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Facile synthesis of cellulose nanofiber nanocomposite as a SERS substrate for detection of thiram in juice
Carbohydrate Polymers 189 (2018) 79–86
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Facile synthesis of cellulose nanofiber nanocomposite as a SERS substrate for detection of thiram in juice
T
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Ziyi Xionga, Mengshi Lina,b, , Hetong Linb, Meizhen Huangc a
Food Science Program, Division of Food System & Bioengineering, University of Missouri, Columbia, MO 65211-5160, USA College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China c Department of Instrument Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Cellulose nanofiber Gold nanoparticles SERS Thiram
There has been growing interest in the use of nanocellulose-based substrate for surface-enhanced Raman spectroscopy (SERS) applications. This study aimed to use cellulose nanofibers (CNF) to develop novel CNFbased nanocomposite as a SERS substrate. CNF were cationized with ammonium ions and then interacted with citrate-stabilized gold nanoparticles (AuNPs) via electrostatic attraction to form uniform nanocomposites. The CNF-based nanostructures were loaded with AuNPs that were firmly adhered on the CNF surfaces, providing a three-dimensional plasmonic SERS platform. A Raman-active probe molecule, 4-aminothiophenol, was selected to evaluate the sensitivity and reproducibility of CNF-based SERS substrate. The intensity of SERS spectra obtained from CNF/AuNP nanocomposite was 20 times higher than that from the filter paper/AuNP substrate. The SERS intensity map demonstrates good uniformity of the CNF/AuNP substrate. CNF/AuNP nanocomposites were used in rapid detection of thiram in apple juice by SERS and a limit of detection of 52 ppb of thiram was achieved. These results demonstrate that CNF/AuNP nanocomposite can be used for rapid and sensitive detection of pesticides in food products.
1. Introduction Nanocellulose is a novel nanomaterial consisting of nano-structured cellulose that exhibits unique properties, including large surface area, high length-to-width ratio, high stiffness, and optical transparency. Among different types of nanocellulose, cellulose nanofibers (CNF) consist of crystalline regions of cellulose that are interconnected with each other through amorphous regions to form nanocellulose with length in micrometer range and width spanning from several nanometers to several hundred nanometers, as a result of strong inter-fiber hydrogen bonding (Abitbol et al., 2016; Ruiz-Palomero, Soriano, & Valcárcel, 2017). CNF has received much attention in recent years for the applications in material science, food packaging, and food analysis for chemical contaminants such as pesticides. Thiram, a sulfur fungicide, has been widely used as a protective agent on food crops, vegetables, and fruits (Sharma, Aulakh, & Malik, 2003). Approximately 165,000 pounds of thiram are applied to 35,000 acres of strawberries, apples, and peaches annually in the U.S. (EPA, 2004). Thiram is slightly toxic by ingestion and inhalation and moderately toxic by dermal absorption (Sharma et al., 2003). Various analytical methods have been used for the determination of thiram,
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including chromatography, polarographic and voltammetric methods, and capillary electrophoresis (Sharma et al., 2003). However, these methods are time-consuming, labor-intensive, and require tedious sample pretreatment. Consequently, there is an increasing interest in developing rapid and sensitive methods with easy sample preparation for detection of thiram in foods. Raman spectroscopy is a spectroscopic technology based on measuring molecular vibrations of analyte molecules. It can provide information about structural properties of molecules by capturing the signals from inelastic light scattering from the incident light on analytes (Cialla et al., 2012; Haynes, McFarland, & Duyne, 2005). Surface-enhanced Raman scattering (SERS) is a surface-sensitive technique that greatly enhances Raman scattering signals of analyte molecules adsorbed on roughened metal surface, which is being increasingly used in medical science, environmental monitoring, and food analysis (Craig, Franca, & Irudayaraj, 2013; Liu et al., 2015). Although the exact mechanism of SERS is not clearly understood, two theories have been widely accepted: electromagnetic and chemical enhancement. The electromagnetic enhancement is considered as the main contributor to the SERS in which the enhancement of Raman signals is due to the excitation of the localized surface plasmon resonance of nanoparticles
Corresponding author at: Food Science Program, Division of Food System & Bioengineering, University of Missouri, Columbia, MO 65211-5160, USA. E-mail address: [email protected] (M. Lin).
https://doi.org/10.1016/j.carbpol.2018.02.014 Received 18 October 2017; Received in revised form 14 January 2018; Accepted 5 February 2018 Available online 08 February 2018 0144-8617/ © 2018 Elsevier Ltd. All rights reserved.
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Fig. 1. TEM images at different magnifications of synthesized AuNPs (A); insert: UV–vis of AuNPs with a peak at 531 nm; unmodified CNF suspension (B); modified CNF coated with AuNPs (C & D).
than 100 nm can be used to support higher loading of nanomaterials and improve homogeneity of the substrate. Our previous study developed CNF-based SERS substrate by placing gold nanoparticles (AuNPs) onto dried CNF sheet (Xiong, Chen, Liou, & Lin, 2017). However, this approach was largely based on gravity-assisted loading of AuNPs on CNF, which led to inhomogeneous distribution of AuNPs mainly on the surface layer of the CNF. To improve the sensitivity and reproducibility of SERS method, a novel approach was taken in this study to modify CNF with ammonium ions and allow them to interact with citrate-stabilized AuNPs, forming a uniform film with firmly adhered nanoparticles. This CNF-based nanocomposite provides a three-dimensional (3D) and highly porous structure as a plasmonic SERS substrate. In addition, the detection of thiram in apple juice was conducted by SERS coupled with CNF/AuNP nanocomposite. Multivariate statistical analysis was used to analyze the SERS spectral data.
Table 1 Zeta potential of different materials. Materials
AuNPs
Original CNF
Modified CNF
ζ-potential (mV)
−3.94
−39.47
+14.77
stimulated by an incident light (Cialla et al., 2012). The regions of the intense local field enhancement are known as “hot-spots”. In general, the SERS detection is performed on a plasmon-active SERS substrate and its fabrication plays a key role in obtaining high SERS performance. Paper-based SERS substrates have been developed to take advantage of natural wrinkles and fibril structures of paper, thus allowing metal nanoparticles to be deposited and arranged on a paper to from large-area SERS “hot-spots” (Li et al., 2016; Wei & White, 2013; Zhang et al., 2012). However, the width of cellulose fibril of paper, which is a range of several to tens of micrometers, limits the SERS performance due to its inhomogeneous distribution of nanoparticles and low signal intensity. To solve this problem, nanocellulose with lateral dimensions of less 80
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Scheme 1. Synthesis of cationic cellulose nanofibers.
