Simultaneous in-situ Extraction and Fabrication of SERS Substrate for

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Simultaneous in-situ Extraction and Fabrication of SERS Substrate for Reliable Detection of Thiram Residue Miao Chen, Wen Luo, Qi Liu, Naiying Hao, Yuqiu Zhu, Minzhuo Liu, Lumin Wang, Hua Yang, and Xiao-Qing Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03940 • Publication Date (Web): 31 Oct 2018 Downloaded from http://pubs.acs.org on November 1, 2018

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Analytical Chemistry

Simultaneous in-situ Extraction and Fabrication of SERS Substrate for Reliable Detection of Thiram Residue

Miao Chen1#, Wen Luo1#, Qi Liu1, Naiying Hao1, Yuqiu Zhu1, Minzhuo Liu1, Lumin Wang1, Hua Yang*,1, Xiaoqing Chen*,1,2

1College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, Hunan, China 2 Key Laboratory of Hunan Province for Water Environment and Agriculture Product Safety, Central South University, Changsha 410083, Hunan, China # The

authors contributed equally to this work

*Corresponding author: Tel:/fax: +86-731-88830833. E-mail address: [email protected] (Hua Yang), [email protected] (Xiaoqing Chen)

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Abstract We report a novel strategy of simultaneous in-situ extraction and fabrication of SERS substrate (IE-SERS) to perform selective and reliable on-site determination of thiram residue in soil, fruits and vegetables. In this protocol, the thiram residue on complex surfaces can facilely diffuse into the solvent (DCM) and specifically bind to AuNPs, affording the SERS substrate through the embedding of the thiram-trapped AuNPs into the cellulose p-toluenesulfonates (CTSA) film through the evaporation of DCM. SERS signals of the specifically prepared CTSA could be used as internal standard to calibrate the absolute signal of thiram, which can avoid the fluctuation of SERS intensities caused by uneven and irregular morphology of SERS substrate. Thus, reliable quantitation of thiram through SERS detection and superior reproducibility in the SERS measurement (RSD=4.21%) were achieved. As for directly sensing the thiram residue in soil, the established method shows strong anti-interference ability and a good linear response from 0.1 to 12 μg/g with a low LOD of 50 ng/g, which is lower than that of all the previously reported methods. The recoveries range from 91.76 to 112.3 % for thiram in paddy soils, indicating that the established IE-SERS method is reliable and applicable to the detection of thiram residue in real soil samples. In addition, the measurement of the residual thiram on strawberry and cucumber surface was also successfully accomplished by this strategy, indicating that the established method also has great potential in the in situ ultrasensitive detection of thiram on irregular fruits and vegetables.

Keywords: surface-enhanced Raman scattering (SERS), in situ extraction, thiram residue, cellulose p-toluenesulfonates, soil


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Introduction Thiram is a dithiocarbamate fungicide, which is widely used as seed dressing to prevent various crop diseases and preservative to preserve mature fruits and vegetables during storage and shipment1-2. However, its overuse would give rise to soil pollution and even seep into groundwater or mix with air dust, causing high toxicity to human skin and mucosa. Likewise, uptake of thiram from fruits and vegetables could induce dyspnea, ataxia, convulsions, and severe fetal malformations3. To date, several techniques have been explored for the detection of thiram residue, such as spectrophotometry combined with solid-phase extraction4-5, chromatographic methods (LCMS)6-7, enzyme-linked immunosorbent assay (ELISA)8, and enzyme inhibition9. However, the necessity of time-consuming sampling and separation procedures, sophisticated detection protocols, unsatisfactory detective sensitivity and high cost significantly limited wide application of these techniques, especially in on-site analysis. Surface enhanced Raman scattering (SERS) featuring rapid readout, rich molecular information, nondestructive data acquisition, and excellent sensitivity has gradually become a promising tool in food safety10-11, environmental monitoring12, and life science13-14. Over recent years, owing to the development of SERS substrate, a variety of SERS-based platforms have been established for onsite rapid detection of thiram residue. One commonly used strategy is to disassociate the thiram residue from surface matrix by adding a droplet of ethanol onto the targeting sampling surface. After completely evaporating the solvent, the designated noble metal nanostructures colloid was spread to this site, allowing for direct record of Raman signals in this area15-17. For instance, Han reported a SERS-based detection of thiram on fruit peels by simply dropping Au@Ag nanoparticles onto the sample surface, realizing the in situ and field-portable SERS assay17. But this method suffers from a significant limitation in quantitative detection due to the varied surface morphologies and uncontrolled particle aggregation. The aggregated nanoparticles were distributed on tiny, rugged and irregular surfaces, causing marked fluctuation in the strength and number of hotspots in the detection volume and variation of the obtained SERS signals. In addition, tissues of fruits and vegetables may cause fluorescence interference during SERS detection. In order to solve this problem, more stable and sensitive SERS probes were developed by using flexible SERS substrates, in which plasmonic nanostructures are constructed onto flexible solid supporter and protected from 3 ACS Paragon Plus Environment


