 is assigned to the vibration of
255
O-H and C-H.43 The bands at 1486 cm-1 is assigned to the bending vibration of CH3, and it was
256
enhanced and displays clear peaks in comparison with the bands in the spectrum of AOH added to
257
the bare AgNPs with adding sodium nitrate.38,41 The bands at 1367 cm-1 is attributed to CH3
258
symmetric bending vibrations.38 The band at 1615 cm-1 is related to the ring stretching mode of
259
C-C.50 These results suggested that the sensitivity of determination was advanced through the
260
modification of the AgNps surface by using pyridine. The improvement of the efficiency of the
261
pyridine modified AgNPs to bind AOH is ascribed to the interaction of the aromatic rings of
262
adsorbed pyridine and AOH. Moreover, due to the formation of a covalent bond through the lone pair
263
of electrons of the nitrogen atom, pyridine interact strongly with metal.48 In addition, it is helpful for
264
the determination of phenols because the formation of hydrogen bonds between the nitrogen atoms
265
of pyridine and the hydroxyl functions contained in phenol.51,52 These above results indicated that the
266
characteristic fingerprint of band at 1252 cm-1 could be used to identify the presence of AOH using
267
the pyridine modified AgNPs substrate.
268 269
3.3. SERS detection of AOH standard solution
270
In this part, the LOD was confirmed by using a series of AOH standard solutions with various
271
concentrations. Fig. 3 shows the concentration-dependent SERS spectra of AOH standard solutions
272
ranging from 3.16 to 316.0 µg/L. As shown in Fig. 3a, the locations and the intensities of the
273
pyridine peaks (1002 cm-1 and 1033 cm-1) remain invariable, which meant that the addition of AOH
274
had no effect on pyridine adsorption and the pyridine was steadily adsorbed on the AgNPs, while the
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 12 of 28
275
SERS signal intensities of the fingerprint Raman bands of AOH was increased with the increase in
276
the concentrations. This was because higher concentrations of AOH standard solutions caused
277
increase in the amount of the AOH molecules conjugated on the AgNPs. For obtaining the
278
quantitative relation of SERS signal intensities with the concentrations, a curve with the SERS
279
intensity at 1252 cm-1, which was one of the most intensive bands was plotted in Fig. 3b, and it was
280
observed that there existed a linear relationship between the intensity and the log concentration of
281
AOH solution in the range of 3.16-316.0 µg/L, which can be described as
282
I = 1453.7 log c - 553.4
(1)
283
where c is the concentration of AOH solution, I is the SERS intensity. The correlation coefficient (R2)
284
of Eq. (1) is 0.9926.
285 286
The limit of detection can be calculated by53 LOD = 3(σ / k)
(2)
287
where LOD is limit of detection, σ is the predicted error in the y-intercept and k is the slope of the
288
regression line based on Eq. (1), LOD could thus be calculated as 1.30 µg/L.
289 290
3.4. Linearity and LOD of HPLC analysis
291
The HPLC method with the 2475 FLR detector was able to measure AOH in less than 15 min,
292
with retention time of 8.7 min (Fig. 4a). The linearity was assessed under the chromatographic
293
conditions described by preparing calibration curve using standard solutions with concentrations of
294
10.0, 50.0, 100.0, 200.0 and 400.0 µg/L. Calibration curve was drawn by linear regression of the
295
least-squares method based on the plotting of peak areas at 8.7 min against concentrations (Fig. 4b).
296
As a result, satisfactory linearity was observed with R2 value as high at 0.9995. The LOD was
297
defined as three times the ratio of the standard deviation of the blank over the slope of the calibration
298
graph, and the LOD of HPLC method was 6.96 µg/L, which was much higher than that of the SERS
299
method.
