Signal-on fluorescent strategy for label-free detection of carbendazim based on gold nanoparticles quenching graphene quantum dots

Signal-on fluorescent strategy for label-free detection of carbendazim based on gold nanoparticles quenching graphene quantum dots | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Signal-on fluorescent strategy for label-free detection of carbendazim based on gold nanoparticles quenching graphene quantum dots Zhifeng Wang, Xiaochen Liu, Yu Huang, Ke Pei, Tingting Feng This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6225961/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract A fluorescence biosensing method based on fluorescence resonance energy transfer between gold nanoparticles (AuNPs) and graphene quantum dots (GQDs) was developed for the rapid detection of carbendazim. Upon adding the aptamer to the AuNPs solution, the aptamer binds to the AuNPs, preventing their aggregation in a high-concentration NaCl solution. Therefore, the AuNPs retain their optical properties and can quench the fluorescence of GQDs. When carbendazim is introduced, it specifically binds to the aptamer on the AuNPs, forming a complex. This interaction causes the AuNPs to lose the protective effect of the aptamer and aggregate in the high-concentration NaCl solution, leading to a loss in their ability to quench GQDs fluorescence, which increases the fluorescence intensity of the solution. This method was applied to recover carbendazim from the leaves of Chinese herbal medicines, achieving recoveries of 90.68%-106.29%. The results demonstrate that the sensor is simple, convenient, and effective. Biological sciences/Biochemistry Physical sciences/Nanoscience and technology Biosensor fluorescence gold nanoparticles graphene quantum dots carbendazim aptamer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction The use of pesticides is one of the important measures to reduce the pest of plant Chinese medicinal materials.[ 1 ]. The use of pesticides is a key measure for reducing crop damage from pests and rodents. Carbendazim, a benzimidazole fungicide, is known for its high efficacy, low toxicity to humans and animals, and broad antibacterial spectrum [ 2 ]. The primary molecular structure of carbendazim is a benzimidazole ring, which is chemically stable and resistant to degradation under natural conditions [ 3 ]. However, excessive use can lead to residues. Studies have shown that carbendazim residues in the environment can harm the reproductive systems of mammals and negatively impact the genetic behaviors of some organisms. Moreover, its degradation products are toxic organic substances [ 4 , 5 ]. Therefore, establishing an analytical method to rapidly detect carbendazim in the field is crucial. Currently, the most common methods for detecting the pesticide carbendazim in China include fluorescence spectrometry [ 6 ], spectrophotometry [ 7 ], electrochemical methods [ 8 ], liquid chromatography [ 9 ], liquid mass spectrometry [ 10 ], and gas chromatography [ 11 ]. Although these methods exhibit high accuracy and stability, they are limited by the high cost and complexity of the instruments, the complicated sample pretreatment process, and the extended detection times. Thus, they are unsuitable for the rapid field detection of pesticide residues. Surface-enhanced Raman spectroscopy [ 12 ] is an emerging spectroscopic detection technology known for its low cost, high accuracy, and fast detection capabilities. However, it still has limitations, such as challenges in preparing consistent substrates, which hinder its broader application and development in various fields [ 13 ]. Enzyme-linked immunosorbent assay offers strong specificity, high sensitivity, low cost, and portability [ 14 , 15 ]. However, the long preparation time and variability in antibody production can lead to inconsistent results between different batches. Additionally, the inherent instability of the antibody proteins and enzymes used in this method further limits its scope of application. An aptamer (Apt) is a single-stranded nucleic acid molecule that can specifically bind to target molecules through in vitro screening technology [ 16 ]. Its affinity and specificity are similar to those of antibodies, allowing it to selectively recognize target molecules. Compared to antibodies, Apts offer significant advantages, such as ease of synthesis, high stability, and low cost [ 17 , 18 ]. Biosensing methods that combine Apts with electrochemistry, fluorescence, and colorimetry have been extensively used in food detection, particularly for the detection of pesticide residues. In this study, a simple and rapid fluorescence sensing method was developed to detect carbendazim in Chinese herbal slices based on the FRET effect between AuNPs and GQDs. The synthesized gold nanoclusters remain dispersed due to the electrostatic repulsion generated by the citrate, which prevents them from aggregating. When NaCl is added, the negative charge of citrate is neutralized, resulting in the aggregation of gold nanoparticles. When Apt is added to AuNPs solution, aptamers can be strongly absorbed on AuNPs surface through the coordination of nitrogen atoms with AuNPs, thus enhancing the stability of AuNPs against salt-induced aggregation [ 6 , 19 , 20 ]. This interaction preserved the optical characteristics of the AuNPs, allowing them to quench the fluorescence of the GQDs. However, when carbendazim is introduced, it specifically binds to the Apt on the AuNPs, forming a carbendazim–Apt complex. This causes the AuNPs to lose the protection of the Apt, leading to their aggregation in the high NaCl concentration and resulting in a failure to quench the GQDs fluorescence. Consequently, the fluorescence intensity of the solution increased. Based on this principle, a fluorescent Apt sensing method was developed to detect carbendazim in traditional Chinese medicine decoction pieces. 