Application of Carboxymethylcellulose @ Au NPs Hydrogel Beads for Detection of Thiram in Three Fruit Juices via Surface-Enhanced Raman Scattering

TL;DR

A new method using hydrogel beads with gold nanoparticles detects thiram pesticide in fruit juices with high sensitivity.

Contribution

A novel CMC @ Au NPs hydrogel bead SERS substrate for trace thiram detection in complex food matrices.

Findings

Detection limits of 0.001–0.002 mg/L thiram in apple, grape, and orange juices.

Sugars and acids in juices interfere with SERS measurements to varying degrees.

The hydrogel bead method is practical, affordable, and expandable for other food compounds.

Abstract

A simple and highly sensitive surface-enhanced Raman spectroscopy (SERS) method has been developed for the detection of thiram residues in fruit juices. Carboxymethyl cellulose (CMC) @ gold nanoparticles (Au NPs) hydrogel beads as SERS substrates were prepared through ionic crosslinking. The obtained porous CMC @ Au NPs hydrogel bead substrates showed excellent sensitivity for the detection of thiram in apple, grape, and orange juices, with detection limits of 0.001, 0.002, and 0.002 mg/L, respectively. The impact of primary non-target components in juices on SERS detection of thiram was investigated, revealing that the presence of sugars and acids caused varying degrees of interference in SERS measurements. This innovative, practical, and affordable CMC @ Au NPs porous hydrogel bead for thiram detection might be readily expanded to analyze a broad spectrum of other compounds found in food goods at trace amounts.

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Full text

1. Introduction

Thiram, a thiocarbamate fungicide with the molecular formula C_6_H_12_N_2_S_4_, is extensively used in agriculture—for seed treatment, foliar spraying on crops, vegetables, and fruits to control diseases like apple black spot and rice blast, as well as an animal repellent—with an annual sales volume of around US$20 million and high residue detection rates in tobacco [1]. Critically, thiram exerts severe toxic effects: acute human exposure causes skin, gastrointestinal, and respiratory irritation, while chronic exposure inhibits NK and T cell activity, impairs immunity, induces neurotoxicity, and disrupts reproductive, hepatic, and renal functions [2]. While thiram is used on plants, its presence extends beyond postharvest fruits and vegetables, as it can be transported into various other products, including juices, milk, honey, and meat [3]. As a result, developing a simple and sensitive method to identify thiram in complicated matrix materials is crucial. Surface-enhanced Raman scattering or spectroscopy (SERS) has become a potent method for the detection of numerous analytes in many food products in recent years. Owing to its great sensitivity and quick “fingerprinting” capabilities [4]. Research has demonstrated that SERS enables the rapid detection of pesticide residues on fruit peels via the paste-and-peel and wiping sampling methods with the aid of various flexible substrates [5,6,7]. Pesticide detection in fruit juices remains limited.

Recently, hydrogel substrates have received considerable attention in SERS due to their thermal shrinkage ability and highly superporous structure [8,9,10]. Jiang et al. prepared gold nanorods in a thermosensitive P (NIPAM-NVP) hydrogel membrane by in situ reduction to detect diquat. The hydrogel membrane, undergoing three cycles of expansion and contraction, demonstrated a concentration capability for diquat with an enrichment factor of approximately 4 [11]. In another study, Kim et al. prepared regenerated cellulose-coated gold nanorod (AuNRs/RC) membranes as SERS substrates by vacuum filtration. The dried AuNRs/RC hydrogel membranes exhibited superior SERS performance compared to their undried counterparts, attributed to effective adsorption of the analyte and increased hot-spot density, which resulted in enhanced SERS activity [12]. Various types of hydrogel substrates have been applied for the detection of thiram. For instance, Liu et al. developed a SERS platform based on Ag NPs-TEMPO-oxidized cellulose nanofiber (TOCNF)/polyacrylamide (PAAM) hydrogel, which enables the detection of thiram on fruit peels with a limit of detection (LOD) of 0.24 mg/L [13]. Yi et al. developed a microneedle (MN) patch-based SERS sensor with silver nanoparticles and a sodium hyaluronate/poly(vinyl alcohol) (HA/PVA) hydrogel, which achieved a LOD of 0.024 mg/L for thiram [14]. Zhang et al. prepared a β-lactoglobulin amyloid fibril-poly (vinyl alcohol)-silver nanoparticle (β-LAFs-PVA-AgNPs) hydrogel SERS substrate, which exhibited a LOD of 0.0371 mg/L for thiram [15].