ACS Paragon Plus Environment
Page 13 of 28
Journal of Agricultural and Food Chemistry
300 301
3.5. Detection of AOH in pear fruit
302
3.5.1 Recovery of AOH spiked in pear fruit
303
To confirm the feasibility of the proposed SERS method, pear fruits were used as a substrate to
304
perform recovery tests and the results are summarized in Table 1. As shown in Table 1, the recovery
305
ranged from 70.22% to 111.10%, and the average recovery of the three spiked levels were greater
306
than 84.05%. The accuracy as expressed as the relative standard deviation (RSD) was assessed using
307
five replicates with the same spiked samples (14.13%-18.46%). The results demonstrated that the
308
SERS method had a good potential for rapid detection of AOH in pear fruit. The results of recovery
309
experiments of HPLC method are also given in Table 1, showing the recoveries of 73.70%-90.00%,
310
the average recovery being higher than 74.70%, and the RSD ranging from 1.89% to 9.76%. These
311
excellent results confirmed that the traditional HPLC method would be useful for the detection of
312
AOH residues in pear fruit. Comparing the results of these two methods, it was easy to find that the
313
determined concentrations of AOH in the spiked samples by SERS method were close to their added
314
concentrations. However, by the HPLC method, the determined concentrations of AOH were much
315
lower than those of AOH added, this was probably due to the loss of AOH in the process of clean up.
316
However the RSD of the HPLC method was far less of that of the SERS method, indicating the
317
HPLC method was more accurate than the SERS method. The reason may be ascribed to that the
318
AOH extract usually contains various impurities, thus introducing difficulties in AOH detection by
319
the SERS method, due to the matrix effect.
320 321
3.5.2 Determination of AOH in real samples
322
To validate the availability of the SERS method for real samples, three kinds of pear fruit were
323
detected by the SERS method. All pear fruit were processed and detected by the procedure above,
324
and the results are presented in Table 2, clearly indicating that AOH was not found in any of the fresh
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
325
fruit, while the rotten fruit and inoculated fruit were positive. For further confirming the above
326
results, HPLC was performed and the results are also shown in Table 2. The comparison of these the
327
two methods showed the consistency, with the RSD of less than 10% between the two methods in
328
detecting the same samples.
329
It was found that the performance of the developed SERS method had some variation in detecting
330
AOH, which probably attributed to the variation in sample treatments and matrix effects. Although
331
the traditional HPLC method had better accuracy, the proposed SERS method still possessed good
332
accuracy with the advantages of high sensitivity, speed and low LOD, cost. SERS took less than 1
333
min to collect the spectra, while HPLC required several minutes for obtaining the results. In addition,
334
a series of complex purification processes were required before HPLC detection. Therefore, the
335
proposed SERS method is advantageous for detecting AOH residues with very low concentration,
336
especially it is suitable for trace detection.
337 338
4. Conclusions
339
A simple SERS method was developed in the current study for rapid detection of AOH in pear
340
fruit based on an accessible AgNPs substrate. AgNPs were modified with pyridine to circumvent the
341
weak affinity of AOH on metallic surface and to improve the sensitivity of detection. To the best of
342
our knowledge, the proposed SERS method has, for the first time, realized the detection of hazardous
343
Alternaria mycotoxins contaminated fruit sample. The SERS method performed satisfactorily with
344
the LOD of 1.30 µg/L in the detection range of 3.16 to 316.0 µg/L. In addition, the established SERS
345
method was successfully used to detect AOH in pear fruit and the results were cross validated against
346
the traditional HPLC method. Furthermore, the proposed method is rapid and results can be available
347
within the hour. Therefore, this SERS detection technique could be a valuable tool for rapid
348
detection of AOH in pear fruit and other agricultural products.
349
ACS Paragon Plus Environment
Page 14 of 28
Page 15 of 28
350
Journal of Agricultural and Food Chemistry
Acknowledgments
351
The authors are grateful to the National Key Technologies R&D Program (2015BAD19B03). This
352
research was also supported by the Collaborative Innovation Major Special Projects of Guangzhou
353
City (201604020007, 201604020057, 201508020097,), the International S&T Cooperation Program
354
of China (2015DFA71150) for its support, the Guangdong Provincial Science and Technology Plan
355
Projects (2015A020209016, 2016A040403040), the Key Projects of Administration of Ocean and
356
Fisheries of Guangdong Province (A201401C04), the International and Hong Kong – Macau -
357
Taiwan Collaborative Innovation Platform of Guangdong Province on Intelligent Food Quality
358
Control and Process Technology & Equipment (2015KGJHZ001), the Guangdong Provincial R & D
359
Centre for the Modern Agricultural Industry on Non-destructive Detection and Intensive Processing
360
of Agricultural Products, the Common Technical Innovation Team of Guangdong Province on
361
Preservation and Logistics of Agricultural Products (2016LM2154) and the Innovation Centre of
362
Guangdong Province for Modern Agricultural Science and Technology on Intelligent Sensing and
363
Precision Control of Agricultural Product Qualities.