Fig. 2. SEM images of CNF (A & B) and filter paper (C & D) consisting of microfibers with coated AuNPs at different magnifications.
2. Experimental section
Fisher Scientific (Fair lawn, New Jersey, U.S.A). CNF were obtained from the University of Maine (USA) and they were made from natural lignocellulosic fibers and the final product was a slurry of about 3 wt% cellulose nanofibers with 90% fines. Organic apple juice (Apple & Eve Organics, 100% apple juice) was purchased from a local grocery store and it was prepared from reconstituted organic apple juice concentrate containing malic acid and ascorbic acid. All chemicals were used as received without further purification and Milli-Q water was used throughout all experiments.
2.1. Chemicals and materials Hydrogen tetrachloroaurate solution (HAuCl4, 30 wt% in dilute HCl), (3-chloro-2-hydroxypropyl)trimethylammonium chloride (CHPTMAC), thiram (analytical standard), 4-aminothiophenol (4-ATP, 97%) and acetonitrile (HPLC Plus, ≥99.9%) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Trisodium citrate dihydrate (Na3Cit·2H2O), sodium hydroxide (Certified A.C.S) and acetone (HPLC grade) and hydrochloric acid (Certified ACS Plus) were purchased from 81
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Scheme 2. The mechanism and analysis process for CNF/AuNP substrate.
Fig. 3. SERS spectra of 1 ppm of 4-ATP on CNF/AuNP nanocomposite and filter paper/AuNPs substrates (A); SERS spectra of different concentrations of 4-ATP measured by CNF/AuNP substrate (B).
2.2. Synthesis of citrate-stabilized AuNPs
2.4. Preparation of the CNF/AuNP nanocomposite
The synthesis of AuNPs was based on a modified citrate reduction method (Frens, 1973). First, 90 mL of Milli-Q water was heated to boiling temperature, followed by addition of 10 mL of HAuCl4 (2.5 mM) with stirring. When the solution was boiled again, 4 mL of Na3Cit·2H2O (0.25% w/v) was then added immediately. The color of the solution was changed from pale yellow to colorless then to black. The solution was kept at the boiling temperature until its color changed to redpurple, and then cooled down with stirring for 30 min. The as-prepared AuNP suspension was stored at 4 °C prior to use.
An aliquot 1 mL of the modified CNF was washed three times to remove chemical residues by centrifuge (Eppendorf 5452 Minispin, Marshall Scientific, Hampton, NH, USA) at 2465 G for 10 min before it was concentrated two times. A volume of 1 mL of the AuNP suspension was concentrated five times by centrifugation at 2465 G for 8 min. The modified CNF and AuNPs with a ratio of 1:1 were mixed well in a microtube by vortex mixer (Fisher Scientific). The CNF/AuNP nanocomposites were then deposited on a gold side for use as a SERS substrate. 2.5. Characterization of the CNF/AuNP nanocomposite
2.3. Modification of CNF
Fourier transform infrared (FTIR) characterization was conducted with a FTIR spectrometer (Nicolet 380, Thermo Fisher Scientific, Waltham, WA, USA) in transmittance mode. Both unmodified and modified CNF with same concentration (5%) underwent centrifugation and precipitated gel-like CNF was collected for FTIR test. Spectra were acquired for a total of 64 scans in the wavenumber range from 400 to 4000 cm−1 with a resolution of 4 cm−1. Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, UK) was employed to measure ζ-potential of AuNPs and CNF. The as-prepared AuNP suspension was used without dilution and the unmodified and modified CNF were diluted to the concentration of 5%. Transmission electron microscopy (TEM) images were acquired for characterizing AuNPs, CNF, and their mixture
Preparation of cationic CNF was performed using (2,3-epoxypropyl) trimethylammonium chloride (EPTMAC) followed a previous published method (Hauser & Tabba, 2001). Briefly, 6 mL of CHPTMAC was transferred into a beaker to make a 50 mL solution with Milli-Q water, followed by the addition of 2 g of NaOH. The solution was kept stirring for 30 min to allow for the formation of EPTMAC. Then 1.25 g of CNF was immersed into the EPTMAC solution with stirring for 2 h, which allowed the epoxide to react with the hydroxyl groups of CNF and grafted ammoniums to the cellulose molecules and hence rendered cationic CNF. The modified CNF was then adjusted to pH 7 using pH meter (Oakton, Australia). 82
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Fig. 4. Optical images of CNF/AuNP nanocomposite (A & D) and corresponding SERS intensity maps (B) at 1075 cm−1 of 5 ppm of 4-ATP; representative SERS spectra of 4-ATP (C) collected from CNF/AuNP nanocomposite marked with squares in B; SERS spectra of 5 ppm of 4-ATP (E) collected from 16 randomly selected positions from the area indicated with red lines in the optical images A and D. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Averaged Raman spectra of thiram with concentration from 0 to 10 ppm (A); second derivative transformation of Raman spectra at 548 cm−1 (B); averaged Raman spectra of thiram in apple juice with concentration from 0 to 10 ppm (C); the plot of the log values of Raman peak intensity at 548 cm−1 versus the log values of concentration of thiram in apple juice (D).
the as-prepared CNF/AuNP nanocomposite on a slide and were then gold sputtered for 60 s prior to SEM imaging.
