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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

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Journal of Agricultural and Food Chemistry

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A simple approach for rapid detection of alternariol in pear fruit by

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SERS with pyridine modified silver nanoparticles

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Ting-tiao Pan1,2,3, Da-Wen Sun1,2,3,4∗, Hongbin Pu1,2,3, Qingyi Wei1,2,3

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School of Food Science and Engineering, South China University of Technology, Guangzhou 510641, China 2

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Academy of Contemporary Food Engineering, South China University of Technology, Guangzhou Higher Education Mega Center, Guangzhou 510006, China

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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

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Food Refrigeration and Computerized Food Technology (FRCFT), Agriculture and Food Science Centre, University College Dublin, National University of Ireland, Belfield, Dublin 4, Ireland

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Abstract: A simple method based on surface-enhanced Raman scattering (SERS) was developed for

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rapid determination of alternariol (AOH) in pear fruit by using an easily prepared silver nanoparticles

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(AgNPs) substrate. The AgNPs substrate was modified by pyridine to circumvent the weaker affinity

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of AOH molecular on sliver surface and improve the sensitivity of detection. Quantitative analysis

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was performed in AOH solutions at concentrations over a range of 3.16-316.0 µg/L, and the limit of

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detection was 1.30 µg/L. The novel method was also applied to detect AOH residues in pear fruit

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purchased from market and those were artificially inoculated with A. alternata. AOH was not found

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in any of the fresh fruit, while it resided in the rotten and inoculated fruits. Finally, the SERS method

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was cross validated against HPLC. It was revealed that SERS method have great potential utility in

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rapid detection of AOH in pear fruit and other agricultural products.

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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


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1. Introduction

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Alternaria is a cosmopolitan fungal genus that includes saprophytic, endophytic and pathogenic

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species. Many Alternaria species are plant pathogens that can damage crops in the field. Moreover,

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they are capable of causing significant postharvest decay of fruits, vegetables, and cereals, resulting

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in quality degradation of the infected products.1 Even worse, some Alternaria species can generate

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diverse toxic secondary metabolites, known as Alternaria mycotoxins, contaminating agricultural

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products during storage.2,3 The occurrence of Alternaria mycotoxins has been reported in fruits,

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vegetables, cereals, and beverages.4-12

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More than 30 Alternaria mycotoxins belonging to different structural groups have been isolated

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from Alternaria species, among which, one of the dibenzopyrone derivatives, alternariol (AOH,

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3,7,9-trihydroxy-1-methyl-6H-dibenzo(b,d)pyran-6-one) is considered to be the most important

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mycotoxins produced in alternata infected pear fruit and other products.13-15 From a toxicological

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point of view, Alternaria mycotoxins are associated with a variety of adverse health effects, and

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AOH is genotoxic, carcinogenic, mutagenic, and cytotoxic in microbial and mammalian cell

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systems.16-19 To date, no in vivo toxicology researches in experimental animals for Alternaria

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mycotoxins have been carried out, however, some symptoms of precancerous changes in esophageal

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mucosa of mice have been presented.20 Although the acute toxicity of Alternaria toxins is low in

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animals, limited evidences are available for long term toxicity effects of them and their synergistic

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effect with other toxins or contaminants.1,20 In addition, many researchers believe that the

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incremental incidence of human esophageal cancer in China was related to the contamination of

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Alternaria toxins, and A. alternata might be one of the etiological factors.17,21

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The traditional methods reported in the literatures for AOH and other Alternaria mycotoxins

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analysis include thin-layer chromatography,22 gas chromatography (GC),23 liquid chromatography

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(LC),13,24,25 and high-performance liquid chromatography (HPLC).11,14,26-34 Among them, HPLC is


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the most commonly used method. Even though these chromatographic methods are specific, accurate,

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they are laborious, time-consuming, and need skilled staff due to a series of complex clean-up and

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pre-concentration steps are required prior to analysis. Besides, these methods are insufficiently

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sensitive to detect nanogram amounts of Alternaria mycotoxins.

