bacterial nanocellulose paper composites for paste-and-read SERS detection of pesticides on fruit surfaces

Journal Pre-proof Silver nanoparticle/bacterial nanocellulose paper composites for paste-and-read SERS detection of pesticides on fruit surfaces Attasith Parnsubsakul, Umphan Ngoensawat, Tuksadon Wutikhun, Thanyada Sukmanee, Chaweewan Sapcharoenkun, Prompong Pienpinijtham, Sanong Ekgasit

PII:

S0144-8617(20)30130-2

DOI:

https://doi.org/10.1016/j.carbpol.2020.115956

Reference:

CARP 115956

To appear in:

Carbohydrate Polymers

Received Date:

10 November 2019

Revised Date:

1 January 2020

Accepted Date:

3 February 2020

Please cite this article as: Parnsubsakul A, Ngoensawat U, Wutikhun T, Sukmanee T, Sapcharoenkun C, Pienpinijtham P, Ekgasit S, Silver nanoparticle/bacterial nanocellulose paper composites for paste-and-read SERS detection of pesticides on fruit surfaces, Carbohydrate Polymers (2020), doi: https://doi.org/10.1016/j.carbpol.2020.115956

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Silver nanoparticle/bacterial nanocellulose paper composites for paste-andread SERS detection of pesticides on fruit surfaces

Attasith Parnsubsakul1,2, Umphan Ngoensawat1,2, Tuksadon Wutikhun3, Thanyada Sukmanee1,2, Chaweewan Sapcharoenkun3, Prompong Pienpinijtham1,2, Sanong Ekgasit1,2* 1

Sensor Research Unit (SRU), Department of Chemistry, Faculty of Science, Chulalongkorn University, 254 Phaya Thai Road, Pathum Wan, Bangkok 10330, Thailand 2

Research Network NANOTEC-CU on Advanced Structural and Functional Nanomaterials, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand 3

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National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani 12120, Thailand * Corresponding author, E-mail: [email protected]

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Graphical abstract

Highlights 

AgNP-BNC paper was fabricated as an eco-friendly disposable, flexible SERS substrate.

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The prepared AgNP-BNCPs have high SERS activity, good reproducibility and stability.

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BNC exhibits potential as an alternative substrate to plastics for SERS applications.

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

SERS active AgNP-BNC paper achieves paste-and-read detection of pesticides on fruits.

ABSTRACT This study aimed to develop an eco-friendly flexible surface-enhanced Raman scattering (SERS) substrate for in-situ detection of pesticides using biodegradable bacterial nanocellulose (BNC). Plasmonic silver nanoparticle- bacterial nanocellulose paper (AgNP-BNCP) composites were prepared by vacuum-assisted filtration. After loading AgNPs into BNC hydrogel, AgNPs were

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trapped firmly in the network of nanofibrous BNCP upon ambient drying process, resulting in 3D SERS hotspots within a few-micron depth on the substrate. The fabricated AgNP-BNCPs exhibited high SERS activity with good reproducibility and stability as demonstrated by the detection of 4-

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aminothiophenol and methomyl pesticide. Due to the optical transparency of BNCP, a direct and rapid detection of methomyl on fruit peels using AgNP-BNCPs can be achieved, demonstrating a simple

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and effective ‘paste-and-read’ SERS approach. These results demonstrate potential of AgNP-BNCP composites for user-friendly in-situ SERS analysis.

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Keywords: bacterial nanocellulose; silver nanoparticles; surface enhanced Raman scattering; flexible

Introduction

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substrate; methomyl; in situ detection

In the past decade, surface-enhanced Raman scattering (SERS) has been extensively exploited for development of highly sensitive detection of pesticides, drugs, explosives, etc., due to its capability of trace-level detection of small-molecules (Liszewska et al., 2019). SERS assay is also promising as a simple, non-destructive, and pretreatment-free methods, thereby, being preferred in rapid field screening. Hence, great effort has been made to fabricate novel and efficient SERS active substrates

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for several applications such as environmental monitoring and safety assessment of foods and agricultural products (Shi, Liu, & Ying, 2018). For producing high-performance SERS active substrates, general strategies mostly rely on either assembly or growth of plasmonic metal nanostructures such as Ag and Au nanoparticles on substrates (Liszewska et al., 2019). Under light excitation, the metallic NPs create surface plasmon polariton resonance, which functions as electromagnetic “hotspots” and provide drastic enhancement of Raman signals from adsorbed analytes in these sites (Ding et al., 2016). This phenomenon, therefore, allows those plasmonic