using a JEOL electron microscope (JEOL 1400, Tokyo, Japan) operated at an accelerating voltage of 120 kV. The TEM samples were prepared by concentrating the as-prepared AuNP suspension 10 times and making 5% unmodified CNF and CNF/AuNP mixture. Scanning electron microscopy (SEM) images were obtained using a FEI Scios DualBeam FIB SEM (Thermo Fisher Scientific, Hillsboro, Oregon, USA) at accelerating voltage of 5 kV. SEM samples were prepared by dropping 2 μL of
2.6. SERS measurement A DXR2 Raman microscope (Thermo Fisher Scientific) was used in this study. This system is equipped with a DXR 785 nm laser source. 83
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anionic surface properties (Sjöström, 1989), which is proven by the ζpotential values shown in Table 1. In addition, no strong chemical forces exist between CNF and AuNPs. Therefore, modification of CNF is a prerequisite for the interactions between anionic CNF and AuNPs. In this study, a novel strategy was taken by grafting positive ammonium ions to CNF to obtain cationic CNF, which allowed attachment of negatively charged AuNPs onto the CNF surface via electrostatic interactions. Scheme 1 illustrates the synthesis of cationic CNF (Guo, Filpponen, Su, Laine, & Rojas, 2016; Hyde, Dong, & Hinestroza, 2007). CHPTMAC reacted with NaOH to form EPTMAC. Epoxy moiety of EPTMAC went through a nucleophilic reaction with CNF under alkaline conditions, producing cationic CNF. Fourier transform infrared (FTIR) spectra of original CNF and corresponding cationic CNF are shown in Fig. S1. A typical spectrum of cationic CNF displays a new band at 1480 cm−1, which is assigned to the trimethyl groups of quaternized ammonium (Guo et al., 2016; Pei, Butchosa, Berglund, & Zhou, 2013). Additionally, the ζ-potential of modified CNF is positive (14.77 mV) (Table 1), which is in agreement with previous observations (Guo et al., 2016; Olszewska et al., 2011). These results indicate the presence of positive charges on the modified CNF surface. Both FTIR spectra and ζ-potential results provide clear evidences of successful introduction of quaternary ammonium groups on the CNF surface. The width of the CNF could be a few nanometers to several hundred nanometers (Fig. 1B). The broad size distribution could be attributed to heterogeneous mechanical treatment and the aggregation of individual fibers. Fig. 1C and D shows TEM images of modified CNF that were mixed with AuNPs. It is clearly observed that cationic CNF was coated with AuNPs due to strengthened interactions between cationic CNF and anionic AuNPs via electrostatic forces. This CNF/AuNP nanocomposite can be used as a SERS substrate. Conventional papers have been used in several studies for loading different metal nanostructures to serve as SERS substrates (Lee, Tian, & Singamaneni, 2010; Li et al., 2016; Wei & Huang, 2017). The spot size of Raman’s laser beam is ∼1 μm that is smaller than both width and length of cellulose fibers in the papers. In addition, the porosity and high roughness of the papers make it a challenge to achieve uniform distribution of nanoparticles on the paper. Compared with normal cellulose, nanocellulose with a fiber width in nanometer range provides much larger surface area for loading metal nanostructures, which could lead to higher sensitivity and improved homogeneity as a SERS substrate. In this study, filter paper coated with AuNPs was prepared and used as a comparison with CNF/AuNP nanocomposite. Fig. 2 displays SEM images of filter/AuNP film and CNF/AuNP nanocomposite at different magnifications. CNF (Fig. 2A and B) shows surface morphology in 2D structures and fibers with a diameter in nanometer size, demonstrating that nanofibers have much larger surface area that can load more AuNPs as compared to the filter paper in micrometer size (Fig. 2C and D).
During measurement, light from the high power (35 mW) laser was directed and focused onto the sample at a microscope stage through a × 10 objective with 25 μm pinhole aperture. Each spectrum was measured once with an accumulation time of 10 s. Spectra data were collected by OMNIC for Dispersive Raman software (Thermo Fisher Scientific). A typical procedure was as follows: 2 μL of as-prepared CNF/AuNP nanocomposite was deposited onto the slide and air-dried. A round-shaped film was thus formed with a diameter of 3 mm, and an aliquot of 2 μL of the sample solution was then placed on the film. The film with samples was air-dried prior to the SERS detection. In this study, 4-ATP was selected as a probing molecule because its thiol group can easily bind to gold, which can be used to evaluate the SERS effect for CNF/AuNP substrate. The reproducibility of the substrate was evaluated by acquiring SERS intensity maps of 5 ppm of 4-ATP solution. 2.7. Detection of thiram by the CNF/AuNP substrate A stock solution (100 ppm) of thiram was prepared by mixing 5 mg of thiram powders in 50 mL of acetone with stirring. The stock solution was subsequently diluted with acetone to reach a series of thiram standard solutions (0.05, 0.1, 0.5, 1, 5, and 10 ppm). Pure acetone solution was used as a control group. For detection of thiram in apple juice, thiram powders were spiked into organic apple juice with magnetic stirring to obtain 100 ppm of thiram in apple juice, from which a series of concentrations (0.05, 0.1, 0.5, 1, 5, and 10 ppm) of apple juice containing thiram were prepared. Apple juice with thiram was used as a control group. The SERS measurement for both pure thiram solution and apple juice samples was conducted as described above. 2.8. Data analysis Calibration curve was established by monitoring SERS intensity as a function of analyte concentration. Each point on the calibration curve represented an average value of seven replicate measurements. Limit of detection was calculated by determining the minimum distinguishable signals using the following expression:
s m = sb + kσsb where Sm is the minimum distinguishable signal, Sb is the Raman signal generated by a blank measurement of the SERS substrate in the absence of the analyte. k is the proportionality constant, and σsb is the standard deviation of blank measurements. The concentration value is determined by substituting the minimum distinguishable signal to the best-fit equation of the calibration curve to determine the limit of detection. 3. Results and discussion 3.1. Fabrication and characterization of CNF/AuNP nanocomposite
3.2. SERS performance of CNF/AuNP substrates AuNPs were selected as building blocks in this study because they are thermally and chemically stable (Chen, Kou, Yang, Ni, & Wang, 2008; Takahashi & Tatsuma, 2010). The preparation of AuNPs was based on the citrate reduction of gold salts in aqueous solution. As a reducing agent, sodium citrate reduced Au (III) in the HAuCl4 solution to Au (0), leading to the formation of AuNPs of certain size. Sodium citrate also served as a stabilizer, imparting negative surface charges to nanoparticles from weakly bound citrate ions, which prevented the aggregation of nanoparticles in solution. For resultant AuNPs, the UV–vis spectra show a narrow peak at wavelength of 531 nm (Insert of Fig. 1A) due to surface plasmon resonance. The TEM image in Fig. 1A shows that AuNPs are in spherical shape. The average size of AuNPs is 40.67 nm with a standard deviation of 7.73 based on measurement of 150 AuNPs. Citrate-reduced AuNPs are negatively charged and CNF have
Scheme 2 shows the mechanism and analysis process using CNF/ AuNP nanocomposite in SERS measurement. Negatively charged AuNPs and positively charged CNF were bound tightly via electrostatic attraction. For individual fibers, many AuNPs were aggregated on the CNF surface, which created SERS “hot-spots” that mostly occur at consecutive NPs and the gaps between the NPs and their attached surface (Satheeshkumar et al., 2017). Two hot-spot zones are shown in the Scheme 2. In addition, CNF consists of countless fibers that can be loaded with numerous AuNPs that would contribute to enhanced Raman scattering signal from analyte molecules. Fig. 3A shows the SERS spectra of 1 ppm of 4-ATP deposited on the CNF/AuNP nanocomposite and a filter paper/AuNPs. The 4-ATP peaks in the SERS spectra are in good agreement with the spectra reported in previous studies (Cialla et al., 2012; Zhang et al., 2015). However, a 84
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increased as the concentration of thiram increased from 0 to 10 ppm. Thiram in apple juice needs to be extracted by organic solvents before the determination by HPLC. In contrast, apple juice samples contaminated with thiram can be directly placed on a CNF/AuNP substrate for SERS measurement. A transparent layer was formed on the substrate after the droplet was dried due to sugars in apple juice. However, this layer caused little interference with spectral collection. Fig. 5C shows the spectra of apple juice spiked with different concentrations of thiram from 0 to 10 ppm, exhibiting characteristic thiram peaks similar to the spectra of thiram in acetone solution. The pure thiram solution yielded much higher Raman intensity than the same concentration of thiram in apple juice. This is because other components in apple juice competed with thiram molecules and blocked the way of thiram molecules binding to AuNPs, resulting in reduced Raman intensity of thiram. Fig. 5C clearly shows characteristic peaks of thiram at 548, 930, 1138, and 1370 cm−1. In addition, an evident peak was observed at 734 cm−1 in all the spectra from 0 to 10 ppm of thiram in apple juice. This interfering band was likely from sugars in apple juice. As shown in Fig. 5D, a good linear correlation (R = 0.9323) was established by plotting the log values of the SERS intensity and 4-ATP concentration. The limit of detection was calculated to be 52 ppb, which is well below the maximum residue limit (MRL) in fruit of 7 ppm set by the U.S. Environmental Protection Agency (EPA). The results show that SERS coupled with CNF/AuNP nanocomposite can be used as a sensitive method to detect pesticides in apple juice.