2. Material and methods 2.1. Reagents and chemicals The FAM-labeled Apt (5'-CGACACAGCGGAGGCCACCCGCCCACCAGCCCCTG CAGCTCCTGTACCTGTGTGTGTG-3') was obtained from Hema Biology Co., Ltd. (Zhejiang, China). GQDs were purchased from Nanjing Xianfeng Nanomaterial Technology Co., Ltd. (Nanjing, China). Na 2 HPO 4 ·12H 2 O, KH 2 PO 4 , NaCl, KCl, carbendazim, dinotefuran, ethiprole, acetamiprid, fipronil, pymetrozine, benomyl and thiophanate standards were purchased from Maikelin Biology Co., Ltd. (Shanghai, China). Phosphate-buffered saline (PBS) (10 mmol/L, pH 7.4), composed of Na 2 HPO 4 ·12H 2 O, KH 2 PO 4 , NaCl and KCl, was prepared and used in subsequent experiments. 2.2. Instrumentation Fluorescence spectra were recorded using an RF-6000 fluorescence spectrophotometer (Shimazu, Japan) with an excitation wavelength of 380 nm and an emission range of 430–600 nm. Both excitation and emission wavelengths were set with a 5-nm bandwidth. A pHS-3E pH meter (Shanghai, China) was used for pH measurements, and a PR124ZH/E electronic balance (Changzhou, China) was used for weighing. An MTH-100 constant-temperature mixing instrument was employed for incubation baths. Dynamic light scattering (DLS) measurement was acquired on a Zetasizer Nano ZS90 analyzer (Malvern Instruments Ltd., UK). 2.3. AuNPs preparation and synthesis The synthesis and preparation of AuNPs were based on methods described by this document [ 21 ], with slight modifications. Initially, 5 mL of 1% (m/m) sodium citrate solution was placed in a brown conical flask wrapped in tin foil and stirred at 180℃. Next, 90 mL of distilled water was added and heated to boiling. Subsequently, 5 mL of 0.2% (m/m) chloroauric acid solution was quickly added, and the mixture was brought back to a boil. The color of the solution gradually changed from colorless to light pink. After continuing to boil for about 10 min, the color of the solution turned to wine red. Heating was then stopped, but the solution was stirred and allowed to continue heating with residual heat for 1 h. Finally, the AuNPs solution was cooled to room temperature and stored at 4℃ in the dark for later use. 2.4. Optimization of experimental conditions To optimize the Apt concentration, Apt solutions of varying concentrations were added to 200 µL of AuNPs solution, mixed, and incubated at 25℃ for 5 min. Subsequently, 22.5 µL of NaCl solution (150 nmol/L) was added and incubated at room temperature for 5 min. Finally, 40 µL of the GQDs suspension (100 µg/mL) was added and incubated at 25℃ for 2 min. The mixture was then brought to a final volume of 500 µL with PBS, and the fluorescence intensity of each sample was measured between 430–600 nm using a fluorescence spectrophotometer. To optimize the NaCl solution, an Apt solution (800 nmol/L) was added to 200 µL of AuNP solution, mixed, and incubated at 25℃ for 5 min. NaCl solutions of varying concentrations were then added and incubated at room temperature for 5 min. Afterward, 40 µL of GQDs suspension (100 µg/mL) was added and incubated at 25℃ for 2 min. The mixture was brought to a final volume of 500 µL with PBS, and the fluorescence intensity of each sample was measured between 430–600 nm using a fluorescence spectrophotometer. To optimize the reaction time between Apt and carbendazim standard solution, a carbendazim standard solution (5 µg/mL) was mixed with 30 µL of Apt solution (800 nmol/L) and incubated at 25℃ for 0, 5, 10, 15, 20, 25, and 30 min. Next, 200 µL of AuNPs solution was added, and the mixture was incubated at 25℃ for 5 min. Subsequently, 22.5 µL of NaCl solution (120 nmol/L) was added and incubated at room temperature for 5 min. Finally, 40 µL of GQDs suspension (100 µg/mL) was added and incubated at 25℃ for 2 min. The final mixture was brought to 500 µL using PBS, and the fluorescence intensity of each sample was measured between 430–600 nm with a fluorescence spectrophotometer. 2.5. Carbendazim detection using a fluorescent biosensor Different concentrations of carbendazim standard solution (1, 2, 3, 4, 5, 6, and 7 µg/mL) and 30 µL of Apt solution (800 nmol/L) were added to a 0.5 mL centrifuge tube and incubated at 25℃ for 20 min. Next, 200 µL of AuNPs solution was added, and the mixture was incubated at 25℃ for 5 min. Subsequently, 22.5 µL of NaCl solution (120 nmol/L) was added and incubated at room temperature for 5 min. Finally, 40 µL of GQDs suspension (100 µg/mL) was added and incubated at 25℃ for 2 min. The final mixture was brought to a volume of 500 µL with PBS, and the fluorescence intensity of each sample was measured between 430–600 nm using a fluorescence spectrophotometer. The excitation wavelength was set to 380 nm, with an emission spectrum range of 430–600 nm. 2.6. Specificity experiment To evaluate the selectivity of the fluorescent Apt sensor, the effects of carbendazim and other potential interfering substances on the fluorescence intensity were investigated. Carbendazim standard solution (5 µg/mL) and various interfering substances (dinotefuran, ethiprole, acetamiprid, fipronil, pymetrozine, benomyl and thiophanate) were incubated with 30 µL of Apt solution (600 nmol/L) at 25℃ for 20 min. Subsequently, 200 µL of AuNPs solution was added, and the mixture was incubated at 25℃ for 5 min. Next, 22.5 µL of NaCl solution (150 nmol/L) was added and incubated at room temperature for 5 min. Finally, 40 µL of GQDs suspension (100 µg/mL) was added and incubated at 25℃ for 2 min. The mixture was then brought to a final volume of 500 µL with PBS, and the fluorescence intensity of each sample was measured between 430–600 nm using a fluorescence spectrophotometer. The excitation wavelength was set to 380 nm, with an emission spectrum range of 430–600 nm. 2.7. Determination of carbendazim in traditional