Among various hydrogels, as an anionic polyelectrolyte and water-soluble cellulose derivative, carboxymethyl cellulose (CMC), stood out as an extensively used adsorbent either alone or in composites due to its excellent characteristics such as hydrophilicity, non-toxicity, affordability, and gel-forming capabilities [16,17,18]. Despite these advantages, current research on CMCs for the manufacture of SERS substrates remains relatively limited. The CMC/Au@AgNRs chip prepared by Hu et al. showed excellent sensitivity for detecting thiram residues in fruits with detection limits of 58 and 78 ppb [19]. Melo et al. synthesized gold nanoparticles (AuNPs) for SERS detection by a one-step method in which CMC acted as a reducing agent and stabilizer. The substrate showed impressive protection against salt-induced aggregation [20]. However, there is a need for more research to explore the potential of 3D CMC hydrogels and their concentration and enrichment capacity in the fabrication of SERS substrates.

Sodium dodecyl sulfate (SDS) functioned as a surfactant that self-assembles into spherical micelles in a water solution, which was encapsulated in the mesh structure during hydrogel crosslinking. The incorporation of SDS was a common practice to improve the porosity of hydrogel spheres, leveraging the superporous structure formed inside the hydrogel after SDS was washed out [21]. For example, Chatterjee prepared a chitosan hydrogel bead (CS/SDS) for Congo red adsorption with sodium dodecyl sulfate, which showed greater adsorption capacity than chitosan hydrogel beads (CS) [22]. The SDS-modified CS beads prepared by Suraya Jabeen et al. showed significantly higher adsorption capacity for the effective removal of rhodamine B compared to various reported adsorbents [23]. Here, SDS was used to enhance the pore size of CMC hydrogel beads, increasing their affinity for the target analyte.

In this study, the objective was to develop a highly efficient CMC SERS substrate for the analysis of hazardous compounds in foodstuffs, capable of not only increasing the hot spot density through shrinkage but also enriching in target analytes. The study was conducted in three main steps. First, CMC @ Au NPs hydrogel beads were obtained by ionic crosslinking. The COO-carboxyl group on the CMC chain was able to electrostatically interact with the multivalent metal cation Al^3+^ to form stable ionic hydrogel beads [24,25]. During the synthesis process, the pore space of the hydrogel beads was expanded by adding SDS to increase their adsorption capacity for the analytes. Next, the density of the analytes and metal nanoparticles was increased by drying and shrinking to increase the SERS signal. Finally, the CMC @ Au NPs hydrogel beads were applied to perform SERS analysis on thiram in three different fruit juice extracts. This technology may provide a foundation for further research that can easily adapt to detecting other pesticides present in food, thus opening up new avenues for the application of SERS.

2. Materials and Methods

2.1. Reagents

Chloroauric acid (≥49%), trisodium citrate (99%), and acetonitrile (GR) were purchased from J&K Scientific Ltd. (Logan, UT, USA). Thiram (≥99%) was acquired from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). Sodium chloride (AR), sodium carboxymethyl cellulose (M.W. 250,000 (DS = 0.9), 1500–3100 mPa.s), and anhydrous magnesium sulfate (99.99%) were purchased from Aladdin reagent company (Shanghai, China). Sangon Biotech Co., Ltd. (Shanghai, China) provided the sodium dodecyl sulfate. Aluminum nitrate nonahydrate (Al(NO_3_)3·9H_2_O) was sourced from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Primary secondary amine (PSA, 40–63 μm) was purchased from ANPEL Scientific Instrument Co., Ltd. (Shanghai, China).

2.2. Preparation of CMC @ Au NPs Solution and CMC @ Au NPs Porous Hydrogel Beads

The preparation process of CMC @ Au NPs solution and CMC @ Au NPs porous hydrogel beads was illustrated in Figure 1. First, certain adjustments were made to Prusty et al.’s approach for fabricating the CMC @ Au NPs solution [26]. Briefly, 0.75 g of carboxymethyl cellulose was added to 50 mL chloroauric acid solution (0.015%, w/w) and was magnetically stirred at 1100 rpm for 4 h and then heated to boiling. Subsequently, 0.75 mL of aqueous trisodium citrate solution (1%, w/w) was added and stirred continuously at 1100 rpm for 40 min before ending the reaction in an ice-water bath. As a result, a solution of carboxymethyl cellulose @ Au NPs could be produced.