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nanocomposite materials function as SERS sensors. In real applications, analytes are commonly found on rough, curved, or non-planar surfaces. By using conventional SERS substrates as colloids or rigid substrates, the analytical procedures require additional extraction processes with appropriate solvents as these substrates are more compatible with

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samples in a liquid form (Shi et al., 2018). Thus, current researches have attempted to develop SERS substrates that allow rapid analysis and user-friendly. Emerging plasmonic nanostructure-decorated

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flexible templates, including petroleum-based polymers such as polyethylene terephthalate (PET)

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(Zuo et al., 2016), polyethylene (PE) (Zhou et al., 2016), polyvinyl chloride (PVC) (Zhong et al., 2018) have distinguished their applicability for in situ detection. Exploiting transparency of plastics, Raman signals of analytes can be detected by directing the incident excitation laser on the SERS

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substrates that were pasted over the examine sites. Hence, the sample collection, preconcentration, and transfer steps can be skipped for this ‘paste-and-read’ SERS approach. As a current trend, the

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growing attentions have been given toward the wearable sensor developments for point-of-care diagnostics (Xu, Lu, & Takei, 2019). For instance, a recent research showcased a cutting-edge

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application of integrating the SERS approach onto a contact lens for real-time monitoring of glucose level in human tears (Jeong et al., 2016). However, those current SERS substrates were mostly based on oxo-degradable plastics, which simply fragment into microplastics after disposed and ended up in landfills, leading to considerable concern about consequent environmental impacts. Since the microplastics are extremely persistent in the environment (e.g., soils, marines, water sources) and cause harm for organisms (Kubowicz & Booth, 2017). Accordingly, in very recent, WHO has begun

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to assess the potential human health risks associated with exposure to microplastics in the environment through drinking-water (World Health Organization, 2019). These issues motivated us to develop an eco-friendly flexible SERS substrate for rapid and efficient analysis of trace chemicals on real sample surfaces. Bacterial nanocellulose (BNC), a natural cellulose synthesized by bacteria, composed of a continuous 3D network of cellulose nanofibers (CNFs) with a width less than 100 nm providing high flexibility, good mechanical property, high purity, and low production cost (Hu, Chen, Yang, Li, & Wang, 2014).

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Recently, BNC-plasmonic nanocomposites, such as AgNP-BNC (Marques, Nogueira, Pinto, Neto, & Trindade, 2008), and AuNP-BNC (Park, Chang, Jeong, & Hyun, 2013), have exhibited excellent performance to be used as an effective SERS platform. Compared to most commonly used cellulose microfibers (e.g., filter paper), the properties of CNFs provide higher NP loading, improved hot-spot

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uniformity, and higher surface area-to-volume ratio (Xiong, Lin, Lin, & Huang, 2018). The plasmonic nanocomposites were capable of sensitive detection of pesticides on contaminated fruit surfaces

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through the extraction process (Liou, Nayigiziki, Kong, Mustapha, & Lin, 2017). Toward more rapid

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detection, BNC-plasmonic nanocomposites also has been developed to fabricate flexible nanopaper SERS substrates for directly swabbing various contaminants on surfaces to the hot-spot of the SERS substrates (Tian et al., 2016; Wei, Rodriguez, Renneckar, Leng, & Vikesland, 2015). However,

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preparation of the plasmonic nanocomposites for ‘paste-and-read’ rapid detection, similar to those aforementioned transparent polymer-based SERS substrates, has not yet been mentioned. Although

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BNC paper (BNCP) substrates have a relatively good optical transparency (Yano et al., 2005). We, therefore, anticipate that facile in-situ SERS analysis on non-planar surfaces without the additional

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processes using green material-based BNC nanocomposites could be achieved. Herein, BNCP was utilized for fabricating a low-cost, environmentally friendly, high-performance, flexible SERS substrate by incorporating AgNPs using a simple vacuum-assisted filtration process. This AgNP-BNCP, which owes a 3D plasmonic hotspot on highly porous structure, provides an excellent SERS activity for detection of small molecules. In addition, our study showed that optical property of the substrate is advantageous for applying paste-and-read SERS method on surfaces.