Chinese medicine decoction pieces Sophora flavescens and yam slices were selected as the traditional Chinese medicine samples. A precise 0.2000 g of powdered TCM decoction pieces was placed in a 2 mL centrifuge tube, and 2 mL of 70% (v/v) methanol solution was added. After ultrasonic treatment for 10 min, the supernatant was collected through centrifugation for 10 min. The filtrate, obtained by passing through a microporous filter membrane, was diluted to 1 µg/mL, yielding the test solution, which was stored at 4℃ in the dark.Carbendazim standard solution (5,6 and 7 µg/mL) is added to the above sample, and were incubated with 30 µL of Apt solution (600 nmol/L) at 25℃ for 20 min. Subsequently, 200 µL of AuNPs solution was added, and the mixture was incubated at 25℃ for 5 min. Next, 22.5 µL of NaCl solution (150 nmol/L) was added and incubated at room temperature for 5 min. Finally, 40 µL of GQDs suspension (100 µg/mL) was added and incubated at 25℃ for 2 min. The mixture was then brought to a final volume of 500 µL with PBS, and the fluorescence intensity of each sample was measured between 430–600 nm using a fluorescence spectrophotometer. Fluorescence detection was conducted, and each sample was measured in triplicate. The recovery rate and the relative standard deviation (RSD) were calculated for each sample. 3. Results and discussion 3.1. Characterization of AuNPs materials As shown in Fig. 1 , AuNPs were characterized using transmission electron microscopy (TEM). The image indicates that the size distribution of the prepared AuNPs is relatively uniform and well dispersed. In order to further study the size distribution and surface properties of gold nanoparticles, the particle size of AuNPs was detected by DLS measurement. It can be seen that the size of AuNPs is about 13 nm [ 22 ]. 3.2 Characterization of GQDs The TEM images in Fig. 2 show that GQDs were spherical in morphology with an average diameter of 14.5 nm. 3.3. Feasibility analysis of the carbendazim fluorescent biosensor To evaluate the feasibility of the proposed method, the fluorescence spectra of the detection system under different conditions were recorded (Fig. 3 ). When only GQDs were present in the solution, the fluorescence intensity was highest (Black line). Upon the addition of AuNPs to the GQDs solution, the fluorescence intensity decreased significantly due to the FRET phenomenon (Purple line). In the presence of AuNPs, GQDs, and NaCl, the fluorescence intensity increased compared to Purple line, indicating that a high NaCl concentration causes AuNPs to aggregate. This aggregation weakens the FRET effect, leading to an increase in fluorescence intensity (Red line). When AuNPs, GQDs, NaCl, and Apt were present, the fluorescence intensity decreased somewhat compared to Blue line. This reduction is due to Apt binding around the AuNPs, protecting them from aggregation and allowing FRET to occur between AuNPs and GQDs (Green line). Blue line shows the fluorescence spectrum for a solution containing AuNPs, GQDs, NaCl, Apt, and carbendazim. Compared to Green line, the fluorescence intensity is slightly increased. This increase occurs because when Apt binds to carbendazim, a stable complex forms, reducing the amount of Apt around the AuNPs. This allows numerous AuNPs to aggregate, decreasing FRET and increasing fluorescence intensity. Overall, these results demonstrate that the proposed fluorescence Apt sensing method is feasible for detecting carbendazim. 3.4. Optimization of experimental conditions 3.4.1. Optimization of aptamer and NaCl concentrations Different concentrations of Apt and NaCl can influence the aggregation of AuNPs, affecting the ability of the biosensor to detect carbendazim. Therefore, optimizing the concentrations of Apt and NaCl is essential. As shown in Fig. 4 (A), the fluorescence intensity (F) gradually stabilizes when the Apt concentration exceeds 600 nmol/L. This stabilization likely occurs because, at higher Apt concentrations, there is sufficient Apt around the AuNPs to protect them from being quenched by GQDs, resulting in a more stable system. For NaCl concentration optimization, Fig. 4 (B) shows that as the NaCl concentration increases to 120 nmol/L, the fluorescence intensity (F) gradually stabilizes. This indicates that AuNPs have fully aggregated at this concentration. Therefore, the optimal Apt and NaCl concentrations in the reaction system are 600 nmol/L and 120 nmol/L, respectively. 3.4.2. Optimization of reaction conditions for carbendazim and Apt The reaction time between Apt and carbendazim affects the stability and sensitivity of the method. As shown in Fig. 5 , the fluorescence intensity (F) of the system reaches its maximum at 25 min. This peak intensity likely results from the optimal binding between Apt and carbendazim, which occurs at this reaction time. Therefore, a reaction time of 25 min is most effective for generating reliable experimental results. 3.5. Analytical performance of the carbendazim fluorescent aptamer sensor 3.5.1. Establishment of the standard curve To investigate the effectiveness of the fluorescent Apt sensor for detecting carbendazim, absorbance changes in the sensor system were measured at varying concentrations of carbendazim standard solution (1, 2, 3, 4, 5, 6, and 7 µg/mL). As shown in Fig. 6 , the fluorescence intensity of the system increased with increasing carbendazim concentrations. Within the concentration range of 0.5–7 µg/mL, the relationship between carbendazim concentration (C) and (F − F 0 )/F 0 (where F indicates the fluorescence intensity after carbendazim addition) was linear. The equation derived is (F − F 0 )/F 0 = 0.2099C carbendazim + 0.3349, with a correlation coefficient of 0.9913. Using the 3σ/s principle, the calculated limit of detection (LOD) is 0.44 µg/mL. These results indicate that the sensor exhibits high sensitivity for carbendazim detection. 