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Recently, some emerging analytical techniques, such as stable isotope dilution assays (SIDAs),5-8

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polymerase chain reaction (PCR),27,33,35 enzyme-linked immunosorbent assay (ELISA),26 and

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molecularly imprinted polymer,36 offer alternative tools for the detection of Alternaria species in

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agricultural products, and could be used as an indirect marker of the presence of toxins. These novel

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techniques usually have the advantage of a high sensitivity, low limit of detection (LOD), and high

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selectivity. However, they also require complex preparation steps prior to analysis. For example,

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PCR method based on the internal transcribed spacer (ITS) genetic marker needs a complex DNA

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extraction and PCR amplification process before analysis.33 In using SIDAs for the determination of

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alternata mycotoxins, the deuterated mycotoxins are first synthesized by palladium catalyzed

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protium-deuterium exchange from the unlabeled toxins.6,7 While in ELISA, polyclonal or (and)

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monoclonal antibodies need to be prepared in advance, which is a complex process that takes a long

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time.26 In the process of molecularly imprinted polymer analysis, template selection and preparation

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is a necessary step, but also an extremely complex step.36

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Surface-enhanced Raman spectroscopy (SERS) is a powerful analytical technique that provides

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molecular-structural information of the target molecule adsorbed on the nanostructured metals. Due

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to the surface plasmon resonance in the visible electromagnetic range, metal nanoparticles, such as

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gold and silver, provide a considerable enhancement of the SERS signal from a molecule located in

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the close vicinity of nanoparticles surface.37 Recent studies showed that SERS based on novel

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nanoparticles substrates have increased in popularity as important tools in toxin detection.38-43

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Therefore, it is our intention to develop a SERS method for the detection of Alternaria mycotoxins,

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considering the advantages such as rapidness, high sensitivity and low cost that the technique can



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offer.

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To the best of our knowledge, this is the first report dealing with the determination of AOH by

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using SERS, and the aim of this study was thus to develop a simple and rapid SERS approach to

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detect AOH in pear fruit. An important issue in the development of the SERS approach is the low

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affinity of target molecules for metallic surfaces. Therefore, a new strategy, which could modify the

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silver nanoparticles (AgNps) by pyridine, was developed to avoid above limitation and improve the

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detection sensitivity in the current study. Finally, the proposed method was tested on the detection of

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AOH. In addition, the occurrence of AOH in pear fruit was analyzed by HPLC method to validate

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the feasibility of the SERS method to identify the samples contaminated with AOH.

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2. Materials and methods

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2.1. Chemicals and reagents

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Silver nitrate (99.9%), trisodium citrate (dihydrate, 98%), pyridine, sodium nitrate, sodium

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chloride, anhydrous sodium sulfate, acetonitrile, methanol, dichloromethane, ethyl acetate,

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methanoic acid, and acetic acid were purchased from Shanghai Aladdin Bio-Chem Technology Co.

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Ltd. (Shanghai, China). Both acetonitrile and methanol are of HPLC grade and the other chemicals

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are of analytical grade. AOH (>98.5%) was purchased from Sigma-Aldrich (Shanghai, China).

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Ultrapure water was prepared by a Milli-Q system (EMD Millipore Co., Billerica, MA, USA). The

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pear fruit were collected from local retail shops in Guangzhou, China.

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2.2. Silver nanoparticles synthesis and characterization

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AgNPs were synthesized with slight modification based on the method available.44 Briefly, 90.0

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mg of silver nitrate was dissolved in 500.0 mL of milli-Q water in a clean flask, the solution was

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stirred and heated to boiling point in a constant temperature heating magnetic whisk (DF-101S,

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Yuhua Instrument Co. Ltd., Yiwu, China). The solution was kept boiling and stirring, 5.0 mL solution


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of trisodium citrate (1%, m/v) was quickly added to the boiling solution. The resulting solution was

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continued to boil and stir for 1 h, and then cooled to 25oC.

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A UV-1800 spectrophotometer (Shimadzu Co., Kyoto, Japan) was applied to measure the visible

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adsorption spectra from 300 to 700 nm with a 1 nm interval. The newly synthesized AgNps were

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diluted five times and detected. The transmission electron microscope (TEM) images were obtained

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on a field-emission high-resolution JEM-1400Plus (JEOL Ltd., Tokyo, Japan) at an acceleration

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voltage of 120 kV. The samples for TEM analysis were prepared by dropping the diluted solution of

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freshly synthesized AgNPs on carbon film (T11023, Beijing Xinxing Braim Technology Co. Ltd,

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Beijing, China) and air dried at 60 oC. Dynamic light scattering (DLS) measurements were

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performed using a two angle particle and molecular size analyzer (Zetasizer Nano ZS, Malvern

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Instruments Ltd., Worcestershire, UK) at 25 °C under a scattering angle of 173° at λ = 633 nm.

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2.3. Standard preparation for SERS

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The AOH stock solution of 1.0×106 µg/L was prepared by diluting AOH standard with methanol

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and kept in darkness at -20 oC. A working solution (1.0×105 µg/L) was prepared by diluting the AOH

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stock solution with methanol. Standards for SERS detection were obtained by diluting working

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solutions with milli-Q water. A series of standards with the concentrations of 3.16, 10.0, 31.6, 100.0,

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and 316.0 µg/L were prepared.