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Lastly, the rapid in-situ detection of methomyl – an example of pesticide – on fruit was therefore demonstrated a practical use of the fabricated SERS substrate. 2. Experimental 2.1 Chemical and materials Tri-sodium citrate dehydrate, rhodamine 6G, and 4-aminothiophenol (4-ATP) were purchased from Sigma-Aldrich. Silver nitrate (purity  99.8%) was purchased from Merck (Thailand). Methomyl was purchased from Dr.Ehrenstorfer GmbH (Germany). Ethanol was used to dissolve 4-ATP while the

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other chemicals were dissolved in deionized (DI) water. All chemicals were used as received without further purification. 2.2 Bacterial nanocellulose (BNC) preparation

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BNC hydrogel was prepared by a procedure described elsewhere (Napavichayanun, Ampawong, Harnsilpong, Angspatt, & Aramwit, 2018). Briefly, Acetobacter xylinum ATCC 23769 (Kasetsart

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University, Thailand) was grown in a static coconut water medium under pH 4.5 supplemented with

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5% (w/v) sucrose, 1% (v/v) acetic acid, 0.5% (w/v) ammonium phosphate at 30°C for ~10 days. A film of BNC hydrogel formed on the surface of the growth medium. To purify the film, the grown

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film was washed by 2% NaOH aqueous solution at 70°C until a neutral pH was obtained. 2.3 Synthesis of citrate-stabilized silver nanoparticles (AgNPs)

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A greyish-yellow colloid of AgNPs were prepared via a citrate-reduction method (Sukmanee, Wongravee, Ekgasit, Thammacharoen, & Pienpinijtham, 2017). Five hundred milliliters of silver

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nitrate aqueous solution (0.018%) was boiled in a 500 mL flask. Then, 10 mL of trisodium citrate solution (1%) was rapidly added into the boiling silver nitrate solution. The colorless mixture turned yellow immediately, indicating the formation of AgNPs. The mixture was vigorously stirred and kept boiling for an hour. The resulting colloid was cooled to room temperature prior to use. The obtained AgNPs were characterized by UV-visible spectroscopy and the optical density at λmax (430 nm) was ~0.64 after a 10x dilution.

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2.4 Fabrication of AgNP-BNCPs The moist BNC films with thickness of 280 m were cut into circular shape with 7.5 cm in diameter, then placed on filter paper (Johnson Grade 304) positioned on a Buchner filter funnel (75 mm diameter). AgNPs were loaded into the BNC film by vacuum-filtration. The as-prepared AgNP colloid with 30 mL, unless otherwise stated, was filtered through the BNC film and the filtrate was recycled repeatedly until appeared colorless. The AgNP-BNC composited films were allowed to dry in a dark place at ambient conditions for a day. Finally, the obtained AgNP-BNCPs were rinsed

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several times with DI water and ethanol, and vacuum-dried again before stored at 4 °C for further experiments. The AgNP density in the SERS active paper can be tuned by varying volume of the filtered AgNPs colloid.

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2.5 SERS measurement

A DXR Raman microscope (Thermo Fisher Scientific) equipped with a 780 nm excitation laser was

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employed using a laser power of 14 mW. The samples were measured through a 10X (NA = 0.25) objective lens with a 25 μm pinhole aperture and a laser spot of 3.1 m. All spectra were presented

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without any spectral correction. A typical procedure for SERS evaluation was as follows: AgNPBNCP was cut to 0.8 × 0.6 cm2 and then soaked in 4-ATP (in ethanol) or methomyl (in DI water)

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solutions of varying concentrations for 24 or 1 h, respectively. SERS measurements were typically performed after a complete evaporation of solvent on the ‘top-side-up’ AgNP-BNCP substrate. To

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demonstrate the paste-and-read SERS method on fruit peels, a solution of 10-6 M methomyl was dropcasted on surfaces of orange and apple. For the detection, 10 µL water was applied onto the

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contaminated fruit peels for helping the analyte extraction. Then, the AgNP-BNCP was pasted over the fruit peels with ‘bottom-side-up’ and subsequently SERS signal was collected. Designation of sides on AgNP-BNCP substrate for SERS measurement was graphically illustrated in Fig. 1. 3. Results and discussion 3.1 Fabrication and optimization of AgNP-BNCPs for high SERS performance