3.5.2. Method specificity Specificity is a crucial indicator for assessing whether the test system can reliably detect carbendazim without interference from other substances (such as similar pesticides) that may be present in actual sample tests. Under optimized conditions, the biosensor was used to detect 100 µg/mL of carbendazim and 50 µg/mL of seven common pesticide residues (dinotefuran, ethiprole, acetamiprid, fipronil, pymetrozine, benomyl and thiophanate), and the corresponding fluorescence intensities were recorded. As shown in Fig. 7 , the fluorescence intensity increased significantly when only carbendazim was present in the system. Conversely, the fluorescence intensity of the other substances remained minimal, even when present at concentrations 10 times higher than carbendazim. These results confirm that the fluorescent Apt sensor has good selectivity for detecting carbendazim in Chinese herbal slices. 3.6. Repeatability and stability of the method To evaluate the repeatability and stability of the method, the carbendazim standard solution (100 µg/mL) was measured in triplicate under optimized conditions, and the corresponding fluorescence intensities were recorded. The (F − F 0 )/F 0 values were calculated, where F represents the fluorescence intensity after adding carbendazim, and F 0 is the fluorescence intensity without carbendazim. As illustrated in Fig. 8 , after 14 d, the fluorescence intensity of the sensor remained at 95%, demonstrating that the constructed fluorescence Apt sensor exhibits good repeatability and stability. 3.7. Chinese medicine actual sample test To assess the practical application of the proposed method, the sensor system was used to detect carbendazim concentrations in herbal slices of Sophora flavescens and yam. First, the sensor was used to detect carbendazim in the Chinese yam and Sophora flavescens, but no detectable carbendazim residues were found in the samples. Then, three different concentrations of carbendazim (5, 6, and 7 µg/mL) were added to the diluted reserve solution in item 2.7. The results are shown in Table 1 . The recoveries of carbendazim in the two sample types ranged from 90.68–106.29%, with RSDs between 0.52% and 1.60%. These results indicate that the method has good practicability and can be extensively used for the rapid detection of carbendazim in traditional Chinese medicine decoction pieces, offering a new approach to determining pesticide residues. Table 1 Spiked recoveries in real samples. Sample Added (µg/mL) Found (µg/mL) Recovery (%) RSD (%, n = 3) Sophora flavescens 0 - - - 5 4.81 96.13 1.60 6 6.38 106.29 1.17 7 7.13 101.85 1.10 Yam 0 - - - 5 5.05 100.95 1.08 6 6.04 100.67 0.52 7 6.35 90.68 0.88 4. Conclusion In this study, we developed a biosensor utilizing FRET between AuNPs and GQDs for the detection of carbendazim pesticide residues in traditional Chinese medicine decoction pieces. Under optimized conditions, the linear range for the fluorescence Apt sensor was 0.5–7 µg/mL, with an LOD of 0.44 µg/mL. The results demonstrate that the biosensor exhibits a broad linear detection range, a low detection limit, and the potential to rapidly detect carbendazim in actual samples of traditional Chinese medicine. This method offers simple operation, a short detection time, and minimal equipment requirements. In addition, this fluorescent Apt sensor could be adapted to detect other chemicals that have corresponding Apts. Declarations Author Contributions: Methodology, Z.W; Writing—original draft, X.L and Y.H.; Writing—review & editing, T.F.; Funding acquisition, T.F. and K.P. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Special Fund for Science and Technology Innovation Teams of Shanxi Province (No. 202304051001044) and the Natural Science Foundation Project of Shanxi Province (No. 202303021221158). Data Availability Statement: All data generated or analysed during this study are included in this published article Conflicts of Interest: The authors declare no conflicts of interest. References Dong, J.; Yang, H.; Li, Y.; Liu, A.; Wei, W.; & Liu, S. Fluorescence sensor for organophosphorus pesticide detection based on the alkaline phosphatase-triggered reaction. Anal Chim Acta 2020, 1131, 102-108. Liang, N.; Hu, X.; Li, W.; Mwakosya, A. W.; Guo, Z.; Xu, Y.; Huang, X.; Li, Z.; Zhang, X.; Zou, X.; & Shi, J. Fluorescence and colorimetric dual-mode sensor for visual detection of malathion in cabbage based on carbon quantum dots and gold nanoparticles. Food Chem 2021, 343, 128494. Wang, S.; Su, L.; Zhang, D.; Shen, G.; Ma, Y. Colorimetric determination of carbendazim based on the specific recognition of aptamer and the poly-diallyldimethylammonium chloride aggregation of gold nanoparticles. Spectrochim Acta A Mol Biomol Spectrosc 2020, 228:117809. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6225961","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":445258545,"identity":"68943868-faaa-44ba-a49b-c9299552b3a1","order_by":0,"name":"Zhifeng Wang","email":"","orcid":"","institution":"Shanxi Provincial People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhifeng","middleName":"","lastName":"Wang","suffix":""},{"id":445258546,"identity":"b4367c4f-0c5a-4bab-8541-1030e3d55102","order_by":1,"name":"Xiaochen Liu","email":"","orcid":"","institution":"Shanxi University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiaochen","middleName":"","lastName":"Liu","suffix":""},{"id":445258547,"identity":"50893fbf-6173-4f73-8951-bd2ce092073d","order_by":2,"name":"Yu Huang","email":"","orcid":"","institution":"Shanxi University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Huang","suffix":""},{"id":445258548,"identity":"f16eb327-6749-44dd-8f64-c5a8cd54a3b5","order_by":3,"name":"Ke Pei","email":"","orcid":"","institution":"Shanxi