aggregation while their surfaces are still accessible to the analytes. These substrates can be attached to rough and irregular (i.e., nonplanar or ripply) surfaces and employed to collect samples directly, offering a noninvasive or minimally invasive method of sample analysis18. Currently, several groups have used flexible SERS substrates to analyze thiram residue19-21. For example, in a “paste and peel off” approach, thiram residue in fruits and vegetables could be directly extracted to the as-prepared flexible SERS substrates and analyzed22. In order to improve sampling efficiency on complex surface, Han designed a gecko-inspired nanotentacle SERS substrate, in which 3D “tentacle” with high density can approach microarea freely and make extensive contact towards almost any surface21. However, due to the uncertainty of reproducibility and the accuracy of measurement results in practical application, limitations also exist in quantitative detection by employing these substrates as it is still a challenging task to achieve uniform distribution of target molecules on the hotspots of the SERS substrate by “paste and peel off”, “press and peeled-off” or swab, respectively. In order to solve this problem, Fang reported a SERS-enabled micropipette for detection of surface thiram residues through a “drop-self-priming measure” detection mode23. Duo to the hydrophobicity of thiram, this method also need to first extract thiram from solid sample by dropping ethanol. Moreover, all these previous works on thiram residue detection are focused on determining the quantity of thiram on the surface of fruits and vegetables, while rarely on the detection of thiram residue in soil. As well known, the components in soil are more complicated than that on the surface of fruits and vegetables, which gives rise to intimidating hurdle towards the selective extraction and detection of thiram. Besides, the surface of soil is too tiny to accomplish direct sampling by traditional colloidal, flexible SERS substrates, or SERS-enabled micropipette. Therefore, these current challenges motivated us to develop a strategy, which can generally achieve in-situ extraction of thiram from different samples including soil, fruits, and vegetables, and uniform target molecules distribution on SERS substrate for the assay of thiram residue. Herein, we report a novel strategy through the simultaneous in-situ extraction and fabrication of SERS substrate (IE-SERS) to perform a selective and reliable on-site determination of thiram residue in soil, fruits and vegetables. The work mode of this strategy is outlined in Scheme 1. Firstly, octadecylamine (ODA)-modified AuNPs (AuNPs-ODA) and cellulose p-toluenesulfonates (CTSA) are well dispersed in dichloromethane (DCM) as the reaction matrix (Au/CTSA). Then, the 4 ACS Paragon Plus Environment

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incubation of Au/CTSA mixture with solid samples would allow the hydrophobic thiram residue to migrate into the reaction matrix. By virtue of displacement reaction between citrate-ODA and thiram, thiram would be uniformly attached onto AuNPs through Au-S bonding. At this stage, thiram can be selectively captured from irregular sample surfaces via in-situ extraction and the following evaporation of DCM leads to the formation of CTSA film. Meantime, the thiram-modified AuNPs would be self-assembled to produce considerable “hot spots” ensuring high enhancement of SERS signals, thus generating a flexible SERS substrate for recording the Raman signals. Since the p-toluenesulfonate moiety in CTSA can used as the internal standard to calibrate the absolute SERS signal of thiram, the fluctuation of SERS intensity caused by uneven and irregular morphology of SERS substrate can be effectively avoided, ensuring the reliability of SERS detection. Finally, the feasibility of practical application of this strategy would be assessed by the quantitative detection of thiram residue in soil, fruits (strawberry) and vegetables (cucumber).

Scheme 1. Schematic representation of simultaneous in-situ extraction and fabrication of SERS substrate in the detection of thiram residue on irregular samples.