slight shift in Raman bands can happen due to a strong polarization occurred at the surface of AuNPs (Gong et al., 2014). The intensity of SERS spectra obtained from CNF/AuNP nanocomposite was found to be nearly 20 times higher than that from the filter paper/AuNP substrate, which proved that SERS performance was greatly enhanced using CNF compared to filter paper because more SERS “hot-spots” were created on CNF/AuNP nanocomposite. To further test the sensitivity of the CNF/AuNP nanocomposite, this substrate was employed for the detection of trace amount of analytes. Fig. 3B shows the averaged SERS spectra (n = 7) of different concentrations of 4-ATP in acetonitrile. The characteristic peaks of 4-ATP were clearly observed in the SERS spectra of 0.05–10 ppm of 4-ATP solutions in the range from 900 to 1700 cm−1. The absence of this peak in the control sample proves that the peak was caused by 4-ATP. There is an increasing trend of Raman intensity of the peak at both 1076 and 1583 cm−1 as the concentration of 4-ATP increased. In addition, the SERS peaks were still distinguishable when the concentration reached 0.05 ppm, indicating that CNF/AuNP nanocomposite could be used for detecting trace amount of analytes. These results demonstrate high sensitivity of CNF/AuNP nanocomposite in SERS measurement. The reproducibility of the SERS substrate is important for quantitative analysis of analyte molecules. In this study, 5 ppm of 4-ATP was used to assess the reproducibility of SERS signals. As indicated in the boxed optical image (Fig. 4A), a SERS map of 4-ATP was collected over a 150 × 150 μm2 area on a CNF/AuNP nanocomposite (Fig. 4B). The SERS intensity map corresponding to the Raman band at 1076 cm−1 exhibits acceptable uniformity with a relative standard deviation (RSD) of 18%, which was calculated from 36 pixels in the map. To visually demonstrate the degree of the difference among different regions, three representative spectra were extracted from low to high intensity region of the SERS map (Fig. 4C). No significant difference was observed, further confirming a small variation in the SERS signals over the mapped area. What’s more, a point-by-point SERS mapping was conducted by measuring 5 ppm of 4-ATP on the CNF/AuNP nanocomposite to further characterize the homogeneity of the substrate (Figs. 4D & E). As shown in Fig. 4D, 16 positions covering a large area (area square, total square) was captured for collecting SERS signals and the corresponding map (Fig. 4E) shows that the SERS enhancement is uniform with a RSD of 25.47%. Based on the data analysis from area and pointto-point maps, the reproducibility is found to be acceptable considering the wide width range of CNF.
4. Conclusion In summary, facile synthesis of CNF/AuNP nanocomposite was achieved and the film was used as a SERS substrate. Compared with filter papers, nanoscale CNF have much larger surface area and allow for more homogeneous and dense loading of AuNPs. AuNPs were adsorbed on the CNF through electrostatic attraction after CNF cationization, creating more “hot-spots” for SERS enhancement. CNF/AuNP nanocomposite was used for measurement of 4-ATP and satisfactory sensitivity and reproducibility were achieved. Detection of thriam in apple juice was conducted by SERS and the limit of detection was 52 ppb. These results demonstrate that CNF/AuNP nanocomposite has a great potential to be used as a SERS substrate in detection of pesticides in food products. Acknowledgements
3.3. Detection of thiram in apple juice by SERS We acknowledge the support from USDA NIFA Multi-state Project (NC1194) and the National Natural Science Foundation of China (Grant Nos. 31728016 and 61775133). We thank Dr. Bongkosh Vardhanabhuti for her assistance in the Zetasizer measurement.