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In this work, AgNPs were employed as a plasmonic nanostructure as it provides stronger Raman enhancement compared to other metal nanoparticles such as AuNPs. As shown in Fig. S1A, a TEM image shows that AgNPs have quasi-spherical shape with an average diameter of 66  27 nm, which is within the optimal size for SERS applications (Stamplecoskie, Scaiano, Tiwari, & Anis, 2011). UVvisible spectrum exhibits absorption maxima at 430 nm (Fig. S1B) representing surface plasmon resonance of AgNPs, which corresponds to the particle size of AgNPs from TEM image (Wan et al., 2013). Fig. 1 schematically illustrated the fabrication process for flexible SERS active AgNP-BNCPs. Owing to the dense 3D network porous structure of BNC, AgNPs were substantially detained within

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the hydrogel film when the AgNPs colloid flowed through the film under a vacuum-filtration. By repeating the filtration cycles, all of AgNPs in the feed colloid could be transferred into the BNC film as indicated by colorlessness of the final filtrate solution. Upon drying under ambient condition,

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AgNPs adsorbed with well-spatial distribution within the BNC nanoporous matrix. Finally, a flexible AgNP-BNCPs for SERS substrate were obtained. It should be noted that the employed fabrication

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process is privilege for BNC compared to filter paper. As it was obviously observable that the filter paper of which was inserted beneath the BNC film, in order to aid spreading of vacuum-pressure in

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the filtration process, appeared no signs of AgNP deposition, as revealed by SEM image (Fig. S2). This is due to relatively large pore size of filter paper (several micrometers), which leads to an

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absolute penetration of the AgNPs. Whereas the pore size of BNCP is in the sub-100 nm range.

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Fig. 1. Schematic illustration of fabrication process for SERS active AgNP-BNCPs via vacuum-

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filtration.

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To optimize fabrication condition for SERS performance, different volumes of AgNP-feed colloids were employed. Amount of AgNPs loaded into the BNCPs increased with an increase in feed volume

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from 10 to 35 mL, as indicated by an increment of the AgNP plasmon absorbance (Fig. 2A). To evaluate their SERS activity, those AgNP-BNCPs were probed using chemisorption of 4-ATP. Fig.

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2B shows SERS spectra of 4-ATP (0.1 mM) absorbed on AgNP-BNCPs under different AgNP loadings. Characteristic Raman peaks of 4-ATP can clearly be detected at 1081 cm-1 (aromatic C–S stretching) and 1583 cm-1 (aromatic C–C stretching) (Chen et al., 2017). As observed in Fig. 2C, the

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intensity of Raman peaks increased with an increase in volumes of feed colloids and reached maximal

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value when the applied volume was 30 mL. Therefore, AgNP-BNCP prepared by feeding 30 mL of AgNP colloid (AgBNCP-30) was selected for further experiments. The increasing SERS enhancement can be attributed to an increase in interparticle plasmonic coupling between closely packed (and/or aggregated) AgNPs (Fraire, Pérez, & Coronado, 2013) on BNCPs as suggested by a consecutive red shift in plasmon resonance wavelength together with a broadening of the LSPR band (Fig. 2A). This explanation was also supported by SEM images of AgNP-BNCP in Fig. 3A and Fig. S3A that the AgNP aggregation can be obviously seen.

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Fig. 2. Optimization of AgNP loading densities for SERS performance. (A) UV-visible spectra of AgNP-BNCP with different volumes of AgNP feed colloids. (B) SERS spectra of 4-ATP (0.1 mM)

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absorbed on AgNP-BNCPs prepared with different volumes of AgNP feed colloids. (C) SERS intensity of 4-ATP (0.1 mM) at 1081 and 1583 cm-1.

AgBNCP-30 was subjected to morphology investigation as representatives of AgNP-BNCPs prepared

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by vacuum-filtration process. A series of SEM images taken from different perspectives, as shown in Fig. 3A-C, revealing localization of the trapped AgNPs on the top few layers of the substrate, which

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corresponded very well with the result of EDX scanning on the entire thickness of the substrate (as observed as green dots in Fig. 3B3). Upon drying process, the thickness of the AgNP-loaded BNC

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hydrogel substantially shrank to ~14 m (about 20-fold) due to the formation of hydrogen bonding among nanofibers. This spatial deformation of the plasmonic BNC composites basically gives rise to

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the increase of SERS hotspot density (Park et al., 2013). For our substrate, it can be ascribed to the induced aggregation of AgNPs as appeared not only at the surface layer (Fig. 3A) but also within the