Experimental Detection of thiram by IE-SERS. For the detail process of synthesis of CTSA (1.1) and AuNPsODA (1.2), the preparation of real samples (1.3), materials (1.4) and instruments (1.5), please see Supporting Information (SI). Firstly, 0.075 g of CTSA was dissolved in 3 mL of DCM. Then 200 μL of CTSA DCM solution, 150 μL of DCM, and 50 μL of AuNPs-ODA DCM solution were mixed and dispersed by means of ultrasonication and used as Au/CTSA reaction matrix for further analysis. 5 ACS Paragon Plus Environment


For the SERS performance, optimization, reproducibility and concentration gradient measurement, SERS sample was prepared as follows: 10 μL of thiram in DCM solution was added into a centrifuge tube. After the DCM was evaporated, the Au/CTSA reaction matrix was added into the tube, which was immediately capped tightly. The mixture was ultrasonicated for 30 s and incubated for 30 min. Then, the Au/CTSA film was removed from the tube with tweezers after the evaporation of DCM. The laser beam was directly focused on the Au/CTSA film and the exposure time was 5 s with 3 accumulations for each measurement. For rapid detection of thiram residue in soil, the detection process is illustrated in Figure 1: 1) 20 mg of soil sample was directly placed into a centrifuge tube without any pretreatment. 2) The Au/CTSA reaction matrix was added into the tube and incubated with the soil sample for 30 min. Thiram in soil diffused to the DCM solution and was attached onto AuNPs, realizing an in-situ extraction process. 3) During the incubation time, the soil sample was precipitated while the Au/CTSA film was fabricated on the inner wall of the tube with the evaporation of DCM. 4) The Au/CTSA film was moved out from the tube with tweezers while the soil still remained in the tube. 5) The laser beam was directly focused on the Au/CTSA film and the exposure time was 5 s with 3 accumulations for each measurement.

Figure 1. Photographs sketch of detection of thiram in soil by the proposed IE-SERS method. For rapid detection of thiram residue on the surface of strawberry and cucumber, as shown in Figure 2: (a) A strawberry or cucumber was placed in a culture. (b) 100 μL of Au/CTSA reaction matrix was dropped on the strawberry or cucumber, the thiram residue diffuse to the DCM solution and was attached onto AuNPs, Au/CTSA film was in situ fabricated with the evaporation of DCM. (c) The Au/CTSA film was peeled off from the surface with tweezers. (d) The film was transferred to the object stage and the laser beam was focused on the film. The exposure time was 5 s with 3 accumulations for each measurement. 6 ACS Paragon Plus Environment

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Figure 2. Photographs sketch of detection of thiram on the surface of strawberry and cucumber by the proposed IE-SERS method.

Results and discussions Fabrication and characterization of Au/CTSA substrate. Cellulose has been employed to develop low-cost SERS substrates by incorporating precious metal nanoparticles due to its extraordinary mechanical, thermal and optical properties24-25. However, its poor solubility either in water or common organic solvents would cause the poor efficiency in the contact of cellulose with colloidal precious metals or metal precursor (such as AuCl4-), resulting in uneven and sparse particle distribution on cellulose. Herein, cellulose p-toluenesulfonates (CTSA) were prepared and used as an ideal template for the fabrication of SERS substrate owing to their advantageous features: (1) Good solubility in DCM allows for mixing with colloidal AuNPs. (2) Facile self-assembly facilitates the formation of transparent film with the evaporation of DCM. (3) Tosyl groups in CTSA serve as internal standard to calibrate the absolute signal of each SERS measurement. (4) Cellulose backbone in CTSA has a low Raman scattering cross section, which is essentially optically transparent and would avoid introducing Raman interference. As shown in Figure S2 (SI), compared with the microcrystalline cellulose (a), the synthesized CTSA can be dissolved in DCM to form a transparent solution (b), which can be fully mixed with the colloidal AuNPs in any ratio. FTIR spectra (Figure S3A, SI) were collected to elucidate the structural changes in the homogeneous reactions of cellulose. FTIR spectra of CTSA (d) show typical absorptions of the cellulose backbone as well as characteristic peaks of the aromatic moieties at 1598, 1550 and 798 cm-1, respectively26. Furthermore, two bands with high intensities at 1350 and 1152 cm-1 (v SO2) indicate the presence of tosyl groups27. To further confirm the element compositions of CTSA, XPS was also performed. From Figure S3B, it can be seen that the typical peaks of O 1s, C 1s, and S 2p are present around 535, 287, and 170 eV, respectively. The peaks at 7 ACS Paragon Plus Environment


283.47, 284.86, 285.16 and 286.75 eV in high-resolution C 1s XPS spectrum (Figure S3C) correspond to C−C/C=C, C−S, O−C−O, and C−O, respectively. In the O 1s XPS spectrum (Figure S3D), the peaks at 531.96 and 532.90 eV are attributed to O−C and O=S. The S 2p spectrum (Figure S3E) has two peaks located at 168.62 and 169.36 eV, which can be assigned to C−S and S=O.