364 365
References
366
(1) Logrieco, A.; Moretti, A.; Solfrizzo, M. Alternaria toxins and plant diseases: an overview of origin, occurrence
367 368 369 370 371 372 373
and risks. World Mycotoxin J. 2009, 2, 129-140. (2) Andersen, B.; Frisvad, J. C. Natural occurrence of fungi and fungal metabolites in moldy tomatoes. J. Agric. Food Chem. 2004, 52, 7507-7513. (3) Scott, P. M.; Zhao, W.; Feng, S.; Lau, B. P. Y. Alternaria toxins alternariol and alternariol monomethyl ether in grain foods in Canada. Mycotoxin Res. 2012, 28, 261-266. (4) Andersen, B.; Nielsen, K. F.; Pinto, V. F.; Patriarca, A. Characterization of Alternaria strains from Argentinean blueberry, tomato, walnut and wheat. Int. J. Food Microbiol. 2015, 196, 1-10.
374
(5) Asam, S.; Konitzer, K.; Rychlik, M. Precise determination of the Alternaria mycotoxins alternariol and
375
alternariol monomethyl ether in cereal, fruit and vegetable products using stable isotope dilution assays.
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
376 377 378 379 380 381 382 383 384
Mycotoxin Res. 2011, 27, 23-28. (6) Asam, S.; Konitzer, K.; Schieberle, P.; Rychlik, M. Stable isotope dilution assays of Alternariol and Alternariol Monomethyl Ether in Beverages. J. Agric. Food Chem. 2009, 57, 5152-5160. (7) Asam, S.; Liu, Y.; Konitzer, K.; Rychlik, M. Development of a stable isotope dilution assay for tenuazonic acid. J. Agric. Food Chem. 2011, 59, 2980-2987. (8) Asam, S.; Rychlik, M. Recent developments in stable isotope dilution assays in mycotoxin analysis with special regard to Alternaria toxins. Anal. Bioanal. Chem. 2015, 407, 7563-7577. (9) da Motta, S.; Soares, L. M. V. Simultaneous determination of tenuazonic and cyclopiazonic acids in tomato products. Food Chem. 2000, 71, 111-116.
385
(10) Lopez-Maestresalas, A.; Keresztes, J. C.; Goodarzi, M.; Arazuri, S.; Jaren, C.; Saeys, W. Non-destructive
386
detection of blackspot in potatoes. by Vis-NIR and SWIR hyperspectral imaging. Food Control 2016, 70,
387
229-241.
388 389 390 391
(11) Patriarca, A.; Azcarate, M. P.; Terminiello, L.; Pinto, V. F. Mycotoxin production by Alternaria strains isolated from Argentinean wheat. Int. J. Food Microbiol. 2007, 119, 219-222. (12) Siegel, D.; Merkel, S.; Koch, M.; Nehls, I. Quantification of the Alternaria mycotoxin tenuazonic acid in beer. Food Chem. 2010, 120, 902-906.
392
(13) Lau, B. P. Y.; Scott, P. M.; Lewis, D. A.; Kanhere, S. R.; Cleroux, C.; Roscoe, V. A. Liquid
393
chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry of the
394
Altemaria mycotoxins alternariol and alternariol monomethyl, ether in fruit juices and beverages. J.
395
Chromatogr. A. 2003, 998, 119-131.
396
(14) Myresiotis, C. K.; Testempasis, S.; Vryzas, Z.; Karaoglanidis, G. S.; Papadopoulou-Mourkidou, E.
397
Determination of mycotoxins in pomegranate fruit and juices using a QuEChERS-based method. Food
398
Chem. 2015, 182, 81-88.