University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Pei","suffix":""},{"id":445258549,"identity":"41767e0c-3717-4eeb-a41f-a69495beb428","order_by":4,"name":"Tingting Feng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABE0lEQVRIie2PsUrEQBCGJyxsmvHOMjb6CgsLyxXL5UFsNgS2OkUQJMXBBYTYnNyrCIJ1ZECbcPcCFrERQYtcZ2HhLmolybUW+xX//MV8MAMQCPxHmA8BEJdumkIvxozdt0MGeqV2CtautI01B1c8F4OKD7/tlei5IiM2qPaHlDTeo7fu7Gm6jK/pNavsqSRQAHN93H/YyE5q8ZIvcW0nZq3PFYFt4cGelL0KKlELytNkpoS5sNEdwaOIStqt4NG7UzhFt5dRlexQZOuUKSaumIqyG8b4sEKooBFkEGcKTGNlQpwJM/BLvGpkV3xSinEjtx+FPhyvNtu2m+texcETF1n5U74x/ese1rlIf0sgEAgE/vAFpwpdJy/kXf8AAAAASUVORK5CYII=","orcid":"","institution":"Shanxi University of Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Tingting","middleName":"","lastName":"Feng","suffix":""}],"badges":[],"createdAt":"2025-03-14 11:53:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6225961/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6225961/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81034811,"identity":"3ac377ed-679d-4843-b2c1-b5bc1363e8bb","added_by":"auto","created_at":"2025-04-21 12:03:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":156207,"visible":true,"origin":"","legend":"\u003cp\u003eTEM characterization and DLS image of AuNPs.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6225961/v1/5d44d66f06a760a69b957dbc.png"},{"id":81034814,"identity":"c62f1e0b-1ec2-48b0-a7fb-11363b1003ba","added_by":"auto","created_at":"2025-04-21 12:03:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":255646,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTEM images of GQDs.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6225961/v1/dd30d8508691dee529c7bed8.png"},{"id":81034813,"identity":"e1b27e50-06fc-4b5e-8436-b3f2650801fa","added_by":"auto","created_at":"2025-04-21 12:03:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":79273,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence spectra of the biosensor under different conditions.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6225961/v1/a02cee3ca99dc754b3f4346b.png"},{"id":81034819,"identity":"5bdda0c6-2a14-48d3-ba1d-faee38ed3372","added_by":"auto","created_at":"2025-04-21 12:03:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":60677,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of (A) Apt and (B) NaCl concentrations on system fluorescence intensities.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6225961/v1/b1cc66bd7e414a07bbca86b0.png"},{"id":81034815,"identity":"28e26ffe-011c-4555-941c-d38b35ef0135","added_by":"auto","created_at":"2025-04-21 12:03:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":29216,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of reaction time between Apt and carbendazim (0, 5, 10, 15, 20, 25, and 30 min) on system fluorescence intensity (GQDs:100 nmol/L, AuNPs, NaCl:120 mmol/L, Apt:10 μmol/L, Carbendazim:5 μg/mL).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6225961/v1/0df0f2176c66316ca8299d2b.png"},{"id":81035920,"identity":"34d75e41-64aa-41f5-aeda-3f05ef6d10af","added_by":"auto","created_at":"2025-04-21 12:11:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":105642,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence intensity and trends at different carbendazim concentrations (From bottom to top, carbendazim concentrations are 1, 2, 3, 4, 5, 6, 7 μg/mL; Apt:10 μmol/L, AuNPs, NaCl:120 mmol/L, GQDs: 100 nmol/L; F and F\u003csub\u003e0\u003c/sub\u003e are the changes in fluorescence intensity of the system before and after the addition of carbendazim).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6225961/v1/e25f02c69cc9a95e404db434.png"},{"id":81036178,"identity":"9eeff904-03af-4822-a949-1bf5eecda93a","added_by":"auto","created_at":"2025-04-21 12:19:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":42245,"visible":true,"origin":"","legend":"\u003cp\u003eSpecificity of the proposed method (carbendazim: 100 μg/mL, fipronil: 1 mg/mL, pymetrozine: 1 mg/mL, ethiprole: 1 mg/mL, dinotefuran:1 mg/mL, acetamiprid:1 mg/mL, benomyl: 1 mg/mL, thiophanate:1 mg/mL).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6225961/v1/40501ffbdc21e7d77b408558.png"},{"id":81034839,"identity":"5ac59512-1a40-4913-933d-8e59aff8c263","added_by":"auto","created_at":"2025-04-21 12:03:37","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":28434,"visible":true,"origin":"","legend":"\u003cp\u003eRepeatability and stability of the method.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6225961/v1/b2e072fa18127d13c10d6a66.png"},{"id":96278908,"identity":"a7f3bed2-6221-4acb-b938-68548db8cd98","added_by":"auto","created_at":"2025-11-19 10:39:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1499913,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6225961/v1/76532e11-e160-4a51-9cfa-61172c504588.pdf"},{"id":81035918,"identity":"95a68b08-9eb4-41e7-ba40-99338f00a5bc","added_by":"auto","created_at":"2025-04-21 12:11:37","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":182564,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1. \u003c/strong\u003eDiagram illustrating the detection mechanism of the fluorescent Apt sensor for carbendazim.\u003c/p\u003e","description":"","filename":"Scheme1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6225961/v1/2e17ba633aed31da14053a79.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Signal-on fluorescent strategy for label-free detection of carbendazim based on gold nanoparticles quenching graphene quantum dots","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe use of pesticides is one of the important measures to reduce the pest of plant Chinese medicinal materials.