Figure S4 shows a standard 13CNMR spectrum with proton-decoupling of CTSA. Resonances assigned to the tosyl methyl and aromatic ring carbons are visible at δ = 20.5 and 124.8-144.5 ppm, respectively. All these XPS and NMR results further confirm the success of the ptoluenesulfonylation of cellulose. To mix with CTSA DCM solution, the citrate-coated AuNPs were transferred from aqueous phase to organic phase (DCM) by modifying ODA through the electrostatic interaction between amino and citric groups. As shown in the photographs of Figure S2 c, the obtained AuNPs-ODA can be dispersed in DCM homogeneously. To identify the structural changes after surface modification of ODA, FTIR spectrum was collected (Figure S3A). Compared with the original AuNPs (b), the most relevant differences in the FTIR spectra of AuNPs-ODA (a) are the new bands at 3350, 3070, and 2985 cm-1. The narrow peak at 3350 cm-1 could be ascribed to the N-H stretching vibration of amino group. The absorptions at 2985 and 3070 cm-1 are attributed to the CH2 and CH3 stretching vibrations of alkyl chain28. The above results indicate that ODA has been successfully modified on AuNPs. Furthermore, the TEM image (Figure S5, SI) reveals that the obtained AuNPs-ODA are well dispersed and the average diameter based on Gaussian estimation is about 43 nm. The synthesized CTSA DCM solution and Au/CTSA DCM mixture were poured into a polytetrafluoroethylene mold, respectively. DCM was evaporated at room temperature, giving a transparent CTSA film (as shown in the inset photographs, Figure 3a) and a dark blue Au/CTSA film (Figure 3b). The UV-vis spectra in Figure S3F (SI) show that the CTSA film has no characteristic absorption peak, the monodispersed AuNPs-ODA displays a strong characteristic band at 536 nm, while the Au/CTSA film exhibits an absorption band at 565 nm. The red shifted absorption band from 536 to 565 nm can be ascribed to the strong coupling of the plasmons between adjacent AuNPs, indicating the formation of the assembled AuNPs in the CTSA film. In order to confirm this hypothesis, SEM observation was performed. The surface of CTSA film is smooth and flat (Figure 3a), which becomes a rough and “hilly terrain” shaped structure (Figure 3b) after the 8 ACS Paragon Plus Environment

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embedding of AuNPs. EDX analysis (Figure 3 c,d) also clearly shows the presence of abundant Au (AuNPs) in Au/CTSA film (Pt was sprayed onto the sample before analysis). The dense AuNPs were self-assembled into CTSA film and numerous “hot spots” were thus formed, which could significantly amplify Raman signals of analytes adsorbed on its surface. Given that it is difficult to directly characterize the morphology of Au/CTSA film by TEM, the Au/CTSA reaction matrix was then dropped onto the copper grid and then the TEM images were collected until DCM was completely evaporated. Figure 3 (e, f) further demonstrate that the AuNPs were self-assembled into the CTSA film. The red arrows indicate the transparent CTSA film.

Figure 3. SEM observation of the CTSA film (a) and Au/CTSA film (b), the scale bar is 20 μm, the inset are the photographs of CTSA film (a) and Au/CTSA film (b); EDX spectrum of CTSA film (c) and Au/CTSA film (d); TEM view of the Au/CTSA mixture that were dropped on the copper grid after the DCM were evaporated (e,f), the red arrows indicate the transparent CTSA film. SERS performance assessment of the Au/CTSA substrate using IE-SERS. To evaluate the performance of the proposed IE-SERS method in the detection of thiram, thiram standard solution (10-4 M) was selected as the probe molecule. For the purpose of comparison, Raman spectra of six samples (Figure 4) including AuNPs-ODA, CTSA film, thiram powder, AuNPs-ODA incubation with thiram, Au/CTSA film after extraction of thiram, and Au/CTSA film were collected and recorded under the same conditions. Obviously, AuNPs-ODA exhibits no Raman signals (Figure 4a), indicating that the modified ODA with low Raman scattering cross section would not introduce Raman interference. After the incubation of AuNPs-ODA with thiram (Figure 4d), the SERS 9 ACS Paragon Plus Environment


