Journal Pre-proof

Sensitive and reliable detection of deoxynivalenol mycotoxin in

pig feed by surface enhanced Raman spectroscopy on silver
nanocubes@polydopamine substrate

Wodaje Addis Tegegne, Menbere Leul Mekonnen, Agaje Bedemo

Beyene, Wei-Nein Su, Bing-Joe Hwang

PII: S1386-1425(19)31331-9
DOI: https://doi.org/10.1016/j.saa.2019.117940
Reference: SAA 117940

Spectrochimica Acta Part A: Molecular and Biomolecular

To appear in:
Spectroscopy

Received date: 17 September 2019

Revised date: 8 December 2019
Accepted
8 December 2019
date:

Please cite this article as: W.A. Tegegne, M.L. Mekonnen, A.B. Beyene, et al., Sensitive
and reliable detection of deoxynivalenol mycotoxin in pig feed by surface enhanced
Raman spectroscopy on silver nanocubes@polydopamine substrate, Spectrochimica Acta
Part A: Molecular and Biomolecular Spectroscopy(2018), https://doi.org/10.1016/
j.saa.2019.117940

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Sensitive and reliable detection of deoxynivalenol mycotoxin in pig feed by surface

enhanced Raman spectroscopy on silver nanocubes@polydopamine substrate

Wodaje Addis Tegegne a, Menbere Leul Mekonnen b, Agaje Bedemo Beyene a, Wei-Nein Su a, *

and Bing-Joe Hwang b,c,

a
Graduate Institute of Applied Science and Technology, National Taiwan University of Science and

Technology, Taipei 10607, Taiwan

b

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Department of Chemical Engineering, National Taiwan University of Science and Technology Taipei

10607, Taiwan

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c
National Synchrotron Radiation Research Center, Hsinchu,30076, Taiwan

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*Corresponding author: Wei-Nien Su (wsu@mail.ntust.edu.tw )
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ABSTRACT
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Deoxynivalenol (DON) is one of the trichothecene mycotoxin, a frequent contaminant of pig

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feed. Surface-enhanced Raman spectroscopy (SERS) is a fast and ultrasensitive analytical tool
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for point-of-need applications to identify molecular fingerprint structures at low concentrations.

However, the use of SERS for analyte detection with flexible and robust structures is still
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challenging. Herein, we have developed core-shell silver nanocubes coated with polydopamine

(Ag NCs@PDA) SERS substrate for the quantitative detection of deoxynivalenol in pig feed.

The Ag NCs@PDA substrate with ultrathin (1.6 nm) PDA shell thickness enhances the absorption of

DON via hydrogen bonding and π–π stacking interactions, as well as improves the stability of the

substrate. The results of the SERS showed a high analytical enhancement factor (AEF) of 1.82x107

and a detection limit (LOD) as low as femtomolar range (0.82 fM). The LOD of the Ag NCs@PDA

substrate for DON detection is 1.8 times lower than the bare Ag NCs. Furthermore, the Ag

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NCs@PDA substrate is stable which retains 88.24 % of the original Raman intensity after storage

for three months. The obtained results demonstrate that the Ag NCs@PDA substrates can realize

label-free detection of deoxynivalenol mycotoxin with high sensitivity, reproducibility, and stability.

Our work proposes a low-cost method for the designing of the SERS sensing device, and has great

potential to be applied in food safety, biomedical sciences, and environmental monitoring.

Keywords: SERS, Silver nanocubes, Polydopamine, Deoxynivalenol, Pig feed.

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1. Introduction

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Deoxynivalenol (DON, also called vomitoxin) is a mycotoxin from the secondary metabolism

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of some species of the genus Fusarium graminearum and Fusarium culmorum. DON
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contamination occurs in cereals for instance oat, corn, rye, wheat, rice, and barley in the
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production place or during storage [1, 2]. Among the mycotoxins, DON is a major problem in

pig health and production [3]. The contact of DON in animal and human during intake of
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polluted food can cause acute and chronic effects such as vomiting, feed refusal, weight loss,

skin irritation, hemorrhage, diarrhea, gastroenteritis, neurotoxicity, embryotoxicity,

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teratogenicity, and immunosuppression [4-6]. Consequently, the food contamination by DON is

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a demanding issue for consumers in every country due to its numerous toxic effects. To ease

DON exposure from food, the European Union has set a maximum limit for DON concentration

which is 0.75 mg kg−1 for cereal products and 1.25 mg kg−1 for cereals. For the time being, the

DON regulation is a vital need of detection techniques with high selectivity and sensitivity [7].