The preparation of the CMC @ Au NPs porous hydrogel beads was done with some changes to the Benhalima et al. technique [27]. Specifically, 0.4 g of sodium chloride and 50 uL of SDS at concentrations of 0.7, 1.4, and 2.1 mol/L were added into 10 mL of CMC @ Au NPs solution at 400 rpm for 1 h. The resulting solution was then extruded as droplets into 20 mL of aluminum nitrate nonahydrate (3%) at 200 rpm, forming smooth, spherical CMC aluminum hydrogel beads immediately. The generated beads were stirred for an additional 30 min and cured overnight at 4 °C. The wet beads were then thoroughly washed several times with alcohol and ultrapure water to remove the surfactant. Eventually, the resulting hydrogel beads were collected and stored in water.

2.3. Characterization of CMC @ Au NPs Solution and CMC @ Au NPs Porous Hydrogel Beads

The UV-vis spectra of the gold nanoparticles incorporated with carboxymethylcellulose were measured in the range of 400–800 nm by a UV-visible spectrophotometer (Evolution220, Thermo Fisher Scientific, Waltham, MA, USA). The optical properties of the nanoparticles were studied based on the location and intensity of the surface plasma absorption peaks. The surface morphology and cross-section of CMC @Au NPs porous hydrogel beads were visually characterized by field emission scanning electron microscopy (FESEM, SU5000, Hitachi, Tokyo, Japan). Acceleration voltage was 10 kV, and working distance was 10 mm.

2.4. Preparation of Thiram Standard Solution and Thiram Juice Samples

A thiram solution (100 mg/L) was made in acetone and subsequently diluted with water to obtain a series of standard solutions. To evaluate the efficacy of CMC @ Au NPs porous hydrogel beads in detecting thiram in juice extracts, different levels of thiram were added to apple juice, grape juice, and orange juice purchased from a supermarket in Shanghai. A modified QuEChERS approach was employed to extract the thiram from the fruit juice [28]. Briefly, 5 mL of acetonitrile, 2 g of anhydrous magnesium sulfate, and 0.3 g of sodium chloride were added to 5 mL of apple juice with different concentrations of thiram. After one minute of vortexing, the mixture was centrifuged for five minutes at 4000 rpm. Subsequently, primary secondary amine (PSA) was separately added to 3 mL of the transferred supernatant, followed by shaking and vortexing for 1 min. Two milliliters of the supernatant were utilized for the subsequent SERS analysis following a five-minute centrifugation at 4000 rpm.

2.5. SERS Analysis

The CMC @ Au NPs porous hydrogel beads were immersed in 2 mL of thiram standard solution or thiram from juice extract for 3 h before collection for Raman spectroscopy. Then they were removed and placed on a magnetic hot plate (PC-4200, Corning, Glendale, AZ, USA) at 45 °C for 1 h to dry for the SERS detection. Raman spectra were acquired by a Nicolet DXR microscope Raman spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) under the following conditions: 633 nm laser source, 10× objective, 7 mW laser source power, 5 exposures and 2 s per scan, 400–1800 cm^−1^. Five spectra were randomly acquired for each sample tested on a substrate, and the average spectrum was used for analysis. Moreover, the conventional Raman spectra were obtained by lightly pressing the material onto a glass plate following the same method.

2.6. Data Analysis

The collected spectra were averaged via Omnic 9.2 software, and the experimental data were processed and analyzed with Origin 2021b. (OriginLab Corporation, Northampton, MA, USA). With the use of SPSS 21.0 software (IBM Corporation, New York, NY, USA), significance analysis (p < 0.05) was carried out based on classic analysis of variance (ANOVA) and Duncan’s multiple range test. Correlation analysis was performed between the concentration of thiram in the standard solution or juice extract and the Raman intensity of the main characteristic peak. To evaluate the enhancement properties of the porous hydrogel beads CMC @ AuNPs, the enhancement factor (EF) of these SERS substrates was calculated according to the following equation.

$$[eqn]$$

where the analyte concentrations in the SERS and normal Raman measurements are represented by C_SERS_ and C0, and the peak intensities in the SERS and normal Raman spectra are represented by I_SERS_ and I0, respectively.