spectrum greatly differs from normal Raman spectrum of thiram powder (Figure 4c), main characteristic vibrational bands at 1139 cm-1 belonging to C-N stretch or CH3 deformation, and 1368 and 1484 cm-1 belonging to CH3 deformation or C-N stretch can be observed respectively, which correspond to the spectral feature (the sulfur atom attached onto AuNPs) of thiram29. Besides, the strongest band at 551 cm-1 attributed to S-S stretch from thiram powder becomes much weaker in the SERS spectrum. The characteristics suggest that the thiram molecule easily forms the resonated radical structure when interacting with the surface of AuNPs, leading to the S-S bond cleavage of thiram, which give rise to two dimethyl residues that are strongly adsorbed onto AuNPs by replacing the original citrate-ODA ligands on the surface30. No Raman signal was detected on bare CTSA film without embedding AuNPs (Figure 4b), whereas intense scattering signals were detected on Au/CTSA film (Figure 4f). The most prominent peaks at 1071 and 1585 cm-1 correspond to the excitation of ring breathing and axial ring deformation modes, which was exclusively registered from the tosyl groups on CTSA31. These significant differences between bare CTSA film and Au/CTSA film indicate that CTSA which located in close proximity to the surface of embedded AuNPs would be exposed to the electromagnetic field produced by laser irradiation and effectively detected through SERS effect. The SERS spectrum of the Au/CTSA film obtained after extraction of thiram is shown in Figure 4d, in which main vibrations of CTSA and thiram were recorded simultaneously and the characteristic bands can be clearly identified. Considering the accuracy and effectiveness, the strongest signal bands of the internal standard CTSA (1071 cm-1) and thiram (1368 cm-1) were finally selected as the assessment standard of intensities in quantitative analysis.

Figure 4. Raman spectra of (a) AuNPs-ODA, (b) CTSA film, (c) thiram powder, (d) AuNPs-ODA incubation with thiram (10-4 M), (e) Au/CTSA film obtained after extraction of thiram (10-4 M), and (f) Au/CTSA film. 10 ACS Paragon Plus Environment

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As well known, the reproducibility of SERS signal is an important factor for the accuracy of quantitative analysis. In the IE-SERS procedure, the AuNPs-ODA and CTSA were uniformly dispersed in DCM, then the thiram molecules were expected to diffuse into DCM and spread evenly on the surfaces of AuNPs during the in-situ extraction. As the solvent evaporated, the thirammodified AuNPs and CTSA were self-assembled into a film in a certain proportion. During SERS detection, the internal standard (CTSA that located close to AuNPs) and thiram coexist in the same physical and chemical environment with the hotspots, and the Raman intensity of thiram could be normalized with respect to the internal standard. In this way, the fluctuation of SERS intensity caused by the uneven and irregular morphology of SERS substrate would be suppressed. In order to verify our supposition, reproducibility experiment was carried out. IE-SERS was performed as described in the experimental section and the corresponding Au/CTSA film was collected. SERS spectra were recorded from 10 different measurement spots, which were repeated in 20 randomly chosen areas on the Au/CTSA film. Twenty representative SERS spectra randomly selected from the 200 individual points are shown in Figure 5a. It is notable that all the SERS active sites show relatively inconsistent Raman intensities, which might be caused by the irregular shape and varied thicknesses of the Au/CTSA film. Induced by the gravity, the film on the bottom is generally thicker than that on the top as shown in Figure 1. Accordingly, the film on the bottom shows higher Raman intensity. However, the relative standard deviation (RSD) of the normalized intensity of thiram (I1368/I1071) integrated of the 200 individual spectra was calculated to be about 4.21 % (Figure 5b), which is better than the results acquired from the SERS substrates prepared by the previously reported advanced nanoengineering processes12,

32-33.

On the basis of these results, it can be

concluded that the proposed IE-SERS exhibits superior reproducibility, which is crucial to practical assays.

Figure 5. (a) SERS spectra of 20 points randomly selected from the 200 individual points on the 11 ACS Paragon Plus Environment