So far, diverse analytical instruments have been employed for the detection of DON and other

mycotoxin in food. For example; gas chromatography-mass spectrometry (GC-MS) [8, 9], high-

performance liquid chromatography (HPLC) [10], high performance liquid chromatography-

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tandem mass spectrometry (HPLC-MS/MS) [11, 12], enzyme-linked immunosorbent assays

(ELISA) [13], and chemiluminescence enzyme immunoassays (CLEIA) [14]. Although these

techniques have a high selectivity and sensitivity, and attain good detection results for the

analysis of mycotoxins in food samples [15]. However, they are expensive, time-consuming,

complex sample pretreatment, and require skilled manpower, which became their bottleneck in

the actual applications [16]. Recently, researchers are focused on SERS for detecting mycotoxins

due to its tremendous sensitivity, non-invasive detection potential, and distinctive fingerprint

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identification of target molecules [17, 18]. Surface-enhanced Raman spectroscopy (SERS) is a

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fast and sensitive molecular vibrational analytical tool for the identification and quantification of

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analyte molecules as low as ultra-trace levels [19-21]. Nowadays, SERS has become a promising
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vibrational spectroscopic tool for the analysis of food safety [22], environmental monitoring

[23], and biomedical science [24]. Furthermore, SERS is a robust, nondestructive technique that
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the SERS signal can be enhanced up to 1012-1015 times as the analytes are located in the small
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gap between noble metal nanoparticles substrates. Such types of substrates are fabricated from

metals having free electrons (mainly Ag, Au, Cu, and Al), and core-shell nanostructures that
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generate high electromagnetic enhancement generally known as “hotspots [25-27]. There are two
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mechanisms of SERS enhancement: chemical enhancement (CE) and electromagnetic

enhancement (EC). Compared to the chemical enhancement, the electromagnetic enhancement

close to the metallic nanostructures is largely involved in SERS activity [28-30].

Nowadays, research interests are focusing on the fabrication of superior plasmonic

nanostructure-based SERS substrates having high hotspots density, which are able to produce

enhanced-SERS signal from the target molecules [31-34]. For example; Yang group reported

silver colloidal-based SERS sensor for DON detection in agricultural products and they reached

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LOD of 10−7 M [35]. Another study by Li et al. detected down to 10−8 M DON in maize sample

on 3D cauliflower SERS substrate [18]. To enhance the Raman signal, SERS substrates with

high reproducibility of hotspots, sensitivity and stability are usually used. Compared with other

spherical silver-nanoparticles, Ag NCs with sharp corner and edges show higher sensitivity and

are able to obtain a Raman enhancement factor (EF) as high as 10 10 [36]. Currently, a mussel-

inspired polydopamine polymer has paying attention as a result of its biocompatibility and

unique adhesive capability [37]. Polydopamine coating of substrates can be achieved through

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self-polymerization of dopamine in a weak basic solution. PDA coated substrate proves superior

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adsorption competence to the target analytes [38, 39].

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To the best of our knowledge, there are no literatures reports regarding DON detection using
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SERS in pig feed. In this study, we developed ultrasensitive SERS substrate using core-shell

silver nanocubes coated with polydopamine (Ag NCs@PDA) for label-free detection of DON in
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pig feed for the first time. The use of PDA is to maintain the stability of the substrate by
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protecting the Ag core from oxidation, and also to absorb the DON via π–π stacking interactions

between PDA structures and π electrons of DON molecules. Our SERS substrate detected ultra-
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trace concentrations of DON with a limit of detection as slow as femtomolar range (0.82 fM).
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2. Experimental section

2.1. Chemicals and materials

Silver trifluoroacetate (CF3COOAg: 98 %), ethylene glycol (EG: 99.8 %), sodium

hydrosulfide (NaHSx.H2O: ≥ 60 % NaHS), hydrochloric acid (HCl: 36-37 %), rhodamine 6G

(R6G: 99 %), dopamine hydrochloride (C8H11NO2: 99 %), tris(hydroxymethyl)aminomethane

hydrochloride (C4H11NO3: 99.5 %), deoxynivalenol (C15H20O6: 98 %), acetone (CH3COCH3: ≥

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99.5 %), ethanol (C2H5OH: ≥ 99.5 %) were purchased from Acros Organics. Poly (vinyl

pyrrolidinone) (PVP: MW ~55000) was purchased from Sigma-Aldrich. Deionized (DI) water

purified by Milli-Q system was utilized for all experimental procedures.