Even though GC/LC–MS is often the gold standard method for pesticide detection, analysis of thiram involves conversion into carbon disulfide (CS2) in an acid medium prior to estimation using spectroscopy and chromatography due to inherent insolubility in common extraction solvents and poor stability (Mujawar, Utture, Fonseca, Matarrita, & Banerjee, 2014), which makes the analysis process complicated. With simple sample preparation, SERS has been considered a powerful tool to detect thiram. Fig. 5A shows the average Raman spectra (n = 7) of different concentrations of thiram in acetone solution from 0 to 10 ppm. The characteristic peaks of thiram can be clearly observed in the Raman spectra. A peak at 548 cm−1 is assigned to SS stretching mode. A peak around 1370 cm−1 is attributed to the CH3 deformation and the CN stretching. The peaks at 1138 cm−1 and 1504 cm−1 are assigned to the CN stretching and CH3 rocking modes (Saute & Narayanan, 2011). Although some noise signals could be detected in the spectra of the control group, which was attributed to acetone, they didn’t cause significant interference to the characteristic peaks of thiram due to high signal-to-noise ratio. Second derivative transformation is commonly used in spectral data analysis to separate the overlapping peaks, eliminate the baseline drifting, and enhance the spectral resolution. It is clearly shown from the second derivation transformation that the intensity of a peak at 548 cm−1 (Fig. 5B)
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Contents lists available at ScienceDirect
Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Facile synthesis of cellulose nanofiber nanocomposite as a SERS substrate for detection of thiram in juice
T
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Ziyi Xionga, Mengshi Lina,b, , Hetong Linb, Meizhen Huangc a
Food Science Program, Division of Food System & Bioengineering, University of Missouri, Columbia, MO 65211-5160, USA College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China c Department of Instrument Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Cellulose nanofiber Gold nanoparticles SERS Thiram
There has been growing interest in the use of nanocellulose-based substrate for surface-enhanced Raman spectroscopy (SERS) applications. This study aimed to use cellulose nanofibers (CNF) to develop novel CNFbased nanocomposite as a SERS substrate. CNF were cationized with ammonium ions and then interacted with citrate-stabilized gold nanoparticles (AuNPs) via electrostatic attraction to form uniform nanocomposites. The CNF-based nanostructures were loaded with AuNPs that were firmly adhered on the CNF surfaces, providing a three-dimensional plasmonic SERS platform. A Raman-active probe molecule, 4-aminothiophenol, was selected to evaluate the sensitivity and reproducibility of CNF-based SERS substrate. The intensity of SERS spectra obtained from CNF/AuNP nanocomposite was 20 times higher than that from the filter paper/AuNP substrate. The SERS intensity map demonstrates good uniformity of the CNF/AuNP substrate. CNF/AuNP nanocomposites were used in rapid detection of thiram in apple juice by SERS and a limit of detection of 52 ppb of thiram was achieved. These results demonstrate that CNF/AuNP nanocomposite can be used for rapid and sensitive detection of pesticides in food products.
1. Introduction Nanocellulose is a novel nanomaterial consisting of nano-structured cellulose that exhibits unique properties, including large surface area, high length-to-width ratio, high stiffness, and optical transparency. Among different types of nanocellulose, cellulose nanofibers (CNF) consist of crystalline regions of cellulose that are interconnected with each other through amorphous regions to form nanocellulose with length in micrometer range and width spanning from several nanometers to several hundred nanometers, as a result of strong inter-fiber hydrogen bonding (Abitbol et al., 2016; Ruiz-Palomero, Soriano, & Valcárcel, 2017). CNF has received much attention in recent years for the applications in material science, food packaging, and food analysis for chemical contaminants such as pesticides. Thiram, a sulfur fungicide, has been widely used as a protective agent on food crops, vegetables, and fruits (Sharma, Aulakh, & Malik, 2003). Approximately 165,000 pounds of thiram are applied to 35,000 acres of strawberries, apples, and peaches annually in the U.S. (EPA, 2004). Thiram is slightly toxic by ingestion and inhalation and moderately toxic by dermal absorption (Sharma et al., 2003). Various analytical methods have been used for the determination of thiram,
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including chromatography, polarographic and voltammetric methods, and capillary electrophoresis (Sharma et al., 2003). However, these methods are time-consuming, labor-intensive, and require tedious sample pretreatment. Consequently, there is an increasing interest in developing rapid and sensitive methods with easy sample preparation for detection of thiram in foods. Raman spectroscopy is a spectroscopic technology based on measuring molecular vibrations of analyte molecules. It can provide information about structural properties of molecules by capturing the signals from inelastic light scattering from the incident light on analytes (Cialla et al., 2012; Haynes, McFarland, & Duyne, 2005). Surface-enhanced Raman scattering (SERS) is a surface-sensitive technique that greatly enhances Raman scattering signals of analyte molecules adsorbed on roughened metal surface, which is being increasingly used in medical science, environmental monitoring, and food analysis (Craig, Franca, & Irudayaraj, 2013; Liu et al., 2015). Although the exact mechanism of SERS is not clearly understood, two theories have been widely accepted: electromagnetic and chemical enhancement. The electromagnetic enhancement is considered as the main contributor to the SERS in which the enhancement of Raman signals is due to the excitation of the localized surface plasmon resonance of nanoparticles
Corresponding author at: Food Science Program, Division of Food System & Bioengineering, University of Missouri, Columbia, MO 65211-5160, USA. E-mail address: [email protected] (M. Lin).
https://doi.org/10.1016/j.carbpol.2018.02.014 Received 18 October 2017; Received in revised form 14 January 2018; Accepted 5 February 2018 Available online 08 February 2018 0144-8617/ © 2018 Elsevier Ltd. All rights reserved.
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Fig. 1. TEM images at different magnifications of synthesized AuNPs (A); insert: UV–vis of AuNPs with a peak at 531 nm; unmodified CNF suspension (B); modified CNF coated with AuNPs (C & D).
than 100 nm can be used to support higher loading of nanomaterials and improve homogeneity of the substrate. Our previous study developed CNF-based SERS substrate by placing gold nanoparticles (AuNPs) onto dried CNF sheet (Xiong, Chen, Liou, & Lin, 2017). However, this approach was largely based on gravity-assisted loading of AuNPs on CNF, which led to inhomogeneous distribution of AuNPs mainly on the surface layer of the CNF. To improve the sensitivity and reproducibility of SERS method, a novel approach was taken in this study to modify CNF with ammonium ions and allow them to interact with citrate-stabilized AuNPs, forming a uniform film with firmly adhered nanoparticles. This CNF-based nanocomposite provides a three-dimensional (3D) and highly porous structure as a plasmonic SERS substrate. In addition, the detection of thiram in apple juice was conducted by SERS coupled with CNF/AuNP nanocomposite. Multivariate statistical analysis was used to analyze the SERS spectral data.
Table 1 Zeta potential of different materials. Materials
AuNPs
Original CNF
Modified CNF
ζ-potential (mV)
−3.94
−39.47
+14.77
stimulated by an incident light (Cialla et al., 2012). The regions of the intense local field enhancement are known as “hot-spots”. In general, the SERS detection is performed on a plasmon-active SERS substrate and its fabrication plays a key role in obtaining high SERS performance. Paper-based SERS substrates have been developed to take advantage of natural wrinkles and fibril structures of paper, thus allowing metal nanoparticles to be deposited and arranged on a paper to from large-area SERS “hot-spots” (Li et al., 2016; Wei & White, 2013; Zhang et al., 2012). However, the width of cellulose fibril of paper, which is a range of several to tens of micrometers, limits the SERS performance due to its inhomogeneous distribution of nanoparticles and low signal intensity. To solve this problem, nanocellulose with lateral dimensions of less 80
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Scheme 1. Synthesis of cationic cellulose nanofibers.