399 400 401 402
(15) Raistrick, H.; Stickings, C. E.; Thomas, R. Studies in the biochemistry of microorganisms. 90. Alternariol and alternariol monomethyl ether, metabolic products of Alternaria tenuis. Biochem. J. 1953, 55, 421-433. (16) Brugger, E. M.; Wagner, J.; Schumacher, D. M.; Koch, K.; Podlech, J.; Metzler, M.; Lehmann, L. Mutagenicity of the mycotoxin alternariol in cultured mammalian cells. Toxicol. Lett. 2006, 164, 221-230.
403
(17) Dong, Z. G.; Liu, G. T.; Dong, Z. M.; Qian, Y. Z.; An, Y. H.; Miao, J. A.; Zhen, Y. Z. Induction of mutagenesis
404
and transformation by the extract of Alternaria alternata isolated from grains in Linxian, China.
ACS Paragon Plus Environment
Page 16 of 28
Page 17 of 28
405 406 407
Journal of Agricultural and Food Chemistry
Carcinogenesis 1987, 8, 989-991. (18) Lehmann, L.; Wagner, J.; Metzler, M. Estrogenic and clastogenic potential of the mycotoxin alternariol in cultured mammalian cells. Food Chem. Toxicol. 2006, 44, 398-408.
408
(19) Schrader, T. J.; Cherry, W.; Soper, K.; Langlois, I.; Vijay, H. M. Examination of Alternaria alternata
409
mutagenicity and effects of nitrosylation using the Ames Salmonella test. Teratogen. Carcin. Mut. 2011, 21,
410
261-274.
411 412 413 414 415 416 417 418 419 420 421 422
(20) Ostry, V. Alternaria mycotoxins: an overview of chemical characterization, producers, toxicity, analysis and occurrence in foodstuffs. World Mycotoxin J. 2008, 1, 175-188. (21) Liu, G. T.; Qian, Y. Z.; Zhang, P.; Dong, W. H.; Qi, Y. M.; Guo, H. T. Etiological role of Alternaria alternata in human esophageal cancer. Chinese Med. J. 1992, 105, 394-400. (22) Scott, P. M. Analysis of agricultural commodities and foods for Alternaria mycotoxins. J. Aoac Int. 2001, 84, 1809-1817. (23) Scott, P. M.; Weber, D.; Kanhere, S. R. Gas chromatography mass spectrometry of Alternaria mycotoxins. J. Chromatogr. A. 1997, 765, 255-263. (24) Azaiez, I.; Giusti, F.; Sagratini, G.; Manes, J.; Fernandez-Franzon, M. Multi-mycotoxins analysis in dried fruit by LC/MS/MS and a modified QuEChERS procedure. Food Anal. Method. 2014, 7, 935-945. (25) Prelle, A.; Spadaro, D.; Garibaldi, A.; Gullino, M. L. A new method for detection of five alternaria toxins in food matrices based on LC-APCI-MS. Food Chem. 2013, 140, 161-167.
423
(26) Ackermann, Y.; Curtui, V.; Dietrich, R.; Gross, M.; Latif, H.; Martlbauer, E.; Usleber, E. Widespread
424
occurrence of low levels of Alternariol in apple and tomato products, as determined by comparative
425
immunochemical assessment using monoclonal and polyclonal antibodies. J. Agric. Food Chem. 2011, 59,
426
6360-6368.
427
(27) Andersen, B.; Smedsgaard, J.; Jorring, I.; Skouboe, P.; Pedersen, L. H. Real-time PCR quantification of the
428
AM-toxin gene and HPLC qualification of toxigenic metabolites from Alternaria species from apples. Int.
429
J. Food Microbiol. 2006, 111, 105-111.
430 431
(28) Delgado, T.; Gomez-Cordoves, C. Natural occurrence of alternariol and alternariol methyl ether in Spanish apple juice concentrates. J. Chromatogr. A. 1998, 815, 93-97.
432
(29) Delgado, T.; Gomez-Cordoves, C.; Scott, P. M. Determination of alternariol and alternariol methyl ether in
433
apple juice using solid-phase extraction and high performance liquid chromatography. J. Chromatogr. A.
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
434
1996, 731, 109-114.