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subsurface, which was evidenced by backscatter SEM image with EDX mapping (Fig. S3). In a previous work that used a similar fabrication method (Tian et al., 2016), the localization and

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irreversible adhesion of Au nanorods on BNC substrate occurred upon filtration and drying processes. In addition, AgNP-loading density of AgBNCP-30 was also determined as ~54.8 mg/cm3 (See Table S1 for all samples). The estimated content of AgNPs loaded on BNCPs was ~3.4 mg (calculated based on the stoichiometric conversion of 0.018% AgNO3 to the 30 mL feed-volume) and the volume of an as-prepared AgBNCP-30 film was 0.062 cm3 (= r2h =   (7.5 cm/2)2  14 m). This study

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therefore emphasize the use of BNC hydrogel as a potent substrate for loading and trapping of

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plasmonic NPs, thus generating a large number of 3D hotspots for SERS detection.

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Fig. 3. Morphological characterization of AgBNCP-30. SEM images of AgBNCP-30 on (A) top-, (B) cross-section-, and (C) bottom- views. (B1) – (B2) Zoom-in images at the near- top and near-bottom

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of the substrate, respectively. (B3) Cross-sectional image with full thickness of AgBNCP-30 overlaid

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with Ag EDX map (green dot distribution).

AgBNCP-30 substrate were further evaluated to assess the SERS performance. By varying different concentrations of 4-ATP from 10-3 to 10-10 M, the intensities of the characteristic peaks from 4-ATP

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molecules decreased with a decrease in 4-ATP concentrations (Fig. 4). Both the characteristic peaks at

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1081 and 1583 cm-1 can be differentiable down to a concentration of 10-9 M, indicating an excellent SERS enhancement of the substrate. We also tested SERS reproducibility for the AgBNCP-30 substrate. SERS spectra of 4-ATP acquired from 20 random positions on the substrate were collected (Fig. 5A), and quantitatively presented the spot-to-spot intensity of the characteristic peak within 10% relative standard deviation (RSD), shown in Fig. 5C. The same piece of the AgBNCP-30 substrate was subsequently flipped to turn the ‘bottom-side-up’ and performed SERS measurement on 20 random positions again. The obtained SERS spectra, as shown in Fig. 5B, exhibited a good

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consistency with those signals previously measured under ‘top-side-up’. The average intensity and %RSD from both sides were comparable (Fig. 5D). These results together demonstrate the uniformity and reproducibility of the SERS substrate. Moreover, Fig. S4 displayed SERS spectra from 4-ATP molecules, which recorded from AgBNCP-30 substrate after storage for 3 months, showing the stable sensitivity comparable to that of the freshly prepared one. These results demonstrate the SERS

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performance of the AgNP-BNCP substrate that is suitable for practical use in sensing applications.

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Fig. 4. SERS spectra of 4-ATP with different concentrations (10-3 to 10-10 M) on AgBNCP-30.

Fig. 5. SERS spectra of 4-ATP (0.1 mM) at random 20 points on AgBNCP-30 collected from (A) ‘top-side-up’ and (B) ‘bottom-side-up’ measurements. (C, D) SERS signal intensities of 4-ATP at 1081 cm−1 extracted from SERS spectra shown in (A) and (B), respectively.

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3.2 Application for methomyl pesticide detection by SERS Methomyl was selected as a target molecule to demonstrate the pesticide detection applications of the SERS substrate. Methomyl is a carbamate pesticide widely used in agriculture due to its broad spectrum activity. Although it is highly toxic to human and livestock (Van Scoy, Yue, Deng, & Tjeerdema, 2013), no study has been reported on the development of SERS substrate for methomyl detection. Fig. 6A shows the SERS spectra of methomyl with different concentrations collected on AgBNCP-30 substrate. Peaks at 674, 940, 1303, and 1420 cm-1 attributed to aliphatic C–S stretching,

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C–H out-of-plane bending, Amide III, and asymmetric CH3 bending, respectively. All band assignments are summarized in Fig. S5 and Table S2. The characteristic SERS peak at 674 cm-1 of methomyl was linearly proportional to the corresponding common logarithm of methomyl concentration ranging from 10-7 to 10-3 M with R2 = 0.979 (Fig. 6B). The detection limit for methomyl

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by the AgBNCP-30 SERS substrate was 3.610-7 M, which is better than several reported values

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using other techniques (Table S3).