To ensure the reliability of the analytical results and the accurate calculation of standard deviations, all experiments in this study were performed in triplicate, and the reported values represented the mean of three independent measurements.

3. Results and Discussion

3.1. Characterization of CMC @ Au NPs Solution and CMC @ Au NPs Porous Hydrogel Beads

Figure 1a–c showed the SEM images of CMC or CMC @ Au NPs porous hydrogel beads and their UV-vis spectra before the crosslinking reaction. CMC @ Au NPs exhibited a narrow extinction band at 532 nm, caused by the surface plasmon resonance peaks of metal nanoparticles immobilized on the CMC chains [29]. In contrast, CMC did not absorb visible light in the range of 400–800 nm. The SEM images, on the other hand, indicated that the metal nanoparticles were anchored on the CMC hydrogel porous spheres with a uniform distribution and moderate density, confirming the successful reduction in HAuCl_4_ to metal nanoparticles and their anchoring on CMC.

As shown in Figure 1d, there were no significant Raman peaks for CMC @ Au NPs porous hydrogel beads. The conventional Raman peaks of thiram at 433, 551, 841, 967, and 1372 cm^−1^ were attributed to C-S-S strain vibrations, S-S stretching vibrations, methyl vibrations, CH_3_N stretching vibrations, and C-N stretching vibrations, respectively. With the use of CMC @ Au NPs porous hydrogel beads, most of the characteristic peaks (e.g., 548 cm^−1^, 1372 cm^−1^) were slightly shifted. The complexation of Au with thiram was identified as the cause of the characteristic peak’s appearance at 1497 cm^−1^, while the peak at 939 cm^−1^ arose due to C=S bond formation [30]. Therefore, 548, 939, 1138, and 1372 cm^−1^ were selected as characteristic SERS peaks for thiram for further study.

3.2. Optimization of SDS Concentration and the Drying Time for CMC @ Au NPs Porous Hydrogel Beads

The capacity of hydrogels to absorb water and adsorb target compounds was significantly enhanced by their porosity [31]. SEM images of the surface and cross-section of CMC @ Au NPs porous hydrogel beads with and without SDS templates are presented in Figure 2. Figure 2a,c shows that CMC @ Au NPs porous hydrogel beads without SDS templates have a relatively smooth surface and dense cross-sectional pores, indicative of the 3D structure of the hydrogel after freeze-drying [32]. In contrast, CMC @ Au NPs porous hydrogel beads containing SDS templates not only feature relatively rough surfaces but also exhibit some open pores with larger openings on their cross-section. This was attributed to the spherical micelles self-assembled by SDS in an aqueous solution, which were encapsulated in the internal hydrogel network during crosslinking and graft copolymerization. The superporous structure formed when SDS was removed by washing with alcohol [21]. Furthermore, the insertion of sodium chloride improved the connectivity of the internal pores [33].

Figure 3a,b demonstrated the effect of SDS concentration on the SERS detection of thiram (1 mg/L) by porous hydrogel CMC @ Au NPs beads. It was evident that the addition of varying doses of SDS showed a 15.5–32.9 times higher intensity at 1372 cm^−1^ compared to the condition without SDS. Specifically, hydrogel beads prepared with 1.4 mol/L SDS showed approximately 1.29–2.13 times higher SERS signals at 1372 cm^−1^ than other additions. This enhancement is attributed to the fact that at lower concentrations of SDS, the primary presence of SDS as free ions does not produce sufficient micelles. As the concentration of SDS increases, the formed micelles act as pore templates, enhancing the pore structure during gelation and facilitating the contact between gold nanoparticles and thiram. However, it should be noted that higher additions of SDS did not further increase the SERS signal of thiram, possibly because the backbone of the porous hydrogel became more brittle and susceptible to collapse as the SDS concentration increased [34]. Moreover, higher concentrations of SDS may interfere with the gelation mechanism, limiting the formation of the 3D network of the hydrogel [33]. This finding was in line with earlier studies conducted by Sudipta Chatterjee et al., who used chitosan hydrogel beads (CSBs) prepared by SDS gelation to affect the adsorption capacity of CSBs on Congo red (CR) anion dye by varying the concentration of SDS [22]. Therefore, CMC @ Au NPs hydrogel beads prepared with 1.4 mol/L SDS were selected as the optimal SERS substrate preparation conditions for the detection of thiram standard solutions.