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The use of pesticides is a key measure for reducing crop damage from pests and rodents. Carbendazim, a benzimidazole fungicide, is known for its high efficacy, low toxicity to humans and animals, and broad antibacterial spectrum [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The primary molecular structure of carbendazim is a benzimidazole ring, which is chemically stable and resistant to degradation under natural conditions [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, excessive use can lead to residues. Studies have shown that carbendazim residues in the environment can harm the reproductive systems of mammals and negatively impact the genetic behaviors of some organisms. Moreover, its degradation products are toxic organic substances [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, establishing an analytical method to rapidly detect carbendazim in the field is crucial.\u003c/p\u003e \u003cp\u003eCurrently, the most common methods for detecting the pesticide carbendazim in China include fluorescence spectrometry [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], spectrophotometry [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], electrochemical methods [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], liquid chromatography [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], liquid mass spectrometry [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and gas chromatography [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Although these methods exhibit high accuracy and stability, they are limited by the high cost and complexity of the instruments, the complicated sample pretreatment process, and the extended detection times. Thus, they are unsuitable for the rapid field detection of pesticide residues. Surface-enhanced Raman spectroscopy [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] is an emerging spectroscopic detection technology known for its low cost, high accuracy, and fast detection capabilities. However, it still has limitations, such as challenges in preparing consistent substrates, which hinder its broader application and development in various fields [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Enzyme-linked immunosorbent assay offers strong specificity, high sensitivity, low cost, and portability [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, the long preparation time and variability in antibody production can lead to inconsistent results between different batches. Additionally, the inherent instability of the antibody proteins and enzymes used in this method further limits its scope of application.\u003c/p\u003e \u003cp\u003eAn aptamer (Apt) is a single-stranded nucleic acid molecule that can specifically bind to target molecules through in vitro screening technology [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Its affinity and specificity are similar to those of antibodies, allowing it to selectively recognize target molecules. Compared to antibodies, Apts offer significant advantages, such as ease of synthesis, high stability, and low cost [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Biosensing methods that combine Apts with electrochemistry, fluorescence, and colorimetry have been extensively used in food detection, particularly for the detection of pesticide residues.\u003c/p\u003e \u003cp\u003eIn this study, a simple and rapid fluorescence sensing method was developed to detect carbendazim in Chinese herbal slices based on the FRET effect between AuNPs and GQDs. The synthesized gold nanoclusters remain dispersed due to the electrostatic repulsion generated by the citrate, which prevents them from aggregating. When NaCl is added, the negative charge of citrate is neutralized, resulting in the aggregation of gold nanoparticles. When Apt is added to AuNPs solution, aptamers can be strongly absorbed on AuNPs surface through the coordination of nitrogen atoms with AuNPs, thus enhancing the stability of AuNPs against salt-induced aggregation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This interaction preserved the optical characteristics of the AuNPs, allowing them to quench the fluorescence of the GQDs. However, when carbendazim is introduced, it specifically binds to the Apt on the AuNPs, forming a carbendazim\u0026ndash;Apt complex. This causes the AuNPs to lose the protection of the Apt, leading to their aggregation in the high NaCl concentration and resulting in a failure to quench the GQDs fluorescence. Consequently, the fluorescence intensity of the solution increased. Based on this principle, a fluorescent Apt sensing method was developed to detect carbendazim in traditional Chinese medicine decoction pieces.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Reagents and chemicals\u003c/h2\u003e \u003cp\u003eThe FAM-labeled Apt (5'-CGACACAGCGGAGGCCACCCGCCCACCAGCCCCTG CAGCTCCTGTACCTGTGTGTGTG-3') was obtained from Hema Biology Co., Ltd. (Zhejiang, China). GQDs were purchased from Nanjing Xianfeng Nanomaterial Technology Co., Ltd. (Nanjing, China). Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e\u0026middot;12H\u003csub\u003e2\u003c/sub\u003eO, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, NaCl, KCl, carbendazim, dinotefuran, ethiprole, acetamiprid, fipronil, pymetrozine, benomyl and thiophanate standards were purchased from Maikelin Biology Co., Ltd. (Shanghai, China). Phosphate-buffered saline (PBS) (10 mmol/L, pH 7.4), composed of Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e\u0026middot;12H\u003csub\u003e2\u003c/sub\u003eO, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, NaCl and KCl, was prepared and used in subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Instrumentation\u003c/h2\u003e \u003cp\u003eFluorescence spectra were recorded using an RF-6000 fluorescence spectrophotometer (Shimazu, Japan) with an excitation wavelength of 380 nm and an emission range of 430\u0026ndash;600 nm. Both excitation and emission wavelengths were set with a 5-nm bandwidth. A