Fig. 6. SERS detection of methomyl pesticide using AgBNCP-30. (A) SERS spectra of methomyl with different concentrations (10-2 to 510-7 M). (B) Plot of corresponding peak intensities at 674 cm−1 versus the logarithm of methomyl concentration.

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3.3 Optical properties of AgNP-BNC paper BNCP has an exceptionally good optical transparency compared to a filter paper, which is the most commonly used material for flexible SERS substrate fabrication. Optical transmittance value for BNCP is higher than 50% in the region 700 to 800 nm, whereas filter paper has the transmittance values of ~1% (Fig. 7A). However, if flexible SERS substrates with optical transparency are needed, plastic films with higher transmittance such as PET (~85%) (Jung, Bae, Park, Yoo, & Bae, 2011), PE (~80%) (Alghdeir, Mayya, & Dib, 2019), and PVC (~88%) (Qi et al., 2016) were typically employed.

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In the previous section (Fig. 5), the Raman intensities collected from both sides (‘top-side-up’ and ‘bottom-side-up’) of the substrate were almost identical, although the AgNPs were located on the top few layers on the top side (illustrated in Fig. 7B). This SERS signal behavior of AgBNCP-30 is similar to other transparent SERS substrates, e.g., a SERS substrate of AuNPs/PMMA/PE composited

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film (Zhong et al., 2014). In addition, the transmittance spectrum of AgBNCP-30 shows that light at the range of the excitation laser wavelength and its Raman scattered light (~800 nm) still can pass

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through the substrate (Fig. 7A). These results together imply that BNCP may potentially be used to

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substitute those petroleum-based plastic substrates for SERS substrate fabrication. Yet, we attributed that the equal SERS intensities in Fig. 5 without the optical loss, despite the transparency of BNCP significantly lower than those plastic transparent substrates, was due to the compensation by the high

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excitation power (14 mW). To confirm this aspect, we compared the SERS intensities of 4-ATP collected from both sides of the SERS substrate under different excitation powers (Fig. 7C). In the

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inset of Fig. 7C, ITop-IBottom/ITop values reveal that an increased laser power could reduce the difference of the SERS signal intensities collected from ‘bottom-side-up’ to ‘top-side-up’ and those intensities

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reach the same values at 14 mW. Moreover, at this laser power, SERS measurement of 4-ATP molecules on AgBNCP-30 SERS substrate covered by pristine BNCP also resulted in the same intensities compared with AgBNCP-30 alone (Fig. 8). Likewise, by replacing the covering BNCP with transparent PE or PVC film, the acquired SERS signal still remained identical. Conversely, the signal was not detectable from the case that used filter paper, which is an opaque substrate, as the cover. Taken together, at a suitable excitation laser power, these results reaffirmed the potential of the

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BNCP-based SERS active substrate for SERS applications similar to those transparent plastic film-

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based ones.

Fig. 7. (A) Optical transmittance of filter paper, BNCP, and AgBNCP-30. (B) Schematic diagram illustrating the difference of AgNPs location between ‘top-side-up’ and ‘bottom-side-up’ on the AgNP-BNCP substrate when performing SERS measurement. (C) Comparison of the SERS

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intensities from 4-ATP at 1081 cm−1 between ‘top-side-up’ and ‘bottom-side-up’ measurements under

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different excitation laser powers. The inset represents the plot of ITop-IBottom/ITop values derived from

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the main figure.

Fig. 8. Comparison of SERS intensity of 4-ATP (0.1 mM) absorbed on AgBNCP-30 substrate between without and with coverage of substrates (BNCP, PE film, PVC film, and filter paper).

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3.4 Paste-and-Read SERS Applications Conventional assays for pesticide detection on nonplanar surfaces involve, at least, extraction and collection steps thus are time-consuming. Toward overcoming this challenge, we demonstrated an in situ detection of methomyl on fruit peels using the AgNP-BNCP throughout a “paste-and-read” SERS method, as the SERS measurement can be promptly performed when the SERS substrate is in a contact with the contaminated site (Fig. 9A). Prior to the measurement of SERS signal, 10 L DI water was dropped onto orange or apple peels, previously deposited with 10-6 M drop-dried