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2.4. Detection of AOH in standard solution by SERS

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In order to provide a reference for the SERS spectra of AOH, 1.0 mL of 1.0×105 µg/L AOH

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standard solution was loaded into a cuvette, and then scanned by a Raman microscope (LabRAM HR,

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HORIBA Scientific, Longjumeau, France) with a 633 nm laser as the excitation source. The SERS

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measurement was carried out within the wavelength range of 300-1800 cm-1. Two accumulations

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were used, and the acquisition time was set to 30 s.



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Prior to the SERS measurement, 80.0 µL of aqueous pyridine solution (0.1 M) was added to 400.0

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µL of AgNps in the centrifuge tube and the mixed solution was stirred for 5 s, and then 400.0 µL of

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standard solution was dropped into the tube and the solution in the tube was mixed for 5 s. Next step,

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80.0 µL of sodium nitrate solution (1 M) was also added to the tube and mixed for 5 s to facilitate

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AgNps aggregation. Finally, the mixed solution was analyzed after these preparations. For SERS

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detection, the SERS spectrum was collected within the range of 300-1800 cm-1 using the Raman

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microscope with a 532 nm laser source at 50 mW laser power for excitation. Two accumulations

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were used, and the acquisition time was set to 30 s. Baseline correction and denoising were fulfilled

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for all measurements. SERS measurement was repeated 3 times for each concentration. The intensity

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value of the peak at 1252 cm-1 was plotted against the log concentration of AOH.

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2.5. Spiked samples determination by SERS

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The applicability and reliability of the proposed SERS method were evaluated by performing a

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recovery test using blank pear fruit. The fruit purchased were confirmed to be negative for AOH by

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China Entry-Exit Inspection and Quarantine Bureau (Guangzhou, China). AOH standard solution

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was injected into the blank pear fruit and the spiked fruit were stored at room temperature for 2 h

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before initiating the extraction process. The fortification levels in the recovery test were 20.0, 50.0,

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and 100.0 µg/kg, and five replicates were performed for each level. AOH extraction procedure was

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adapted based on previously published methods.26,32 Briefly, 5.0 g of sample was transferred into a

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50-mL centrifuge tube, and then 4.0 g of sodium chloride, 10.0 mL of water and 15.0 mL of

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acetonitrile (with 1% acetic acid) were added. The tube was first shaken in an oscillator for 10 min,

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then was centrifuged for 5 min at 5000 rpm. The upper organic solvent was transferred into a flask,

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while the underlying substance was re-extracted two times with the same solvent composition. All of

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the upper organic solvent portions were pooled and evaporated (vacuum-rotary evaporation, 40 oC)

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to nearly dry, then was concentrated to dryness using a nitrogen flow, and the residue was dissolved


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in 1.0 mL of methanol, and this solution was diluted by 9.0 mL of milli-Q water, the diluted solution

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was detected directly by SERS method as described above, without further purification. The recovery

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(%) was calculated as measured concentration divided by fortification level.

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2.6. SERS determination of AOH in real samples

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The validated SERS method was then used for determination the presence of the AOH and

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quantification its amount in the pear fruit. In total, 10 pear fruit (5 fresh fruit and 5 rotten fruit) were

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analyzed. All the samples were stored at 4 oC prior to analysis. Moreover, AOH production

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measurement was performed on the 10 pear fruit artificially inoculated with A. alternata at the

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concentration of 1.0×106 cfu/mL, which were kept at room temperature for 2 and 8 days.45 The

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extraction of AOH from the fruit samples was performed as per procedure described previously.

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2.7. Comparative analysis by HPLC

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2.7.1 Extraction and cleanup

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The extraction of AOH from the fruits was performed following the same procedure described for

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SERS analysis. The cleanup was conducted by using the extract solution by gravity onto an Oasis

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HLB 3 cc (60 mg) extraction cartridge (Waters Co., Milford, MA, USA), and the cartridge was first

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conditioned and equilibrated by 5.0 mL of methanol and milli-Q water, respectively, then washed

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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

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Supelclean LC-NH2 SPE Tubes (Supelco Inc., Bellefonte, PA, USA). After that, the tubes were

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conditioned with 5.0 mL of dichloromethane and the lower end of the HLB cartridge was connected

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to the tubes, this series was then washed with 5.0 mL of dichloromethane. Finally, AOH was eluted

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with 7.0 mL of 1% methanoic acid in methanol:ethyl acetate (1:1, v/v). The eluent was transferred

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into a flask and evaporated (vacuum-rotary evaporation, 40 oC) to nearly dry, which was then

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concentrated to dryness using a nitrogen flow, and the residue was reconstituted in 5.0 mL of



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acetonitrile:water (2:3, v/v). The reconstituted solution was filtrated through 0.22 µm PTFE filters

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(EMD Millipore Co., Bedford, MA, USA) and moved to the autosampler vials for the instrumental

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analysis by an Acquity® ArcTM HPLC system (Waters Co., Milford, MA, USA).