2.2. Characterization of Ag NCs @PDA

JASCO V-550 spectrophotometer over a wavelength range of 300-700 nm were used for the

measurement of the UV-Vis absorption spectra of the uncoated Ag NCs and polydopamine

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coated Ag NCs. The X-ray diffraction (XRD) pattern was measured on a Bruker D2 Phaser X-

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ray diffractometer with a Cu Kα radiation source (λ=1.5406 Å) operated at a scan rate of

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0.05o/step for 2 theta ranges from 30o to 90o. The morphologies of Ag NCs and Ag NCs@PDA

were characterized by JEOL, JSM-6500F Field Emission Scanning Electron Microscopy (FE-
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SEM) and Philips CM 300 High Resolution Transmission Electron Microscopy (HRTEM))
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operating at 300 kV. The elemental compositions of core-shell Ag NCs@PDA were analyzed
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with Energy Dispersive X-ray Spectroscopy (EDX, JSM-6500F). The FT-IR measurements were

performed using Digi lab FTS-3500 (Bio-Rad IR spectrometer) in the form of potassium
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bromide pellets.
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2.3. SERS spectrum measurement

The SERS spectra of the samples were analyzed by UniDRON Raman spectrometer with 633

nm laser excitation wavelength. A 50x objective was used to focus the laser beam to a spot size

of 1 µm and the working temperature of the charge coupled device (CCD) detector is −60 oC.

SERS analyses were measured with an exposure time of 2 s and number of accumulations 20

times by illuminating 3 mW laser power at the sample. The Raman peak of a silicon reference

standard at 520 cm−1 was used to calibrate the spectrometer. The SERS mapping was performed

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from 20 µm x 20 µm area with a step size of 2 µm, an exposure time of 2 s, and number of

accumulation 10 times.

2.4. Synthesis of Ag NCs

The silver nanocubes were synthesized using polyol method [40, 41], with some modification

using CF3COOAg as a source of silver and ethylene glycol (EG) as both a reducing agent and a

solvent for the preparation of all regents. In brief, in a 100 mL round bottom flask, 20 mL EG

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was heated at 152 oC in an oil bath under magnetic stirring for 50 min. After that, NaHSx.H2O

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(240 µL, 3 mM) was added quickly into the reaction flask and after 2 min, HCl (2 mL, 3 mM)

and PVP (5 mL, 20 mg/mL−1) solutions were injected respectively. After another 2 min,
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CF3COOAg (1.6 mL, 282 mM) was injected quickly to the reaction mixture. Except during the
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addition of reagents, the flask was capped with a glass stopper. After the addition of CF3COOAg,
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the transparent solution changed from whitish color to light yellow, reddish orange and then

green-ochre. After 30 min, the reaction flask was cooled in an ice-water bath then centrifuged at
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12000 rpm for 20 min and washed with acetone, ethanol, and water to remove excess reagents.
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The obtained Ag NCs were re-dispersed in 4 mL of deionized water.

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2.5. Synthesis of Ag NCs@PDA

The Ag NCs@PDA colloids were prepared through self-polymerization of dopamine in

alkaline solution. In brief, dopamine (0.01 mg/mL, 1 mL) dissolved in 10 mM Tris-HCl buffer

(pH=8.5) was added in to 2 mL of as-synthesized Ag NCs colloidal solution and stirred

magnetically at room temperature for 2 h. Then, the PDA coated Ag NCs were collected by

centrifugation at 12,000 rpm, washed with ethanol and deionized water. The synthesized Ag

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NCs@PDA was re-dispersed in 2 mL deionized water for further use. To control the thickness of

PDA shell, different concentration of dopamine (0.01, 0.02, 0.04, and 0.06 mg/mL) were added.

2.6. Preparation of standard solutions and SERS measurements

Standard solutions of R6G (10−3 M, 10−6 M, 10−8 M, and 10−9 M) and DON (10−3 M, 10−6 M,

10−9 M, 10−12 M, and 10−15 M) were prepared by dissolving in ethanol-water (1:1 v/v). For SERS

measurement, 60 µL of Ag NCs@PDA colloidal solution was dropped on a 6 mm x 6 mm

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Advantec® quantitative ashless filter paper. Then, 10 µL of each standard was added on to the

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Ag NCs@PDA substrate and dried at room temperature. SERS measurements were taken at five

random spots.
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2.7. SERS detection of deoxynivalenol (DON) in pig feed sample
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Pig feed (5 g) were grounded by mortar and were placed into a 50 mL centrifuge tube. Then,