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methomyl, to help the pesticide diffusion to the hotspot and the conformal contact between the fruit surfaces and the SERS substrate. According to the AgNP location, AgBNCP-30 was directly pasted ‘bottom-side-up’ onto the target surface (Fig. 7B), facilitating the adsorption of the methomyl molecules to the plasmonic nanostructures. Fig. 9B-C presented normal Raman and SERS spectra,

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which were acquired by different detection methods. As expected, without using the SERS substrate, characteristic signal of residual methomyl on the fruit peels was not observed (red lines). Whereas, the

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main Raman bands of methomyl at 674, 940, 1303, and 1420 cm-1 were clearly observed (blue lines)

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when the contaminated fruit peels contacted with the AgNP-BNCP, indicating its capability of being used for in-situ SERS detection. We further compared the performance to the conventional “extractand-read” method by following the similar procedure, but the extract solution was collected and

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transferred to the ‘top-side-up’ SERS substrate. Those obtained SERS spectra (pink lines) were also consistent with ones of the “paste-and-read” SERS method. This achievement for the rapid past-and-

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read SERS is due to the optical property of the AgNP-BNCP and the laser power, as discussed in the previous section. This work aims to illustrate potential uses of BNCP to achieve user-friendly SERS

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active materials for rapid, facile, and on-site detection that are sustainable for long-term environmental impacts. However, the sensitivity improvement could be further circumvented by changing the plasmonic nanostructures of which the LSPR wavelength matches to the excitation light (Su, Ma, Dong, Jiang, & Qian, 2011).

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Fig. 9. (A) Schematic diagram of “paste-and-read” SERS detection of methomyl on fruit peels using

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AgNP-BNCPs. Normal Raman and SERS spectra of methomyl on (B) orange and (C) apple peels acquired by without (red) and with AgBNCP-30, using “paste-and-read” (blue) and “extract-andread” (pink) method. Note: black line presents normal Raman spectra of the fruit peels in the absence

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of methomyl. 4. Conclusions

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We successfully developed a fully biodegradable, flexible SERS substrate based on BNCP

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composited with AgNPs by using a simple vacuum-filtration method for rapid detection of pesticide on fruit peel. AgNPs were directly adhered onto the 3D networked nanofibrous structure of BNCP, resulting in good SERS activity and signal uniformity as evaluated by 4-ATP molecule. The good

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performance of the substrate for detection of methomyl carbamate pesticide was also demonstrated. Moreover, the optical property of the AgNP-BNCP displays its potential for in situ SERS detection as

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demonstrated via direct detection of methomyl on fruit peels. Using this effective paste-and-read SERS method is simple, rapid, and low-cost for practical uses as well as promising for developing

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novel SERS active substrates based on green material selection in the future. Acknowledgement

This work is supported by Ratchadapisek Somphot Fund for Postdoctoral Fellowship, Chulalongkorn University. This work has also been supported by the Research Network NANOTEC (RNN) program of the National Nanotechnology Center (NANOTEC), NSTDA, Ministry of Higher Education,

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Science, Research and Innovation (MHESI), Thailand. We thank Prof. Pornanong Aramwit, Faculty of Pharmaceutical Science, Chulalongkorn University for providing BNC hydrogel. References Alghdeir, M., Mayya, K., & Dib, M. (2019). Characterization of nanosilica/low-density polyethylene nanocomposite materials. Journal of Nanomaterials, 2019, 8. Chen, R., Zhang, L., Li, X., Ong, L., Soe, Y. G., Sinsua, N., . . . Shen, W. (2017). Trace analysis and chemical identification on cellulose nanofibers-textured SERS substrates using the “coffee ring”

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effect. ACS Sensors, 2(7), 1060-1067. Ding, S.-Y., Yi, J., Li, J.-F., Ren, B., Wu, D.-Y., Panneerselvam, R., & Tian, Z.-Q. (2016). Nanostructure-based plasmon-enhanced Raman spectroscopy for surface analysis of materials.

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Fraire, J. C., Pérez, L. A., & Coronado, E. A. (2013). Cluster size effects in the surface-enhanced

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Highlights AgNP-BNC paper was fabricated as an eco-friendly disposable, flexible SERS substrate.

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The prepared AgNP-BNCPs have high SERS activity, good reproducibility and stability.

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BNC exhibits potential as an alternative substrate to plastics for SERS applications.

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SERS active AgNP-BNC paper achieves paste-and-read detection of pesticides on fruits.

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Graphical abstract

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