pHS-3E pH meter (Shanghai, China) was used for pH measurements, and a PR124ZH/E electronic balance (Changzhou, China) was used for weighing. An MTH-100 constant-temperature mixing instrument was employed for incubation baths. Dynamic light scattering (DLS) measurement was acquired on a Zetasizer Nano ZS90 analyzer (Malvern Instruments Ltd., UK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. AuNPs preparation and synthesis\u003c/h2\u003e \u003cp\u003eThe synthesis and preparation of AuNPs were based on methods described by this document [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], with slight modifications. Initially, 5 mL of 1% (m/m) sodium citrate solution was placed in a brown conical flask wrapped in tin foil and stirred at 180℃. Next, 90 mL of distilled water was added and heated to boiling. Subsequently, 5 mL of 0.2% (m/m) chloroauric acid solution was quickly added, and the mixture was brought back to a boil. The color of the solution gradually changed from colorless to light pink. After continuing to boil for about 10 min, the color of the solution turned to wine red. Heating was then stopped, but the solution was stirred and allowed to continue heating with residual heat for 1 h. Finally, the AuNPs solution was cooled to room temperature and stored at 4℃ in the dark for later use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Optimization of experimental conditions\u003c/h2\u003e \u003cp\u003eTo optimize the Apt concentration, Apt solutions of varying concentrations were added to 200 \u0026micro;L of AuNPs solution, mixed, and incubated at 25℃ for 5 min. Subsequently, 22.5 \u0026micro;L of NaCl solution (150 nmol/L) was added and incubated at room temperature for 5 min. Finally, 40 \u0026micro;L of the GQDs suspension (100 \u0026micro;g/mL) was added and incubated at 25℃ for 2 min. The mixture was then brought to a final volume of 500 \u0026micro;L with PBS, and the fluorescence intensity of each sample was measured between 430\u0026ndash;600 nm using a fluorescence spectrophotometer.\u003c/p\u003e \u003cp\u003eTo optimize the NaCl solution, an Apt solution (800 nmol/L) was added to 200 \u0026micro;L of AuNP solution, mixed, and incubated at 25℃ for 5 min. NaCl solutions of varying concentrations were then added and incubated at room temperature for 5 min. Afterward, 40 \u0026micro;L of GQDs suspension (100 \u0026micro;g/mL) was added and incubated at 25℃ for 2 min. The mixture was brought to a final volume of 500 \u0026micro;L with PBS, and the fluorescence intensity of each sample was measured between 430\u0026ndash;600 nm using a fluorescence spectrophotometer.\u003c/p\u003e \u003cp\u003eTo optimize the reaction time between Apt and carbendazim standard solution, a carbendazim standard solution (5 \u0026micro;g/mL) was mixed with 30 \u0026micro;L of Apt solution (800 nmol/L) and incubated at 25℃ for 0, 5, 10, 15, 20, 25, and 30 min. Next, 200 \u0026micro;L of AuNPs solution was added, and the mixture was incubated at 25℃ for 5 min. Subsequently, 22.5 \u0026micro;L of NaCl solution (120 nmol/L) was added and incubated at room temperature for 5 min. Finally, 40 \u0026micro;L of GQDs suspension (100 \u0026micro;g/mL) was added and incubated at 25℃ for 2 min. The final mixture was brought to 500 \u0026micro;L using PBS, and the fluorescence intensity of each sample was measured between 430\u0026ndash;600 nm with a fluorescence spectrophotometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Carbendazim detection using a fluorescent biosensor\u003c/h2\u003e \u003cp\u003eDifferent concentrations of carbendazim standard solution (1, 2, 3, 4, 5, 6, and 7 \u0026micro;g/mL) and 30 \u0026micro;L of Apt solution (800 nmol/L) were added to a 0.5 mL centrifuge tube and incubated at 25℃ for 20 min. Next, 200 \u0026micro;L of AuNPs solution was added, and the mixture was incubated at 25℃ for 5 min. Subsequently, 22.5 \u0026micro;L of NaCl solution (120 nmol/L) was added and incubated at room temperature for 5 min. Finally, 40 \u0026micro;L of GQDs suspension (100 \u0026micro;g/mL) was added and incubated at 25℃ for 2 min. The final mixture was brought to a volume of 500 \u0026micro;L with PBS, and the fluorescence intensity of each sample was measured between 430\u0026ndash;600 nm using a fluorescence spectrophotometer. The excitation wavelength was set to 380 nm, with an emission spectrum range of 430\u0026ndash;600 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Specificity experiment\u003c/h2\u003e \u003cp\u003eTo evaluate the selectivity of the fluorescent Apt sensor, the effects of carbendazim and other potential interfering substances on the fluorescence intensity were investigated. Carbendazim standard solution (5 \u0026micro;g/mL) and various interfering substances (dinotefuran, ethiprole, acetamiprid, fipronil, pymetrozine, benomyl and thiophanate) were incubated with 30 \u0026micro;L of Apt solution (600 nmol/L) at 25℃ for 20 min. Subsequently, 200 \u0026micro;L of AuNPs solution was added, and the mixture was incubated at 25℃ for 5 min. Next, 22.5 \u0026micro;L of NaCl solution (150 nmol/L) was added and incubated at room temperature for 5 min. Finally, 40 \u0026micro;L of GQDs suspension (100 \u0026micro;g/mL) was added and incubated at 25℃ for 2 min. The mixture was then brought to a final volume of 500 \u0026micro;L with PBS, and the fluorescence intensity of each sample was measured between 430\u0026ndash;600 nm using a fluorescence spectrophotometer. The excitation wavelength was set to 380 nm, with an emission spectrum range of 430\u0026ndash;600 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Determination of carbendazim in traditional Chinese medicine decoction pieces\u003c/h2\u003e \u003cp\u003eSophora flavescens and yam slices were selected as the traditional Chinese medicine samples. A precise 0.2000 g of