Fig. 2. SEM images of CNF (A & B) and filter paper (C & D) consisting of microfibers with coated AuNPs at different magnifications.
2. Experimental section
Fisher Scientific (Fair lawn, New Jersey, U.S.A). CNF were obtained from the University of Maine (USA) and they were made from natural lignocellulosic fibers and the final product was a slurry of about 3 wt% cellulose nanofibers with 90% fines. Organic apple juice (Apple & Eve Organics, 100% apple juice) was purchased from a local grocery store and it was prepared from reconstituted organic apple juice concentrate containing malic acid and ascorbic acid. All chemicals were used as received without further purification and Milli-Q water was used throughout all experiments.
2.1. Chemicals and materials Hydrogen tetrachloroaurate solution (HAuCl4, 30 wt% in dilute HCl), (3-chloro-2-hydroxypropyl)trimethylammonium chloride (CHPTMAC), thiram (analytical standard), 4-aminothiophenol (4-ATP, 97%) and acetonitrile (HPLC Plus, ≥99.9%) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Trisodium citrate dihydrate (Na3Cit·2H2O), sodium hydroxide (Certified A.C.S) and acetone (HPLC grade) and hydrochloric acid (Certified ACS Plus) were purchased from 81
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Scheme 2. The mechanism and analysis process for CNF/AuNP substrate.
Fig. 3. SERS spectra of 1 ppm of 4-ATP on CNF/AuNP nanocomposite and filter paper/AuNPs substrates (A); SERS spectra of different concentrations of 4-ATP measured by CNF/AuNP substrate (B).
2.2. Synthesis of citrate-stabilized AuNPs
2.4. Preparation of the CNF/AuNP nanocomposite
The synthesis of AuNPs was based on a modified citrate reduction method (Frens, 1973). First, 90 mL of Milli-Q water was heated to boiling temperature, followed by addition of 10 mL of HAuCl4 (2.5 mM) with stirring. When the solution was boiled again, 4 mL of Na3Cit·2H2O (0.25% w/v) was then added immediately. The color of the solution was changed from pale yellow to colorless then to black. The solution was kept at the boiling temperature until its color changed to redpurple, and then cooled down with stirring for 30 min. The as-prepared AuNP suspension was stored at 4 °C prior to use.
An aliquot 1 mL of the modified CNF was washed three times to remove chemical residues by centrifuge (Eppendorf 5452 Minispin, Marshall Scientific, Hampton, NH, USA) at 2465 G for 10 min before it was concentrated two times. A volume of 1 mL of the AuNP suspension was concentrated five times by centrifugation at 2465 G for 8 min. The modified CNF and AuNPs with a ratio of 1:1 were mixed well in a microtube by vortex mixer (Fisher Scientific). The CNF/AuNP nanocomposites were then deposited on a gold side for use as a SERS substrate. 2.5. Characterization of the CNF/AuNP nanocomposite
2.3. Modification of CNF
Fourier transform infrared (FTIR) characterization was conducted with a FTIR spectrometer (Nicolet 380, Thermo Fisher Scientific, Waltham, WA, USA) in transmittance mode. Both unmodified and modified CNF with same concentration (5%) underwent centrifugation and precipitated gel-like CNF was collected for FTIR test. Spectra were acquired for a total of 64 scans in the wavenumber range from 400 to 4000 cm−1 with a resolution of 4 cm−1. Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, UK) was employed to measure ζ-potential of AuNPs and CNF. The as-prepared AuNP suspension was used without dilution and the unmodified and modified CNF were diluted to the concentration of 5%. Transmission electron microscopy (TEM) images were acquired for characterizing AuNPs, CNF, and their mixture
Preparation of cationic CNF was performed using (2,3-epoxypropyl) trimethylammonium chloride (EPTMAC) followed a previous published method (Hauser & Tabba, 2001). Briefly, 6 mL of CHPTMAC was transferred into a beaker to make a 50 mL solution with Milli-Q water, followed by the addition of 2 g of NaOH. The solution was kept stirring for 30 min to allow for the formation of EPTMAC. Then 1.25 g of CNF was immersed into the EPTMAC solution with stirring for 2 h, which allowed the epoxide to react with the hydroxyl groups of CNF and grafted ammoniums to the cellulose molecules and hence rendered cationic CNF. The modified CNF was then adjusted to pH 7 using pH meter (Oakton, Australia). 82
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Fig. 4. Optical images of CNF/AuNP nanocomposite (A & D) and corresponding SERS intensity maps (B) at 1075 cm−1 of 5 ppm of 4-ATP; representative SERS spectra of 4-ATP (C) collected from CNF/AuNP nanocomposite marked with squares in B; SERS spectra of 5 ppm of 4-ATP (E) collected from 16 randomly selected positions from the area indicated with red lines in the optical images A and D. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Averaged Raman spectra of thiram with concentration from 0 to 10 ppm (A); second derivative transformation of Raman spectra at 548 cm−1 (B); averaged Raman spectra of thiram in apple juice with concentration from 0 to 10 ppm (C); the plot of the log values of Raman peak intensity at 548 cm−1 versus the log values of concentration of thiram in apple juice (D).
the as-prepared CNF/AuNP nanocomposite on a slide and were then gold sputtered for 60 s prior to SEM imaging.