10 mL of methanol-water (50 %, v/v) was pippetted into the pig feed samples and vortexed for 5
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minutes. The pig feed sample was sonicated for 30 min followed by centrifugation at 12000 rpm
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for 20 min. The supernatant was transferred into a clean centrifuge tube. 60 µL of Ag

NCs@PDA substrate were loaded on 6 mm x 6 mm Advantec® quantitative ashless filter paper

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by ultrasonication for 30 min. Finally, 10 µL of pig feed extract was spiked on the substrate,

then 10 µL of DON standard sample with varying concentrations (10−3 M, 10−6 M, 10−9 M, 10−12

M, and 10−15 M) were pipetted and dried at room temperature. SERS spectra of all the samples

were measured from 5 different points on the substrate.

3. Results and discussion

3.1. Characterization of Ag NCs and Ag NCs@PDA

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The morphology and size of Ag NCs@PDA and Ag NCs were characterized by HRTEM and

SEM. The HRTEM image (Fig. 1a) showed almost uniform and well-defined nanocubes. The

corresponding size distribution histogram revealed the synthesized silver nanocubes have an

average edge length of 50 nm (Fig. 1b) with average PDA shell thickness of 1.6 nm (Fig. 1c)

measured from twelve nanocubes of Fig. 1a by Gatan Microscopy Suite software. It is to note

that the high coating tendency of polydopamine simplifies the preparation process and makes it more

facile than coating with SiO2 [40-42]. Fig. S1a illustrates the SEM image of the Ag NCs on silicon

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wafer and Fig. S1b demonstrates the SEM image of the Ag NCs@PDA on filter paper. As can

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be seen from the SEM, the nanocubes were well assembled due to the fibrous structure of paper

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collects the nanoparticles in a better way than the silicon wafer. The EDX spectrum demonstrates
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the element composition of the synthesized Ag NCS@PDA substrate (Fig. S1c). The spectrum

presents the existence of C, N, O and Ag, indicating the successful synthesis of Ag NCS@PDA.
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The carbon, nitrogen, and oxygen peaks were attributed to PDA. The phase of the as-synthesized
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silver nanocubes was determined by X-ray diffractometer.

The X-ray diffraction pattern (Fig. 2a) of the Ag NCs confirms the formation of highly
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crystalline silver nanocubes. The five diffraction peaks of the 2θ values located at 38.1o, 44.2o,
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64.4o, 77.4o, and 81.9o which corresponding to (111), (200), (220), (311) and (222)

crystallographic planes respectively, which indicates that silver nanocubes are face-centered

cubic (fcc) structure from JCPDS (card. No. 04-0783). The absence of impurity peaks indicates

the high purity of the as-prepared silver nanocubes.

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(a) (b) (c)

(d) (e) (f) (g)

2.4 nm

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1.6 nm

3.3 nm

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Fig. 1. (a) HRTEM image of Ag NCs@PDA substrates; (b) histogram of corresponding particle
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size distribution; (c) Thickness distribution of the Ag NCs@PDA shell that corresponds to fig
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(a); and HRTEM images of Ag NCs@PDA with different concentrations of dopamine: (d) 0.01;

(e) 0.02; (f) 0.04; and (g) 0.06 mg mL−1.

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The localized surface plasmon resonance of noble metal nanostructures depend on their
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shape, size, and dielectric environment [43-46]. Fig. 2b demonstrates the UV-vis spectra of Ag
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NCs and Ag NCs@PDA. The maximum UV-vis absorption band at 452 nm is assigned to the

major LSPR peak of (100) planes of the face-centered cubic of silver metal and the additional

two shoulder peaks at 356 nm and 390 nm corresponds to the LSPR bands of the corners and

edges of silver nanocubes, respectively, which proves the successful synthesis of Ag NCs [47,

48]. After coating with polydopamine, the position of the plasmon resonance band of Ag

NCs@PDA was slightly red-shifted from 452 nm to 459 nm which is ascribed to the alteration in

the local refractive index of the contiguous medium of Ag NCs, as a result of polydopamine shell

creation [42, 49, 50]. Polydopamine showed a maximum UV-vis absorption peak at 285 nm
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(Fig.S2). Absence of PDA peak in the spectra of Ag NCs@PDA confirms the successful coating

of PDA on the surface of Ag NCs.