435
(30) Dzuman, Z.; Zachariasova, M.; Lacina, O.; Veprikova, Z.; Slavikova, P.; Hajslova, J. A rugged
436
high-throughput analytical approach for the determination and quantification of multiple mycotoxins in
437
complex feed. Talanta 2014, 121, 263-272.
438 439
(31) Koesukwiwat, U.; Sanguankaew, K.; Leepipatpiboon, N. Evaluation of a modified QuEChERS method for analysis of mycotoxins in rice. Food Chem. 2014, 153, 44-51.
440
(32) Magnani, R. F.; De Souza, G. D.; Rodrigues, E. Analysis of alternariol and alternariol monomethyl ether on
441
flavedo and albedo tissues of tangerines (Citrus reticulata) with symptoms of alternaria brown spot. J.
442
Agric. Food Chem. 2007, 55, 4980-4986.
443
(33) Pavon, M. A.; Luna, A.; de la Cruz, S.; Gonzalez, I.; Martin, R.; Garcia, T. PCR-based assay for the detection
444
of Alternaria species and correlation with HPLC determination of altenuene, alternariol and alternariol
445
monomethyl ether production in tomato products. Food Control 2012, 25, 45-52.
446
(34) Vaclavik, L.; Zachariasova, M.; Hrbek, V.; Hajslova, J. Analysis of multiple mycotoxins in cereals under
447
ambient conditions using direct analysis in real time (DART) ionization coupled to high resolution mass
448
spectrometry. Talanta 2010, 82, 1950-1957.
449
(35) Pavon, M. A.; Gonzalez, I.; Rojas, M.; Pegels, N.; Martin, R.; Garcia, T. PCR detection of Alternaria spp. in
450
processed foods, based on the internal transcribed spacer genetic marker. J. Food Protect. 2011, 74,
451
240-247.
452
(36) Abou-Hany, R. A. G.; Urraca, J. L.; Descalzo, A. B.; Gomez-Arribas, L. N.; Moreno-Bondi, M. C.; Orellana, G.
453
Tailoring molecularly imprinted polymer beads for alternariol recognition and analysis by a screening with
454
mycotoxin surrogates. J. Chromatogr. A. 2015, 1425, 231-239.
455
(37) Gao, R.; Choi, N.; Chang, S. I.; Kang, S. H.; Song, J. M.; Cho, S. I.; Lim, D. W.; Choo, J. Highly sensitive
456
trace analysis of paraquat using a surface-enhanced Raman scattering microdroplet sensor. Anal. Chim.
457
Acta. 2010, 681, 87-91.
458 459
(38) Hassanain, W. A.; Izake, E. L.; Schmidt, M. S.; Ayoko, G. A. Gold nanomaterials for the selective capturing and SERS diagnosis of toxins in aqueous and biological fluids. Biosens. Bioelectron. 2017, 91, 664-672.
460
(39) Li, A. K.; Tang, L. J.; Song, D.; Song, S. S.; Ma, W.; Xu, L. G.; Kuang, H.; Wu, X. L.; Liu, L. Q.; Chen, X.;
461
Xu, C. L. A SERS-active sensor based on heterogeneous gold nanostar core-silver nanoparticle satellite
462
assemblies for ultrasensitive detection of aflatoxinB1. Nanoscale 2016, 8, 1873-1878.
ACS Paragon Plus Environment
Page 18 of 28
Page 19 of 28
Journal of Agricultural and Food Chemistry
463
(40) Liu, J. Z.; Hu, Y. J.; Zhu, G. C.; Zhou, X. M.; Jia, L.; Zhang, T. Highly sensitive detection of zearalenone in
464
feed samples using competitive surface-enhanced Raman scattering immunoassay. J. Agric. Food Chem.
465
2014, 62, 8325-8332.
466
(41) Muller, C.; Glamuzina, B.; Pozniak, I.; Weber, K.; Cialla, D.; Popp, J.; Pinzaru, S. C. Amnesic shellfish
467
poisoning biotoxin detection in seawater using pure or amino-functionalized Ag nanoparticles and SERS.
468
Talanta 2014, 130, 108-115.