Au/CTSA film and (b) the normalized intensity of thiram (I1368/I1071) distribution. In IE-SERS procedure, the Au/CTSA reaction matrix was incubated with the samples to extract thiram. After the solvent (DCM) was evaporated, the obtained Au/CTSA film was directly used for SERS detection. A noteworthy fact is that the amount of AuNPs-ODA and CTSA in DCM would significantly affect the structure and SERS property of the assembled Au/CTSA film. On one hand, if there was insufficient CTSA in reaction matrix, CTSA was unable to serve as a template to fix the assembled AuNPs with the evaporation of DCM. When it was taken out, the CTSA hardly maintained the structure of AuNPs layer and the fabricated film would be destroyed. Furthermore, due to lack of sufficient CTSA, the Raman signal of CTSA would be too weak to serve as the inner standard molecules to calibrate the absolute signal of each SERS measurement. On the other hand, if there was not enough AuNPs-ODA in the reaction matrix, the AuNPs were unable to provide sufficient reaction sites to capture thiram molecules during in situ extraction. Besides, the AuNPs in Au/CTSA film were sparsely assembled and hardly acquired strong electromagnetic field enhancement, leading to the decreased SERS activity. Therefore, the amount of the AuNPs-ODA and CTSA in reaction matrix was optimized to achieve the stronger normalized intensity of thiram (I1368/I1071). Different amounts of AuNPs-ODA including 10, 50, 100, 150 and 200 μL were added to the reaction matrix, while the amount of CTSA (200 μL) remained constant and the final total volume of the reaction matrix was 400 μL by adding the solvent (DCM). Thiram standard solution (10-4 M) was selected as the probe molecule and the IE-SERS was performed as described in the experimental section. Consequently, all the Au/CTSA films were obtained and the corresponding SERS spectra were accordingly collected (Figure S6a, SI). Compared to other conditions, the reaction mixture containing 50 μL of AuNPs-ODA showed the highest normalized intensity of thiram (I1368/I1071) (Figure S6b) and was used in the following tests. Direct sampling and detection of thiram in soil samples by IE-SERS. The real soil sample (located in Changsha) was calcinated at 500 °C for 3 h to remove the organic residues. To test the effect of other soil components on the detection of thiram, real soil sample, real soil sample spiked with thiram, calcinated soil sample, and calcinated soil sample spiked with thiram were detected by the proposed IE-SERS method respectively, and the corresponding SERS spectra were collected. As shown in Figure S7, the calcinated soil sample exhibits characteristic Raman bands of CTSA 12 ACS Paragon Plus Environment

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(a), which is similar to that in Figure 4 f. Expectedly, after being spiked with thiram, the characteristic peaks of thiram and CTSA could be observed in the spiked calcinated soil sample (b). Other peaks are hardly detectable in the real soil sample, except the characteristic peaks for thiram and CTSA (c), indicating that other components in soil are undetectable by the IE-SERS method. After spiking thiram into the real soil, intense scattering signals of thiram and CTSA were detected (d) whereas no other Raman signal was detected. It is evident that the SERS spectra of thiram in real soil (d) are quite similar to that in calcinated soil (b). Besides, the intensity ratio of thiram to CTSA (1368 cm-1/1071 cm-1, 0.298) is larger than that in calcinated soil (0.246), possibly due to the existence of the inherent thiram. Therefore, in order to avoid the interface of the inherent thiram, different amounts of thiram (0.1 to 36 μg/g) were added into the calcinated soil sample and used as standard samples. As shown in Figure 6a, primary vibrations of thiram and CTSA were recorded simultaneously in each SERS spectrum except the blank. The intensity ratio of thiram to CTSA (1368 cm-1/1071 cm-1) increased with the increased concentrations of thiram (Figure 6b), indicating that the normalized intensity of thiram was proportional to the amount of thiram extracted from soil onto the surfaces of the embedded AuNPs. When the amount of thiram is more than 14.4 μg/g, the rising trend of the normalized intensity slows down. The normalized intensity of thiram versus the concentration of thiram shows a correlation coefficient of 0.9950 in the range of 0.1 to 12 μg/g. And the corresponding LOD (3σb/s) is 50 ng/g. These results illustrate the high determination sensitivity of IE-SERS toward thiram in soil.

Figure 6. (a) SERS spectra of different concentrations of thiram in soil and (b) the variation in intensity ratio of vibration located at 1368 cm-1 and 1071 cm-1 with different thiram concentrations. We employed seed dressing (parathion-methyl), pesticides (2,4-D, chlorpyrifos), other soil organic pollutants (methylene blue, malachite green, naphthoquinone, p-nitrophenol) and 13 ACS Paragon Plus Environment