The influence of drying time on the SERS detection of thiram (1 mg/L) by porous hydrogel CMC @ Au NPs beads was illustrated in Figure 3c,d. It could be seen that as the hydrogel beads underwent continuous drying, the SERS signal of thiram experienced significant enhancement. Specifically, the intensity of thiram at 1372 cm^−1^ based on the porous hydrogel CMC @ Au NPs beads increased significantly with the drying time, reaching approximately 160 times higher at 60 min than at 10 min. This observation could be elucidated by the fact that as the hydrogel beads continued to shrink, the SERS intensity increased with the number of Au NPs and molecules per unit area [11]. Indeed, the enhanced effect of the SERS signal was determined by the density of SERS hot spots located in the nanogaps between the particles [35]. Therefore, CMC @ Au NPs porous hydrogel beads with 1h of drying time were deemed the optimal SERS substrate for the detection of thiram standard solutions.

The selected CMC @ Au NPs porous hydrogel beads exhibited a remarkable EF of 5.57 × 10^6^ for thiram, calculated based on the characteristic peak of 1372 cm^−1^ for the 1 mg/L thiram standard. To evaluate the homogeneity of the chosen CMC @ Au NPs porous hydrogel beads regarding SERS effects for a pesticide, the strength of characteristic peaks in the spectrum of thiram (939, 1139, 1372 cm^−1^) collected at 5 random points on the substrate was assessed. The intra-reproducibility of thiram was shown to have a relative standard deviation (RSD) of 6.26%, 6.05%, and 6.21%.

As shown in Figure 4, Raman spectra of thiram standards were collected using the optimized CMC @ Au NPs porous hydrogel beads at concentrations ranging from 0.0002 to 10 mg/L. The characteristic peaks at 1497, 1372, 1138, 939, and 548 cm^−1^ were distinctly visible in the spectra, with their intensities progressively increasing with the concentration of the standard solution. Particularly noteworthy was the clear visibility of the peak at 1372 cm^−1^ even at a low concentration of 0.0002 mg/L. The concentration of the standard solution and the Raman intensities of the two primary characteristic peaks (1372, 548 cm^−1^) showed a discernible linear connection (R^2^ = 0.982–0.985, RMSE = 4.95–14.74 mg/L). Based on these results, the optimized CMC @ Au NPs porous hydrogel beads were used for further analysis of thiram in fruit juices.

3.3. Effect of Organic Components on SERS Sensitivity of Thiram Based on CMC @ Au NPs Porous Hydrogel Beads

The application of SERS as a detection technique in complex food products can be challenging due to interference from extraneous factors, such as food matrices. To investigate the effect of major organics in fruit juices on thiram detection using porous hydrogel CMC @ Au NPs beads as a substrate, simple one-way simulation experiments were designed to isolate the effects of individual organic compounds. Two acids (citric acid and malic acid) and four sugars (pectin, sucrose, fructose, and glucose) were chosen as representative organic compounds present in fruit juices. The effects of each of these compounds on the SERS signal of thiram were detected separately.

Figure 5 shows that the presence of sugars and organic acids in fruit juices had a significant impact on the SERS signal of thiram. In comparison to the standard 1 mg/L solution, for sugars (Figure 5a), the intensity of the characteristic peak decreased significantly with increasing sugar addition, possibly due to sugars conflicting with the adsorption of thiram by the porous hydrogel CMC @ Au NPs beads [36]. Among these sugars, fructose caused the largest interference with SERS detection of thiram, while pectin had a relatively minor effect, presumably because of structural differences. For the organic acids, as shown in Figure 5b, the intensity of the characteristic peak decreased at lower concentrations with an increasing amount added. However, when the concentration of both acids was increased to 1%, the 3D structure of the porous hydrogel beads CMC @ Au NPs collapsed and could not undergo drying to shrink, probably due to the structure of the organic acids reacting with CMC [37].