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2.7.2. Standard preparation

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A stock solution of 1.0×106 µg/L was prepared by diluting AOH standard with methanol and kept

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in darkness at -20 oC. A working solution (1.0×104 µg/L) was prepared by diluting the AOH stock

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solution with methanol. AOH standard solutions for HPLC calibration and recovery test were

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prepared by diluting the working solution with methanol. A series of standard solutions with the

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concentrations of 10.0, 50.0, 100.0, 200.0, and 400.0 µg/L were prepared.

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2.7.3 HPLC conditions

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All analyses were conducted using the HPLC system equipped with a 2475 FLR detector (Waters

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Co., Milford, MA, USA), which excites at wavelength of 339 nm and emits at wavelength of 404 nm.

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ChromQuest software (Version 4.2, ThermoQuest Italia S.p.A., Milano, Italy) was used to manage

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the HPLC data acquisition and processing. An XBridge® BEH-C18 (Waters Co., Milford, MA, USA)

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column (100 × 2.1 mm, 2.5 µm) was used as chromatographic column. The mobile phase consisted

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of two eluents (eluent A: water and eluent B: acetonitrile). A gradient program with a flow rate of

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0.204 mL/min was used, starting with 85% A and 15% B, reaching 70% B after 8.82 min and then

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maintained for 1.47 min. Afterward, the gradient was returned to 15% B in 0.3 min and allowed to

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equilibrate for 4.41 min. The column temperature was set to 35 oC and the injection volume was set

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to 10 µL. For quantitative analysis, an external calibration curve was used. AOH standard solutions

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with the concentrations of 10.0, 50.0, 100.0, 200.0, and 400.0 µg/L were used for construction of

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five-point calibration curves, and the peak areas versus concentrations were plotted.

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2.7.4 Recovery test

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Blank pear fruits, previously analyzed with negative result for the presence of AOH, were injected

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with working solutions to reach 20.0, 50.0, and 200.0 µg/kg of AOH. Spiked pear fruit were prepared

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and analyzed using the same procedure described for the pear fruit, i.e., extraction, cleanup and

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HPLC analysis. Recovery tests were based on quintuplicate spiking and triplicate analysis. The fruit

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samples detected by SERS previously were also analyzed by HPLC.

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3. Results and discussion

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3.1. Characterization of AgNPs

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AgNPs are widely used as SERS substrate because they are easier to synthesize, in addition,

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different batches of AgNPs are basically the same in shape, size, and size dispersion. As illustrated in

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the UV-visible spectra of AgNPs (Fig. 1a), the wavenumber of maximum absorption peak was

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414.6±0.9 nm and the full width at half-maximum was 116.1±0.1 nm, which suggesting that the

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shape and size of AgNPs were similar from batch to batch. Besides, the UV-visible spectra were

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overlapped, which indicated an excellent repeatability of AgNPs synthesis in various batches. DLS

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analysis was performed to measure the diameter of AgNPs and the result is shown in Fig. 1b,

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indicating that the average hydrodynamic diameter of the bare AgNPs was 48.9 nm, and the diameter

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increased to 49.7 nm after pyridine modification (Fig. 1c). The TEM images of the bare AgNPs and

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the modified AgNPs are shown in Fig. 1d, e and Fig. 1f. As shown in Fig. 1e, the diameters of bare

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AgNPs range from 26.8 nm to 63.3 nm. Compared with the bare AgNPs (Fig. 1e), the modified

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AgNPs had a silver core with a diameter of about 49 nm and about 2 nm thick shell (Fig. 1f) due to

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that the multilayer pyridine molecules were tightly adsorbed on nanostructured surfaces of sliver. The

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adsorption is favored by the heteroatom of nitrogen in the molecular structure.46,47 Pyridine is a

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functional Raman reporter, its molecular structure contains nitrogen atom, which cannot only interact

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with the AgNPs but can also conjugate with the AOH molecule, leading to the tight adsorption on the



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surface of AgNPs and as many as possible number of the AOH molecules adsorbed on AgNPs.40,48

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Therefore, the surface of AgNPs with a thin pyridine shell could circumvent the weak affinity of

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AOH on metallic surfaces and to improve the sensitivity of detection. Moreover, due to that the shell

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was thin enough, AOH molecules tightly adsorbed on the surface of AgNPs.