compound with disulfide bond (2,2'-dipyridyl disulfide) as interfering substances to evaluate the selectivity of the developed IE-SERS method. The soil sample was spiked with thiram and these interfering substances (20 μg/g) and detected by IE-SERS approach. For comparison purposes, SERS spectra of thiram and 2,2'-dipyridyl disulfide detected by IE-SERS approach and the interfering substances were also collected. The experimental results are shown in Figure 7, we can easily identify the characteristic Raman shifts of 2,2'-dipyridyl disulfide (998 cm-1), thiram (1368 cm-1) and CTSA (1071 cm-1) in the spiked soil sample, whereas no Raman signals for other interfering substances were detected (indicated by the dotted lines). This could be explained by the unique properties of IE-SERS that disulfide bond in the target molecule breaks spontaneously upon exposure to the Au surface to replace the citrate-ODA groups binding to AuNPs via stronger Au-S bonds. But other compounds are unable to bind to AuNPs due to the absence of active groups (disulfide bond), which would stay too far away from hotspots to give a SERS response. Although both 2,2'-dipyridyl disulfide and thiram can be detected by the IE-SERS method, the characteristic bands of thiram and CTSA can be clearly identified and won’t be disturbed by the signature band of 2,2'-dipyridyl disulfide (indicated by the gray rectangles). On the basis of these results, it can be concluded that IE-SERS demonstrates good selectivity and anti-interference capability in the thiram analysis of soil samples.

Figure 7. SERS spectra of the spiked soil sample, thiram, and 2,2'-dipyridyl disulfide detected by IE-SERS approach and the interfering substances.


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With the calibration curves in hand, spike-and-recovery experiments were performed to evaluate the practicality of the proposed IE-SERS method. Paddy soil located in Changsha (20 mg) spiked with thiram standards at three different concentrations (0.5 μg/g, 5 μg/g, 10 μg/g) were employed as spiked soil sample. IE-SERS was performed as described in the experimental section. As demonstrated in Table S1 (SI), the related recoveries are in a range of 91.76-112.3 %. The variation coefficients are in the range of 1.02-4.56 %, indicating that the established IE-SERS method is reliable and could be applied to the detection of thiram residue in soil. Table S2 (SI) shows the performance comparison of this method with other thiram detection methods for soil samples. It is worth noting that UV spectrophotometry5,

34,

ion mobility

spectrometer35, and HPLC36 method have some problems in common: time-consuming and sophisticated detection procedures, unsatisfactory detection sensitivity and high cost. The more sensitive method is the SERS based on triangular silver nanoplates (TSNPs)37. However, as TSNPs are unstable in the untreated soil extract, the soil extract needs to be treated with PSS to neutralize the charges of the positively charged substances before SERS detection. In this work, novel IESERS approach was established to detect thiram in soil. The LOD of this method is lower than that of all other methods. Furthermore, this method can directly detect thiram in soil without any other pretreatment process, which has strong anti-interference ability and a comparable quantification range. Direct sampling and detection of thiram in fruits and vegetables by the IE-SERS. To illustrate the analytical capability of the IE-SERS method for surface thiram residue, 10 μL of thiram DCM solution at different concentrations was added into the centrifuge tube and used as standard samples after DCM was evaporated. Then the thiram residue in the tube was detected by the IESERS method as described in experimental section and the corresponding SERS spectra were collected from randomly selected positions on the Au/CTSA films. As shown in Figure S8A (SI), primary vibrations of thiram and CTSA were recorded simultaneously in each SERS spectrum. As the above concentration gradient experiments of thiram in soil, the normalized intensity of thiram was consistent with thiram content value although the change of SERS intensity has no regulation with the increased concentrations of thiram. As shown in Figure S8B (SI), the intensity ratio of vibration located at 1368 cm-1 and 1071 cm-1 versus the concentration of thiram shows a correlation 15 ACS Paragon Plus Environment