469
(42) Temur, E.; Zengin, A.; Boyaci, I. H.; Dudak, F. C.; Torul, H.; Tamer, U. Attomole sensitivity of staphylococcal
470
enterotoxin B detection using an aptamer-modified surface-enhanced Raman scattering probe. Anal. Chem.
471
2012, 84, 10600-10606.
472 473 474 475
(43) Yuan, J.; Sun, C. W.; Guo, X. Y.; Yang, T. X.; Wang, H.; Fu, S. Y.; Li, C. C.; Yang, H. F. A rapid Raman detection of deoxynivalenol in agricultural products. Food Chem. 2017, 221, 797-802. (44) Lee, P. C.; Meisel, D. Adsorption and surface-enhanced Raman of dyes on silver and gold sols. J. Phys. Chem.1982, 86, 3391-3395.
476
(45) Pan, T. T.; Pu, H. B.; Sun, D.-W. Insights into the changes in chemical compositions of the cell wall of pear
477
fruit infected by Alternaria alternata with confocal Raman microspectroscopy. Postharvest Biol. Tec. 2017,
478
132, 119-129.
479
(46) Hu, J. W.; Zhao, B.; Xu, W. Q.; Li, B. F.; Fan, Y. G. Surface-enhanced Raman spectroscopy study on the
480
structure changes of 4-mercaptopyridine adsorbed on silver substrates and silver colloids. Spectrochim.
481
Acta A. 2002, 58, 2827-2834.
482
(47) Pagliai, M.; Bellucci, L.; Muniz-Miranda, M.; Cardini, G.; Schettino, V. A combined Raman, DFT and MD
483
study of the solvation dynamics and the adsorption process of pyridine in silver hydrosols. Phys. Chem.
484
Chem. Phys. 2006, 8, 171-178.
485 486
(48) Malynych, S.; Luzinov, I.; Chumanov, G. Poly(vinyl pyridine) as a universal surface modifier for immobilization of nanoparticles. J. Phys. Chem. B. 2002, 106, 1280-1285.
487
(49) Voiciuk, V.; Valincius, G.; Budvytyte, R.; Matijoska, A.; Matulaitiene, I.; Niaura, G. Surface-enhanced Raman
488
spectroscopy for detection of toxic amyloid β oligomers adsorbed on self-assembled monolayers.
489
Spectrochim. Acta A. 2012, 95, 526-532.
490
(50) Li, R. P.; Yang, G. H.; Yang, J. L.; Han, J. H.; Liu, J. H.; Huang, M. J. Determination of melamine in milk
491
using surface plasma effect of aggregated Au@SiO2 nanoparticles by SERS technique. Food Control. 2016,
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
492
68, 14-19.
493
(51) Chien, W. L.; Yang, C. M.; Chen, T. L.; Li, S. T.; Hong, J. L. Enhanced emission of a pyridine-based
494
luminogen by hydrogen-bonding to organic and polymeric phenols. Rsc Adv. 2013, 3, 6930-6938.
495
(52) De Bleye, C.; Dumont, E.; Hubert, C.; Sacre, P. Y.; Netchacovitch, L.; Chavez, P. F.; Hubert, P.; Ziemons, E. A
496
simple approach for ultrasensitive detection of bisphenols by multiplexed surface-enhanced Raman
497
scattering. Anal. Chim. Acta. 2015, 888, 118-125.
498 499
(53) Schmit, V. L.; Martoglio, R.; Carron, K. T. Lab-on-a-bubble surface enhanced Raman indirect immunoassay for Cholera. Anal. Chem. 2012, 84, 4233-4236.
ACS Paragon Plus Environment
Page 20 of 28
Page 21 of 28
Journal of Agricultural and Food Chemistry
Table 1. Detection of AOH spiked in pear fruit by SERS and HPLC
500
Methods
SERS
HPLC
Spiked concentration (µg/kg)
Detected concentration (µg/kg)
Recovery (%)
RSD (%)
20 50 100 20 50 200
16.81 42.61 89.49 14.94 42.10 171.25
71.30-94.00 70.80-100.36 70.22-111.10 73.70-75.70 78.38-90.00 81.01-89.24
14.13 16.28 18.46 1.89 9.76 4.13
501
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
502
Table 2. Comparison of AOH detection by SERS and HPLC in real samples
AOH content (µg/kg)
Fruit types (n=5)
503
Page 22 of 28
1
2
3
4
5
S
H
S
H
S
H
S
H
S
H
Fresh
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
2 days infected 8 days infected
8.31 8.62
ND 8.40
9.42 9.63
7.55 ND
9.31 11.12
7.69 10.68
6.08 9.40
ND 8.90
6.70 12.92
ND 11.24
Rotten
13.93
11.47
17.81
17.41
18.35
19.48
13.14
13.03
22.96
22.62
Note: ND represents not detected, S and H represent SERS method and HPLC method, respectively.