3.2. FT-IR spectra analysis

As can be seen in Fig. 2c, the FTIR spectra of the as-synthesized Ag NCs, PDA and Ag

NCs@PDA were recorded to confirm the chemical modification of the PDA on the surface of

silver nanocubes. No characteristic peaks were observed in the FTIR spectrum of silver

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nanocubes, demonstrating the successful synthesis of Ag NCs. Four characteristic absorption

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bands located at 3374, 1612, 1502, and 1284 cm−1 were detected in the FTIR spectra of PDA and

Ag NCs@PDA.
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(111) (a) (b)
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Intensity (a.u)

(200)
(220) (311)
Ag NCs (222)
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Ag (JCPDS)
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30 40 50 60 70 80 90
2 (degree)
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(c)

Fig. 2. (a) XRD pattern of Ag NCs; (b) UV-vis spectra of Ag NCs and Ag NCs@PDA; and (c)

FT-IR spectra of (a) Ag NCs@PDA; (b) PDA; and (c) Ag NCs.

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The absorption peak at 3374 cm−1 corresponds to the N−H and O−H stretching vibrations of

the PDA. Similarly, the band at 1612 cm−1 stand for N−H bending vibrations and C=C stretching

vibrations of the aromatic ring of indole or indoline structures in PDA while the absorption band

at 1502 cm−1 corresponds to the skeletal vibration of the PDA benzene ring. Moreover, the peak

at 1284 cm−1 represents C−N and C−O stretching vibrations in PDA molecules [51, 52]. The

result confirms the successful synthesis of core-shell Ag NCs@PDA substrate.

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3.3. Effect of PDA shell thickness

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It is known that SERS activity of core-shell substrate decreases as the thickness of the shell

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increases. We optimized the effect of PDA shell thickness on the SERS performance of the

substrate by adding different concentrations of polydopamine on the Ag NCs. Fig.1 (d-g) shows
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the HRTEM image of 1.6, 2.4, 3.3 and 4.6 nm PDA shell thickness that corresponds to 0.01,
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0.02, 0.04 and 0.06 mg/mL dopamine, respectively. The SERS activities of the synthesized

substrate were assessed using R6G as a probe molecule. Fig. 3a demonstrates the effect of PDA
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shell thickness on the SERS activity of the substrate. The SERS activity of the substrate
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decreases as the shell thickness of dopamine increases.

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3.4. SERS performance of the substrate

Fig. 3b illustrates the typical SERS spectra of various concentrations of R6G molecules using

Ag NCs@PDA (1.6 nm) substrate. The result reveals that R6G is detected down to 1 nM. The

SERS peaks of R6G at 612 cm−1 (Table S1) represents C–C–C ring in-plane bending vibration,

772 cm−1 can be correspond to C–H out of the plane bending, 1126 and 1184 cm−1 can be C–H

in-plane bending, 1364, 1512 and 1648 cm−1 are C–C stretching of the aromatic ring, while the

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peaks at 1312 and 1571 cm−1 ascribing to the N–H in plane bending vibrations [41, 53]. The

analytical enhancement factor (AEF) was determined using the following equation [54]:

AEF = (ISERS/CSERS)/(IRaman/CRaman)

where CRaman and CSERS represents the concentrations of R6G in normal Raman (0.1 M) and

SERS (1 µM) measurements, respectively (Fig. 3b and S3), while IRaman and ISERS are the normal

Raman and SERS intensities of the selected peak obtained on silicon wafer at 612 cm−1,

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respectively. Therefore, the average analytical enhancement factor of monodispersed Ag

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NCs@PDA substrate loaded on filter paper was found to be 1.82x10 7, which was one order of

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magnitude higher than bare Ag NCs (Fig. S4). This result indicates ultrathin PDA coated sliver

nanocubes can be applied as a sensitive SERS substrate. Interestingly, the SERS EF of Ag

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NCs@PDA loaded on filter paper is also higher than the corresponding silicon wafer. The
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increase in sensitivity is due to the improved assemblage of anisotropic Ag NCs@PDA to get

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more hotspots density in the case of filter paper. In the earlier study, a related trend with the

SERS intensity of Ag@SiO2 NCs and uncoated Ag NCs was found [53]. However, the AEF of
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Ag NCs@PDA achieved in this study is higher than the value reported in the case of Ag@SiO 2
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NCs [55], indicating the improved SERS enhancement of the Ag NCs@PDA. This high

sensitivity and enhancement factor can be attributed to the existence of chemical enhancement

between PDA and R6G as a result of high adsorption performance of polydopamine through π–π

stacking interactions and hydrogen bonding in addition to electromagnetic enhancement of the Ag

NCs.