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3.2. SERS activity of AOH on pyridine modified AgNPs

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In order to confirm the SERS activity of pyridine modified AgNPs for the sensitive detection of

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AOH, the SERS spectra of various solutions were given and compared. As shown in Fig. 2, the

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SERS spectra of AOH added to the bare AgNPs (a) and to the modified AgNPs (b) without the

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addition of sodium nitrate did not elicit distinctive SERS bands, and their intensities were extremely

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low. Comparing Fig. 2b with Fig. 2c (the spectrum of the modified AgNPs), it can be seen that the

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addition of AOH did not introduce a new Raman peak, that is to say, the modified AgNPs without the

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addition of sodium nitrate cannot be used directly for the detection. The spectra of AOH added to the

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bare and modified AgNPs with adding sodium nitrate were also collected (Fig. 2d, e). Sodium nitrate

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was added to promote the AgNps close together, which could increase the signal of target.48

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Although an apparent spectral change could not be discerned after the AgNPs was modified by

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pyridine, some new peaks appeared, especially the ones at 1002 and 1033 cm-1 (in black dotted line),

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these two peaks also appeared in the spectrum of pyridine solution (Fig. 2f). Thus, they are likely

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assigned to pyridine ring breathing vibrational mode and in-plane deformation vibrational mode,

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respectively.40 These two peaks showed different intensities in Fig. 2e and Fig. 2g (the spectrum of

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the modified AgNPs with the addition of sodium nitrate), probably due to the different fluorescence

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backgrounds of these two solutions, which have different effects on the spectra. As shown in Fig. 2d,

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low SERS signal of AOH was obtained when detecting AOH with the bare AgNPs, while the

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modified AgNPs exhibited very strong enhancement effect (Fig. 2e). The reasons may be attributed

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to the weak affinity of AOH molecules on the surface of AgNPs. Compared to the Fig. 2g, the


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spectra of pyridine modified AgNPs showed very strong characteristic bands about 1173, 1252, 1298,

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1367, and 1615 cm-1 after the addition of AOH solution, which could be attributed to the vibration

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modes of AOH molecules by comparison with the spectrum of AOH solution (Fig. 2h). The band at

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1173 cm-1 is assigned to the β (C-H) ring.49 The intense peaks at 1298 cm-1 corresponds to the

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stretching C-H vibration of benzene ring, while the band at 1252 cm-1 is assigned to the vibration of

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O-H and C-H.43 The bands at 1486 cm-1 is assigned to the bending vibration of CH3, and it was

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enhanced and displays clear peaks in comparison with the bands in the spectrum of AOH added to

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the bare AgNPs with adding sodium nitrate.38,41 The bands at 1367 cm-1 is attributed to CH3

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symmetric bending vibrations.38 The band at 1615 cm-1 is related to the ring stretching mode of

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C-C.50 These results suggested that the sensitivity of determination was advanced through the

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modification of the AgNps surface by using pyridine. The improvement of the efficiency of the

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pyridine modified AgNPs to bind AOH is ascribed to the interaction of the aromatic rings of

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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

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the determination of phenols because the formation of hydrogen bonds between the nitrogen atoms

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of pyridine and the hydroxyl functions contained in phenol.51,52 These above results indicated that the

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characteristic fingerprint of band at 1252 cm-1 could be used to identify the presence of AOH using

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the pyridine modified AgNPs substrate.

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3.3. SERS detection of AOH standard solution

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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

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ranging from 3.16 to 316.0 µg/L. As shown in Fig. 3a, the locations and the intensities of the

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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



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SERS signal intensities of the fingerprint Raman bands of AOH was increased with the increase in

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the concentrations. This was because higher concentrations of AOH standard solutions caused

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increase in the amount of the AOH molecules conjugated on the AgNPs. For obtaining the

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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

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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.

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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.

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3.4. Linearity and LOD of HPLC analysis

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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

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method.


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3.5. Detection of AOH in pear fruit

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3.5.1 Recovery of AOH spiked in pear fruit

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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

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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%,

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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



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


Page 14 of 28

Page 15 of 28

350


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

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366

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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



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.


Page 23 of 28


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.



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.


Page 24 of 28

Page 25 of 28


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.



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.


Page 26 of 28

Page 27 of 28


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.



Graphic 146x113mm (220 x 220 DPI)


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