using a JEOL electron microscope (JEOL 1400, Tokyo, Japan) operated at an accelerating voltage of 120 kV. The TEM samples were prepared by concentrating the as-prepared AuNP suspension 10 times and making 5% unmodified CNF and CNF/AuNP mixture. Scanning electron microscopy (SEM) images were obtained using a FEI Scios DualBeam FIB SEM (Thermo Fisher Scientific, Hillsboro, Oregon, USA) at accelerating voltage of 5 kV. SEM samples were prepared by dropping 2 μL of
2.6. SERS measurement A DXR2 Raman microscope (Thermo Fisher Scientific) was used in this study. This system is equipped with a DXR 785 nm laser source. 83
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anionic surface properties (Sjöström, 1989), which is proven by the ζpotential values shown in Table 1. In addition, no strong chemical forces exist between CNF and AuNPs. Therefore, modification of CNF is a prerequisite for the interactions between anionic CNF and AuNPs. In this study, a novel strategy was taken by grafting positive ammonium ions to CNF to obtain cationic CNF, which allowed attachment of negatively charged AuNPs onto the CNF surface via electrostatic interactions. Scheme 1 illustrates the synthesis of cationic CNF (Guo, Filpponen, Su, Laine, & Rojas, 2016; Hyde, Dong, & Hinestroza, 2007). CHPTMAC reacted with NaOH to form EPTMAC. Epoxy moiety of EPTMAC went through a nucleophilic reaction with CNF under alkaline conditions, producing cationic CNF. Fourier transform infrared (FTIR) spectra of original CNF and corresponding cationic CNF are shown in Fig. S1. A typical spectrum of cationic CNF displays a new band at 1480 cm−1, which is assigned to the trimethyl groups of quaternized ammonium (Guo et al., 2016; Pei, Butchosa, Berglund, & Zhou, 2013). Additionally, the ζ-potential of modified CNF is positive (14.77 mV) (Table 1), which is in agreement with previous observations (Guo et al., 2016; Olszewska et al., 2011). These results indicate the presence of positive charges on the modified CNF surface. Both FTIR spectra and ζ-potential results provide clear evidences of successful introduction of quaternary ammonium groups on the CNF surface. The width of the CNF could be a few nanometers to several hundred nanometers (Fig. 1B). The broad size distribution could be attributed to heterogeneous mechanical treatment and the aggregation of individual fibers. Fig. 1C and D shows TEM images of modified CNF that were mixed with AuNPs. It is clearly observed that cationic CNF was coated with AuNPs due to strengthened interactions between cationic CNF and anionic AuNPs via electrostatic forces. This CNF/AuNP nanocomposite can be used as a SERS substrate. Conventional papers have been used in several studies for loading different metal nanostructures to serve as SERS substrates (Lee, Tian, & Singamaneni, 2010; Li et al., 2016; Wei & Huang, 2017). The spot size of Raman’s laser beam is ∼1 μm that is smaller than both width and length of cellulose fibers in the papers. In addition, the porosity and high roughness of the papers make it a challenge to achieve uniform distribution of nanoparticles on the paper. Compared with normal cellulose, nanocellulose with a fiber width in nanometer range provides much larger surface area for loading metal nanostructures, which could lead to higher sensitivity and improved homogeneity as a SERS substrate. In this study, filter paper coated with AuNPs was prepared and used as a comparison with CNF/AuNP nanocomposite. Fig. 2 displays SEM images of filter/AuNP film and CNF/AuNP nanocomposite at different magnifications. CNF (Fig. 2A and B) shows surface morphology in 2D structures and fibers with a diameter in nanometer size, demonstrating that nanofibers have much larger surface area that can load more AuNPs as compared to the filter paper in micrometer size (Fig. 2C and D).
During measurement, light from the high power (35 mW) laser was directed and focused onto the sample at a microscope stage through a × 10 objective with 25 μm pinhole aperture. Each spectrum was measured once with an accumulation time of 10 s. Spectra data were collected by OMNIC for Dispersive Raman software (Thermo Fisher Scientific). A typical procedure was as follows: 2 μL of as-prepared CNF/AuNP nanocomposite was deposited onto the slide and air-dried. A round-shaped film was thus formed with a diameter of 3 mm, and an aliquot of 2 μL of the sample solution was then placed on the film. The film with samples was air-dried prior to the SERS detection. In this study, 4-ATP was selected as a probing molecule because its thiol group can easily bind to gold, which can be used to evaluate the SERS effect for CNF/AuNP substrate. The reproducibility of the substrate was evaluated by acquiring SERS intensity maps of 5 ppm of 4-ATP solution. 2.7. Detection of thiram by the CNF/AuNP substrate A stock solution (100 ppm) of thiram was prepared by mixing 5 mg of thiram powders in 50 mL of acetone with stirring. The stock solution was subsequently diluted with acetone to reach a series of thiram standard solutions (0.05, 0.1, 0.5, 1, 5, and 10 ppm). Pure acetone solution was used as a control group. For detection of thiram in apple juice, thiram powders were spiked into organic apple juice with magnetic stirring to obtain 100 ppm of thiram in apple juice, from which a series of concentrations (0.05, 0.1, 0.5, 1, 5, and 10 ppm) of apple juice containing thiram were prepared. Apple juice with thiram was used as a control group. The SERS measurement for both pure thiram solution and apple juice samples was conducted as described above. 2.8. Data analysis Calibration curve was established by monitoring SERS intensity as a function of analyte concentration. Each point on the calibration curve represented an average value of seven replicate measurements. Limit of detection was calculated by determining the minimum distinguishable signals using the following expression:
s m = sb + kσsb where Sm is the minimum distinguishable signal, Sb is the Raman signal generated by a blank measurement of the SERS substrate in the absence of the analyte. k is the proportionality constant, and σsb is the standard deviation of blank measurements. The concentration value is determined by substituting the minimum distinguishable signal to the best-fit equation of the calibration curve to determine the limit of detection. 3. Results and discussion 3.1. Fabrication and characterization of CNF/AuNP nanocomposite
3.2. SERS performance of CNF/AuNP substrates AuNPs were selected as building blocks in this study because they are thermally and chemically stable (Chen, Kou, Yang, Ni, & Wang, 2008; Takahashi & Tatsuma, 2010). The preparation of AuNPs was based on the citrate reduction of gold salts in aqueous solution. As a reducing agent, sodium citrate reduced Au (III) in the HAuCl4 solution to Au (0), leading to the formation of AuNPs of certain size. Sodium citrate also served as a stabilizer, imparting negative surface charges to nanoparticles from weakly bound citrate ions, which prevented the aggregation of nanoparticles in solution. For resultant AuNPs, the UV–vis spectra show a narrow peak at wavelength of 531 nm (Insert of Fig. 1A) due to surface plasmon resonance. The TEM image in Fig. 1A shows that AuNPs are in spherical shape. The average size of AuNPs is 40.67 nm with a standard deviation of 7.73 based on measurement of 150 AuNPs. Citrate-reduced AuNPs are negatively charged and CNF have
Scheme 2 shows the mechanism and analysis process using CNF/ AuNP nanocomposite in SERS measurement. Negatively charged AuNPs and positively charged CNF were bound tightly via electrostatic attraction. For individual fibers, many AuNPs were aggregated on the CNF surface, which created SERS “hot-spots” that mostly occur at consecutive NPs and the gaps between the NPs and their attached surface (Satheeshkumar et al., 2017). Two hot-spot zones are shown in the Scheme 2. In addition, CNF consists of countless fibers that can be loaded with numerous AuNPs that would contribute to enhanced Raman scattering signal from analyte molecules. Fig. 3A shows the SERS spectra of 1 ppm of 4-ATP deposited on the CNF/AuNP nanocomposite and a filter paper/AuNPs. The 4-ATP peaks in the SERS spectra are in good agreement with the spectra reported in previous studies (Cialla et al., 2012; Zhang et al., 2015). However, a 84
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increased as the concentration of thiram increased from 0 to 10 ppm. Thiram in apple juice needs to be extracted by organic solvents before the determination by HPLC. In contrast, apple juice samples contaminated with thiram can be directly placed on a CNF/AuNP substrate for SERS measurement. A transparent layer was formed on the substrate after the droplet was dried due to sugars in apple juice. However, this layer caused little interference with spectral collection. Fig. 5C shows the spectra of apple juice spiked with different concentrations of thiram from 0 to 10 ppm, exhibiting characteristic thiram peaks similar to the spectra of thiram in acetone solution. The pure thiram solution yielded much higher Raman intensity than the same concentration of thiram in apple juice. This is because other components in apple juice competed with thiram molecules and blocked the way of thiram molecules binding to AuNPs, resulting in reduced Raman intensity of thiram. Fig. 5C clearly shows characteristic peaks of thiram at 548, 930, 1138, and 1370 cm−1. In addition, an evident peak was observed at 734 cm−1 in all the spectra from 0 to 10 ppm of thiram in apple juice. This interfering band was likely from sugars in apple juice. As shown in Fig. 5D, a good linear correlation (R = 0.9323) was established by plotting the log values of the SERS intensity and 4-ATP concentration. The limit of detection was calculated to be 52 ppb, which is well below the maximum residue limit (MRL) in fruit of 7 ppm set by the U.S. Environmental Protection Agency (EPA). The results show that SERS coupled with CNF/AuNP nanocomposite can be used as a sensitive method to detect pesticides in apple juice.