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(a) (b)

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Fig. 3. (a) Effect of PDA shell thickness on the SERS activity; and (b) SERS spectra of different

concentrations of R6G.
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3.5. Quantitative SERS detection of DON
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To further reveal the SERS performance, the sensitivity of the substrate for the quantitative

analysis of deoxynivalenol was studied by analyzing the SERS data obtained from
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deoxynivalenol standard solutions. Fig. 4a depicts the typical SERS spectra of DON external
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standards obtained with the SERS signal intensity versus DON concentration. The spectra reveal
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the sensitivity of the substrate for DON quantification down to femtomolar level. The

characteristic SERS intensity at 786 cm−1 increase as the DON concentration increases. The

SERS intensity at 786 cm−1 corresponding to (O–H) + (C–H) bending vibration was considered

to demonstrate the calibration curve for the quantitative detection of DON. As can be seen from

Fig. 4b, a linear calibration curve obtained in the range of 1 fM to 1 mM with a coefficient of

determination (R2) of 0.9958 proves the suitability of the method in quantitative determinations.

The limit of detection (LOD) and limit of quantification (LOQ) of Ag NCs@PDA were

determined from the linear regression of the calibration curve [56], using the equations LOD = 3

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Sb/s and LOQ = 10 Sb/s, respectively, where Sb standard error and S is the slope of the calibration

curve [57]. Thus, the LOD and LOQ values were 0.82 fM and 2.71 fM, respectively. The limit of

detection obtained for the coated Ag NCs was better than the uncoated Ag NCs (LOD =1.46 fM)

determined from Fig. S5a and b. The increase in sensitivity is due to the π-π stacking

interactions between the aromatic polydopamine structures and the π-electron of DON

molecules, and the presence of hydrogen bonding [37, 39, 58].

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(a) (b)

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Fig. 4. (a) SERS spectra of DON standards using Ag NCs@PDA; and (b) Calibration curve of
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the SERS intensity versus the logarithm of DON concentrations. Error bar indicates standard

deviation from five measurements.

3.6. Reproducibility and stability of SERS substrate

High reproducibility is an important criterion for molecule detection in SERS sensor. To

assess the reproducibility of the prepared SERS substrate, SERS spectra of 5 µL of 1 µM DON

solution both substrate-to-substrate and spot-to-spot variation of the SERS intensity at 786 cm−1

band was analysed. Fig. 5a demonstrates the variation of different substrates, where SERS

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intensities of 1 µM DON at 786 cm−1 band was measured from seven different Ag NCs@PDA

substrates. The relative standard deviation (RSD) of the peak intensity was calculated as 12.59

%. Further, the uniformity or spot-to-spot variation on the same substrate was also confirmed.

The SERS intensities of 1 µM DON at 786 cm−1 band obtained from 10 randomly chosen spots

on the same substrate were shown in Fig. 5b. The RSD of the SERS intensities was 8.06 %,

indicating the repeatability of the method. This RSD value confirms that silver nanocubes

functionalized with ultrathin shell polydopamine utilized as a reproducible SERS substrate. The

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excellent reproducibility indicates the uniform distribution of hotspots in substrate. In addition,

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the SERS-signal uniformity of the substrate was demonstrated by SERS mapping (Fig. 5c) of 1

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nM DON, which was recorded from an area of 20 µm x 20 µm with a step of 2 µm. The RSD of
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the 400 spots were found to be 17.69 %, which also proves the reproducibility of the substrate.

The 2D point-by-point SERS mapping shows a uniform spatial distribution of SERS intensity.
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The homogenous distribution of the hotspot and SERS intensity proves the improved assembly
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of the substrate.

The SERS stability of Ag NCs and Ag NCs@PDA substrate was checked using 1µM DON
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over a period of three months. The substrate was stored at room temperature and measured every
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fifteen days interval (0, 15, 30, 45, 60, 75, and 90 days). Fig. 5d shows the plot of the percent

stability of Ag NCs and Ag NCs@PDA substrate versus different detection time. The SERS

intensity of bare Ag NCs dropped significantly in air, declining 45.37 % after three months of

storage, while for the Ag NCs@PDA substrate 88.24 % of the initial SERS intensity could be

maintained after 90 days, indicating good stability. The high stability of the AgNCs@PDA

substrate may be attributed to the fact that polydopamine coating protected the silver nanocubes

from oxidation, which hinders the entrance of oxygen in to the silver nanocubes [59, 60]. The as-

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synthesized Ag NCs@PDA substrates demonstrated excellent reproducibility and high stability

to be used for realistic applications.