coefficient of 0.9958 in the range of 0.1 to 10 μM. And the corresponding LOD (3σb/s) is 0.06 μM. To illustrate the sampling efficiency of the IE-SERS method and its potential in food safety detection, quantitative detections of thiram residues on fresh fruit and vegetable peels (strawberry and cucumber) with a rather rugged and irregular surface were carried out without any other pretreatment process. First, a droplet (10 μL) of the thiram sample at different concentrations (0.1, 0.8, 2, 8 μM) in DCM was spread on the strawberry and cucumber peels. Afterward, DCM was completely evaporated at room temperature to simulate the situation of real samples. Subsequently, the Au/CTSA reaction matrix was added on the narrow sample peels to extract the analytes. As the rough surface of sample peels renders the difficulty in the effective sampling or detection by the commonly used swabbing or “paste and peel off” approach, the reaction matrix is so malleable that it can easily access every part on the peels to allow the target thiram molecules to bind to AuNPs. Upon the complete evaporation of DCM, the laser spots were focused on the self-assembled Au/CTSA film to collect the corresponding Raman signals. Since the film was separated from the peels, the fluorescence interference caused by the peel can be effectively avoided. The spectra from tests on strawberry and cucumber are shown in Figures S9 and Figure S10 (SI). It is obvious that the primary vibrations of thiram and CTSA were recorded simultaneously in each SERS spectrum, which are similar to that in standard samples (Figure S8A). Besides, the reproducibility of the SERS signal was evaluated from 10 randomly selected positions within each Au/CTSA film. As indicated in Figure S11 and Figure S12 (SI), for the different concentrations of thiram, the relative standard deviations of the SERS signal intensity were found to be in a range of 1.84-4.25 %, indicating that thiram molecules were uniformly distributed on the AuNPs surface by virtue of the advantage of IE-SERS approach. The corresponding quantitative results calculated by IE-SERS method according to the calibration curve in Figure S8B are shown in Tables 1. Indeed, there are large negative error values of the calculated results against the real concentration of thiram in samples, which stem from the penetration of thiram on peels especially for high concentration ones38. The error values ranged from -37.6 to 25 %, indicating the high sampling efficiency of this method. All these results exhibit that the proposed IE-SERS method is suitable for application in the determination of thiram in fruits and vegetables.


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Table 1. Quantitative result of thiram on strawberry and cucumber peel by the IE-SERS methoda Strawberry Sample

Cucumber

Cact

Ccal

RSD

Ecal

(μM)

(μM)

(%, n=3)

(%)

1

0.1

0.125

4.10

25.0

2

0.8

0.894

1.67

3

2

1.76

3.02

4

8

5.98

3.89

-25.3

Sample

Cact

Ccal

RSD

Ecal

(μM)

(μM)

(%, n=3)

(%)

1

0.1

0.116

3.25

16.0

11.8

2

0.8

0.709

2.84

-11.4

-12.0

3

2

1.45

3.06

-27.5

4

8

4.99

4.03

-37.6

aC act

is the actual concentration of thiram in samples. Ccal is the corresponding quantitative results calculated by IE-SERS method according to the calibration curve in Figure S8. RSD is the relative standard deviation of the quantitative detection result. Ecal is the error of the calculated results against the real concentration of thiam in samples.

Conclusions In summary, we developed a novel strategy by realizing simultaneous in situ extraction and fabrication of SERS substrate (IE-SERS), which was utilized for reliable on-site SERS quantitative detection of thiram residue in soil, fruits and vegetables. AuNPs-ODA and CTSA in DCM served as a reaction matrix, which can intimately contact with the complex surface and extract thiram to attach onto AuNPs during in situ extraction. The evaporation of DCM promoted the self-assembly of thiram-trapped AuNPs into the CTSA film to form the SERS substrate. By utilizing CTSA as internal standard to calibrate the absolute signal of thiram, the fluctuation of SERS intensity caused by uneven and irregular morphology of SERS substrate can be maximally reduced, thus exhibiting superior reproducibility in the SERS measurement (RSD=4.21%). By virtue of these incomparable features, the proposed IE-SERS approach is particularly suitable for efficient sampling (especially for tiny, rugged and irregular surface), in situ enrichment, and rapid and reliable detection of thiram residue. Following the line of the design, the developed IE-SERS method have been successfully used for the quantitative detection of thiram residue in paddy soils, strawberry and cucumber. Prominently, the proposed strategy of simultaneous in situ extraction and fabrication of SERS substrate can circumvent many drawbacks found in the existing substrate-detection mode by using pre-synthesized SERS substrate-extract analytes, which could broaden SERS applications in rapid and field analysis. ASSOCIATED CONTENT Supporting Information 17 ACS Paragon Plus Environment


Synthesis of CTSA and AuNPs-ODA, materials and instruments, photographs, FTIR spectra, XPS spectrum, 13CNMR spectrum, TEM images, Raman spectrum, and Tables. AUTHOR INFORMATION Corresponding Authors *Phone/Fax: +86-731-88830833. E-mail: [email protected]. *E-mail: [email protected]. ORCID Hua Yang: 0000-0002-5518-5255 Xiaoqing Chen: 0000-0002-8768-8965 Notes The authors declare no competing financial interest. Acknowledgement We gratefully acknowledge the financial support from National Natural Science Foundation of China (21475152 &21576296). References 1.

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TOC


Simultaneous in-situ Extraction and Fabrication of SERS Substrate for

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