slight shift in Raman bands can happen due to a strong polarization occurred at the surface of AuNPs (Gong et al., 2014). The intensity of SERS spectra obtained from CNF/AuNP nanocomposite was found to be nearly 20 times higher than that from the filter paper/AuNP substrate, which proved that SERS performance was greatly enhanced using CNF compared to filter paper because more SERS “hot-spots” were created on CNF/AuNP nanocomposite. To further test the sensitivity of the CNF/AuNP nanocomposite, this substrate was employed for the detection of trace amount of analytes. Fig. 3B shows the averaged SERS spectra (n = 7) of different concentrations of 4-ATP in acetonitrile. The characteristic peaks of 4-ATP were clearly observed in the SERS spectra of 0.05–10 ppm of 4-ATP solutions in the range from 900 to 1700 cm−1. The absence of this peak in the control sample proves that the peak was caused by 4-ATP. There is an increasing trend of Raman intensity of the peak at both 1076 and 1583 cm−1 as the concentration of 4-ATP increased. In addition, the SERS peaks were still distinguishable when the concentration reached 0.05 ppm, indicating that CNF/AuNP nanocomposite could be used for detecting trace amount of analytes. These results demonstrate high sensitivity of CNF/AuNP nanocomposite in SERS measurement. The reproducibility of the SERS substrate is important for quantitative analysis of analyte molecules. In this study, 5 ppm of 4-ATP was used to assess the reproducibility of SERS signals. As indicated in the boxed optical image (Fig. 4A), a SERS map of 4-ATP was collected over a 150 × 150 μm2 area on a CNF/AuNP nanocomposite (Fig. 4B). The SERS intensity map corresponding to the Raman band at 1076 cm−1 exhibits acceptable uniformity with a relative standard deviation (RSD) of 18%, which was calculated from 36 pixels in the map. To visually demonstrate the degree of the difference among different regions, three representative spectra were extracted from low to high intensity region of the SERS map (Fig. 4C). No significant difference was observed, further confirming a small variation in the SERS signals over the mapped area. What’s more, a point-by-point SERS mapping was conducted by measuring 5 ppm of 4-ATP on the CNF/AuNP nanocomposite to further characterize the homogeneity of the substrate (Figs. 4D & E). As shown in Fig. 4D, 16 positions covering a large area (area square, total square) was captured for collecting SERS signals and the corresponding map (Fig. 4E) shows that the SERS enhancement is uniform with a RSD of 25.47%. Based on the data analysis from area and pointto-point maps, the reproducibility is found to be acceptable considering the wide width range of CNF.
4. Conclusion In summary, facile synthesis of CNF/AuNP nanocomposite was achieved and the film was used as a SERS substrate. Compared with filter papers, nanoscale CNF have much larger surface area and allow for more homogeneous and dense loading of AuNPs. AuNPs were adsorbed on the CNF through electrostatic attraction after CNF cationization, creating more “hot-spots” for SERS enhancement. CNF/AuNP nanocomposite was used for measurement of 4-ATP and satisfactory sensitivity and reproducibility were achieved. Detection of thriam in apple juice was conducted by SERS and the limit of detection was 52 ppb. These results demonstrate that CNF/AuNP nanocomposite has a great potential to be used as a SERS substrate in detection of pesticides in food products. Acknowledgements
3.3. Detection of thiram in apple juice by SERS We acknowledge the support from USDA NIFA Multi-state Project (NC1194) and the National Natural Science Foundation of China (Grant Nos. 31728016 and 61775133). We thank Dr. Bongkosh Vardhanabhuti for her assistance in the Zetasizer measurement.
Even though GC/LC–MS is often the gold standard method for pesticide detection, analysis of thiram involves conversion into carbon disulfide (CS2) in an acid medium prior to estimation using spectroscopy and chromatography due to inherent insolubility in common extraction solvents and poor stability (Mujawar, Utture, Fonseca, Matarrita, & Banerjee, 2014), which makes the analysis process complicated. With simple sample preparation, SERS has been considered a powerful tool to detect thiram. Fig. 5A shows the average Raman spectra (n = 7) of different concentrations of thiram in acetone solution from 0 to 10 ppm. The characteristic peaks of thiram can be clearly observed in the Raman spectra. A peak at 548 cm−1 is assigned to SS stretching mode. A peak around 1370 cm−1 is attributed to the CH3 deformation and the CN stretching. The peaks at 1138 cm−1 and 1504 cm−1 are assigned to the CN stretching and CH3 rocking modes (Saute & Narayanan, 2011). Although some noise signals could be detected in the spectra of the control group, which was attributed to acetone, they didn’t cause significant interference to the characteristic peaks of thiram due to high signal-to-noise ratio. Second derivative transformation is commonly used in spectral data analysis to separate the overlapping peaks, eliminate the baseline drifting, and enhance the spectral resolution. It is clearly shown from the second derivation transformation that the intensity of a peak at 548 cm−1 (Fig. 5B)
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