1000 a.u RSD = 12.59% (a) 1000 a.u RSD = 8.06% (b)

Intensity (a.u)
Intensity (a.u)

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1 2 3 4 5 6 7 A B C D E F G H I J
Substrates Spots

(c) -p (d)
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Fig. 5. The reproducibility of Ag NCs@PDA using 1 μM DON; (a) substrate-to-substrate (The

error bar represents standard deviation, N=5); (b) Between spots within the same substrate; (c)

Raman mapping of 1 nM DON; and (d) Stability of the SERS substrates using 1 μM DON.

3.7. Detection of DON in pig feed

The performance of the SERS substrate for practical applications was assessed. These SERS

substrates were applied to DON detection in pig feed. Fig. 6(a) presents the corresponding SERS

spectra of DON spiked-pig feed at different concentrations (10−3, 10−6, 10−9, 10−12, and 10−15 M),

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From the DON spectra, the SERS signal of DON at 786 cm−1 were detected down 10−15 M, which

is much lower than the lowest detection limit 10−7 M reported for Ag colloidal substrate [35], and

8.4x10−8 M using 3D-Nanocauliflower substrate reported by [18]. All the characteristics peaks

were obtained in the SERS spectrum of 10−15 M DON (Table S2), signifying the high detection

sensitivity of Ag NCs@PDA substrate. This result confirmed that the SERS substrate based on

ultrathin PDA coating of silver nanocubes can be applied to the ultrasensitive detection of

deoxynivalenol in pig feed down to femtomolar level (as low as 1fM).

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The SERS-activity of the SERS substrate was evaluated by comparing our detection limit

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with other reported values. From Table 1, it is clearly seen that our SERS substrate achieves a

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lower limit of detection than the reported LOD values, which proves its potential use in the
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ultrasensitive detection of DON. As seen in the examples of R6G dye and DON mycotoxin, Ag

NCs coated with thin polydopamine have demonstrated noticeably enhanced sensitivity, LOD,
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stability as well as high enhancement factor. In addition to the electromagnetic enhancement of

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common SERS substrates, we believe that polydopamine coating has strong affinity towards

DON and it results in high adsorption of these molecules. The π–π stacking interaction and
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hydrogen bonding between PDA and DON produce ultrahigh sensitivity as the consequence of the
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presence of chemical enhancement. Finally, this work shows an interesting approach to enhance the

performance of Ag SERS substrate with a thin organic coating which has been characterized with

details. Unlike inorganic coating, taking advantages of electronic and orbital coupling between

organic coating and analyte molecules in various π–π stacking systems, as well as the resultant

chemical enhancement effect, opens a new route in developing sensitive and reliable SERS-based

analysis.

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(a) (b)

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Fig. 6. (a) SERS spectra of DON in pig feed using Ag NCs@PDA substrate; and (b) Schematic

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illustration of π–π stacking interaction between PDA and DON molecules.
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Table. Comparison of LOD values of our SERS substrate with literature values in
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DON analysis.

Substrate LOD (M) Reference

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3D-Nanocauliflower
8.4x10−8 [18]
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(PDMS@AAO@AuNPs)
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Ag NPs 1x10−7 [35]

Ag NCs@PDA 0.82x10−15 This work

4. Conclusion

In summary, core-shell silver nanocubes coated with ultrathin polydopamine were synthesized

as a sensitive and rapid method to detect deoxynivalenol in pig feed. Ultrathin coating of Ag

NCs with polydopamine improves adsorption ability of deoxynivalenol. The Ag NCs@PDA

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substrate exhibited a high enhancement factor (1.82x10 7) and a very lower limit of detection

which is 0.82 fM (0.243 pg L−1). The enhanced SERS performance could be attributed to the

fibrous structure of the filter paper that improves the uniformity of the substrate, the π-π stacking

interactions between the aromatic PDA structures and the π-electrons of DON molecules, as well

as the presence of hydrogen bonding. Moreover, the as-synthesized substrates show promising

potential in practical applications with excellent reproducibility and high stability. This Ag

NCs@PDA substrate demonstrates exceptional qualities for label free and rapid detection of

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other mycotoxins in food and feed samples.