3.4. Application of CMC @ Au NPs Porous Hydrogel Beads for the Analysis of Thiram in Fruit Juice

As a simple, fast, and cost-effective pretreatment sample, the QuEChERS method was widely used for extracting pesticide residues from fruits and vegetables [28]. The two phases of this procedure were extraction and purification, during which the extraction time can be shortened to within 20 min. In extraction, NaCl promoted phase separation between the aqueous and organic phases, while anhydrous MgSO_4_ was used to remove excess water from the sample. In order to successfully remove non-target materials from apple juice, it is necessary to optimize the dosage of the adsorbent in purification [38,39]. As shown in Figure 6a,b, the SERS signal of thiram extracted from juice was observed to be influenced by the adsorbent dosage. Optimal detection was obtained when PSA was added in amounts of 0.15 g (apple juice), 0.20 g (grape juice) and 0.15 g (orange juice). The different amounts of adsorbent required may be due to different concentrations of organic acids, sugars, colorants and other non-target substances present in the different juices. In addition, due to its three-dimensional cross-linked network structure, the CMC hydrogel acted as a physical molecular sieve and effectively hindered the penetration of residual macromolecular substances during the adsorption process [40].

The SERS spectra of thiram in the extracts of three different juices are shown in Figure 6d–f. Apart from a slight red/blue shift in some characteristic peaks, there was no significant change in the position of these characteristic peaks compared to the thiram standard. These subtle shifts in the characteristic peak positions were linked to the orientation of the analyte molecules on the surface of the substrate, as well as the functional groups of the molecules attached to the substrate [41]. The signal intensity of all characteristic peaks gradually decreased as the concentration of thiram in the juice decreased. The lowest concentrations of thiram measured in apple, grape and orange juice samples based on this method were 0.001, 0.002 and 0.002 mg/L, respectively. These values were significantly lower than the maximum residue threshold for thiram in agricultural fruit and vegetable products (5 mg/L) set by the US Environmental Protection Agency [42], as well as the majority of hydrogel substrates currently used for thiram detection. For instance, the Au@Ag-CMC hydrogel membrane prepared by Hu et al. had a limit of detection of 0.058 mg/L [19], while sodium hyaluronate/poly(vinyl alcohol) and the silver nanoparticles (HA/PVA) hydrogel prepared by Yi et al. had a limit of detection of 0.024 mg/L [13]. Furthermore, the LODs obtained with the SERS method in this study were also compared with those achieved by conventional official analytical methods. As shown in Table 1, our SERS-based approach exhibited a markedly lower LOD, demonstrating its superior sensitivity for thiram detection.

Therefore, the three characteristic peaks (around 1378, 1145, and 557 cm^−1^) were selected with the concentration (log value) of thiram to establish the SERS calibration curve as presented in Table 2. The SERS intensity of these characteristic peaks showed a linear increase with the concentration of thiram (log value) ranging from 0.001 to 10 mg/L (R^2^ = 0.886–0.994, RMSE = 2.73–9.48 mg/L). However, the linear equations for the same characteristic peak in the three fruit juices varied, indicating a strong effect of the sample matrices on the SERS signal for thiram. This implies that an equation for linear regression based on the SERS intensity of a characteristic peak for the group of thiram-infected foods may not be directly applicable to another group of contaminated foods. These findings suggest that the combination of porous hydrogel CMC @ Au NPs beads and SERS technology holds promise for the effective detection of thiram in different juices.

4. Conclusions

In this work, a method showing great potential for detecting banned or restricted pesticides in fruit juices was presented, using novel porous hydrogel CMC @ Au NPs beads synthesized by ionic crosslinking as SERS substrates. The obtained porous hydrogel CMC @ Au NPs beads were highly sensitive, with a Raman EF signal of 5.57 × 10^6^ under optimized conditions. The effect of matrix composition on the detection of thiram in fruit juices by porous hydrogel beads CMC @ Au NPs was investigated, and the samples were pretreated using the QuEChERS method. The lowest detectable concentration of thiram was found to be 0.001 mg/L in apple juice and 0.002 mg/L in both grape and orange juice. The CMC @ Au NPs porous hydrogel beads prepared in combination with the SERS analytical method can enhance the SERS signal for the target analytes of the hydrogel, and the optimized simple QuEChERS pretreatment and low-cost, facile hydrogel preparation process reduce equipment costs, enabling the application of this technology to small-scale juice producers. Nevertheless, this study has several limitations: the method is only validated for thiram, lacking systematic evaluation for other common fruit juice pesticides; large-scale hydrogel bead preparation needs better uniformity of pore size and gold nanoparticle loading; and hydrogel storage stability is not systematically studied. Future research will address these limitations, and this work thus offers a reliable, cost-effective SERS-based approach for thiram detection in fruit juices and holds significant promise for sensitive and selective detection of pesticides in diverse fruit juice matrices.