ACS Paragon Plus Environment
Page 23 of 28
Journal of Agricultural and Food Chemistry
504
Figure caption
505
Fig. 1. (a) The UV-visible spectra acquired from 300 to 700 nm for three different batches of AgNps. The average
506
maximum absorption value was 403 ± 1.8 nm and the FWHM was 74 ± 4.4 nm, (b) & (c) the average
507
hydrodynamic diameter of the bare AgNPs and the pyridine modified AgNPs detected by dynamic light
508
scattering, (d) & (e) the TEM images of the bare AgNPs at low and high magnification, and (f) the TEM
509
images of the pyridine modified AgNPs at high magnification.
510
Fig. 2. (a) & (b) Raman spectra of AOH added to the bare and pyridine modified AgNPs, (c) Raman spectrum of
511
the pyridine modified AgNPs, (d) & (e) Raman spectra of AOH added to the bare and pyridine modified
512
AgNPs with adding sodium nitrate, (f) Raman spectrum of pyridine solution, (g) Raman spectrum of the
513
pyridine modified AgNPs with adding sodium nitrate, and (h) Raman spectrum of AOH solution.
514
Fig. 3. AOH detection based on SERS with pyridine modified AgNPs (n=3). (a) SERS spectra of AOH standard
515
solutions with various concentrations ranging from 3.16 to 316.0 µg/L, and (b) calibration curve of SERS
516
peak intensities at 1251.88 cm-1 with the concentrations.
517 518
Fig. 4. (a) HPLC analysis of AOH standard solution; and (b) calibration curve of the peak area at 8.7 min with the concentrations.
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
519 520 521
Fig. 1. (a) The UV-visible spectra acquired from 300 to 700 nm for three different batches of AgNps. The average
522
maximum absorption value was 403 ± 1.8 nm and the FWHM was 74 ± 4.4 nm, (b) & (c) the average
523
hydrodynamic diameter of the bare AgNPs and the pyridine modified AgNPs detected by dynamic light scattering,
524
(d) & (e) the TEM images of the bare AgNPs at low and high magnification, and (f) the TEM images of the
525
pyridine modified AgNPs at high magnification.
ACS Paragon Plus Environment
Page 24 of 28
Page 25 of 28
Journal of Agricultural and Food Chemistry
526 527 528
Fig. 2. (a) & (b) Raman spectra of AOH added to the bare and pyridine modified AgNPs, (c) Raman spectrum of
529
the pyridine modified AgNPs, (d) & (e) Raman spectra of AOH added to the bare and pyridine modified AgNPs
530
with adding sodium nitrate, (f) Raman spectrum of pyridine solution, (g) Raman spectrum of the pyridine modified
531
AgNPs with adding sodium nitrate, and (h) Raman spectrum of AOH solution.
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
532 533 534 535
Fig. 3. AOH detection based on SERS with pyridine modified AgNPs (n=3). (a) SERS spectra of AOH standard
536
solutions with various concentrations ranging from 3.16 to 316.0 µg/L, and (b) calibration curve of SERS peak
537
intensities at 1251.88 cm-1 with the concentrations.
ACS Paragon Plus Environment
Page 26 of 28
Page 27 of 28
Journal of Agricultural and Food Chemistry
538 539 540 541
Fig. 4. (a) HPLC analysis of AOH standard solution; and (b) calibration curve of the peak area at 8.7 min with the
542
concentrations.
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Graphic 146x113mm (220 x 220 DPI)
ACS Paragon Plus Environment
Page 28 of 28