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Competing interests statement

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The authors have no conflicts of interests to declare.
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Acknowledgement
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The financially supports from the Ministry of Science and Technology (MoST) (MOST 108-
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3116-F-011-001 -CC1, 107-2923-E-011 -002, 107-2119-M-002-033-, 106-2923-E 011-005, 105-

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3113-E-011-001, 105-ET-E-011-004-ET, 104-2923-M-011-002-MY3, 104-2911-1-011- 505-

MY2, 103-2221-E-011-156-MY3), the U2RSC program (MOE 1080059), the Applied Research
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Center for Thin-Film Metallic Glass from the Featured Areas Research Center Program within

the framework of the Higher Education Sprout Project by the Ministry of Education of Taiwan,

Taiwan’s Deep Decarbonization Pathways toward a Sustainable Society Project (AS-KPQ-106-

DDPP) from Academia Sinica as well as the facilities of support from National Taiwan

University of Science and Technology (NTUST) and National Synchrotron Radiation Research

Centre (NSRRC) are also acknowledged.

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Wodaje Addis Tegegne is a PhD student in the Graduate Institute of Applied Science and
Technology at National Taiwan University of Science and Technology, Taiwan. He received his
Bachelor degree in Chemistry from Ambo University, Ethiopia in 2009 and joined Ambo
University, Department of Chemistry as a graduate assistant. He received master’s degree in
Chemistry (Analytical) from Ambo University, Ethiopia in 2014. Then, he worked as lecturer of
Analytical chemistry in Ambo University till 2017. Currently, he is conducting his PhD under
Prof. Wei-Nien Su and Prof. Bing-Joe Hwang, focusing on the Development of Surface Enhanced
Raman Spectroscopy Methods for the Detection of Food and Feed Toxins.

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Menbere Leul Mekonnen received his PhD in Chemical Engineering from National Taiwan

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University of Science and Technology under the supervision of Professor Bing-Joe Hwang. He is
mainly interested in Surface Enhanced Raman Spectroscopy (SERS) and Nanoplasmonics in

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general. Dr. Menbere is currently Assistant Professor of Analytical Chemistry at Addis Ababa
Science and Technology University in Ethiopia.
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Agaje Bedemo Beyene is a PhD student in the Graduate Institute of Applied Science and
Technology, at National Taiwan University of Science and Technology, Taiwan. He received his
Bachelor in Chemistry (2004) and master’s degree in Analytical Chemistry (2007) both from
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Addis Ababa University. Upon completing his undergraduate study, he was offered a tenure
track position and served the Chemistry Department at Addis Ababa University as graduate
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assistant (2004-2005), assistant lecturer (2005-2007) and lecturer (2007- ). Currently, he is

conducting his PhD under Prof. Wei-Nien Su and Professor Hsieh-Chih Tsai, focusing on SERS-
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based immunoassay for early detection of cancer.

Wei-Nien Su is currently an associate professor of the Graduate Institute of Applied Science and
Technology at National Taiwan University of Science and Technology, Taiwan. His research
interests are rooted in materials synthesis and characterization of nanocatalysts for various
electrochemical devices and energy applications. Prof. Su is currently involved in various
international and national research programs. He received his PhD from the Wolfson School,
Loughborough University (UK) and his Diplom-Ing. in Chemical Engineering from Universität
Stuttgart (Germany).

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Bing-Joe Hwang studied Chemical Engineering and received his PhD in 1987 at the National
Cheng Kung University, Taiwan. Since 2006, he has served as a chair professor at the National
Taiwan University of Science and Technology. His research work has spanned a wide range of
subjects, from electrochemistry to spectroscopy, interfacial phenomena, materials science and
theoretical chemistry. He has established both experimental and computational strategies for
the development of new materials and for understanding interfacial phenomena. His work has
led to a better understanding of electrochemical reaction mechanisms and to an improved
ability to predict the properties of potential new materials for batteries, fuel cells and
biosensors.

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Author contributions

Wodaje Addis Tegegne prepared samples, carried out experiments, and drafted the manuscript.

Menbere Leul Mekonnen and Agaje Bedemo Beyene assisted in performing experiments,

characterization, and data analysis. Bing-Joe Hwang and Wei-Nien Su conceived the research

concept, discussed and reviewed the results, data analysis, and manuscript preparation.

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Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:

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Graphical abstract

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Highlights

 Ag NCs@PDA SERS substrate detects deoxynivalenol (DON) ranging from 10−3 to 10−15 M.
 Thin PDA shell enhances the absorption of DON through π–π stacking interactions.
 Core-shell Ag nanocubes -PDA and filter paper allow flexible and robust detection.
 The SERS enhancement factor was estimated as 1.82x107.
 The limit of detection down to femtomolar range (0.82x10−15 M).

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