powdered TCM decoction pieces was placed in a 2 mL centrifuge tube, and 2 mL of 70% (v/v) methanol solution was added. After ultrasonic treatment for 10 min, the supernatant was collected through centrifugation for 10 min. The filtrate, obtained by passing through a microporous filter membrane, was diluted to 1 \u0026micro;g/mL, yielding the test solution, which was stored at 4℃ in the dark.Carbendazim standard solution (5,6 and 7 \u0026micro;g/mL) is added to the above sample, and were incubated with 30 \u0026micro;L of Apt solution (600 nmol/L) at 25℃ for 20 min. Subsequently, 200 \u0026micro;L of AuNPs solution was added, and the mixture was incubated at 25℃ for 5 min. Next, 22.5 \u0026micro;L of NaCl solution (150 nmol/L) was added and incubated at room temperature for 5 min. Finally, 40 \u0026micro;L of GQDs suspension (100 \u0026micro;g/mL) was added and incubated at 25℃ for 2 min. The mixture was then brought to a final volume of 500 \u0026micro;L with PBS, and the fluorescence intensity of each sample was measured between 430\u0026ndash;600 nm using a fluorescence spectrophotometer. Fluorescence detection was conducted, and each sample was measured in triplicate. The recovery rate and the relative standard deviation (RSD) were calculated for each sample.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Characterization of AuNPs materials\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e, AuNPs were characterized using transmission electron microscopy (TEM). The image indicates that the size distribution of the prepared AuNPs is relatively uniform and well dispersed. In order to further study the size distribution and surface properties of gold nanoparticles, the particle size of AuNPs was detected by DLS measurement. It can be seen that the size of AuNPs is about 13 nm [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Characterization of GQDs\u003c/h2\u003e \u003cp\u003eThe TEM images in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e show that GQDs were spherical in morphology with an average diameter of 14.5 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Feasibility analysis of the carbendazim fluorescent biosensor\u003c/h2\u003e \u003cp\u003eTo evaluate the feasibility of the proposed method, the fluorescence spectra of the detection system under different conditions were recorded (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). When only GQDs were present in the solution, the fluorescence intensity was highest (Black line). Upon the addition of AuNPs to the GQDs solution, the fluorescence intensity decreased significantly due to the FRET phenomenon (Purple line). In the presence of AuNPs, GQDs, and NaCl, the fluorescence intensity increased compared to Purple line, indicating that a high NaCl concentration causes AuNPs to aggregate. This aggregation weakens the FRET effect, leading to an increase in fluorescence intensity (Red line). When AuNPs, GQDs, NaCl, and Apt were present, the fluorescence intensity decreased somewhat compared to Blue line. This reduction is due to Apt binding around the AuNPs, protecting them from aggregation and allowing FRET to occur between AuNPs and GQDs (Green line). Blue line shows the fluorescence spectrum for a solution containing AuNPs, GQDs, NaCl, Apt, and carbendazim. Compared to Green line, the fluorescence intensity is slightly increased. This increase occurs because when Apt binds to carbendazim, a stable complex forms, reducing the amount of Apt around the AuNPs. This allows numerous AuNPs to aggregate, decreasing FRET and increasing fluorescence intensity. Overall, these results demonstrate that the proposed fluorescence Apt sensing method is feasible for detecting carbendazim.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Optimization of experimental conditions\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e3.4.1. Optimization of aptamer and NaCl concentrations\u003c/h2\u003e \u003cp\u003eDifferent concentrations of Apt and NaCl can influence the aggregation of AuNPs, affecting the ability of the biosensor to detect carbendazim. Therefore, optimizing the concentrations of Apt and NaCl is essential. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e(A), the fluorescence intensity (F) gradually stabilizes when the Apt concentration exceeds 600 nmol/L. This stabilization likely occurs because, at higher Apt concentrations, there is sufficient Apt around the AuNPs to protect them from being quenched by GQDs, resulting in a more stable system. For NaCl concentration optimization, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e(B) shows that as the NaCl concentration increases to 120 nmol/L, the fluorescence intensity (F) gradually stabilizes. This indicates that AuNPs have fully aggregated at this concentration. Therefore, the optimal Apt and NaCl concentrations in the reaction system are 600 nmol/L and 120 nmol/L, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e3.4.2. Optimization of reaction conditions for carbendazim and Apt\u003c/h2\u003e \u003cp\u003eThe reaction time between Apt and carbendazim affects the stability and sensitivity of the method. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the fluorescence intensity (F) of the system reaches its maximum at 25 min. This peak intensity likely results from the optimal binding between Apt and carbendazim, which occurs at this reaction time. Therefore, a reaction time of 25 min is most effective for generating reliable experimental results.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Analytical performance of the carbendazim fluorescent aptamer sensor\u003c/h2\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e3.5.1. Establishment of the standard curve\u003c/h2\u003e \u003cp\u003eTo investigate the effectiveness of the fluorescent Apt sensor for detecting carbendazim, absorbance changes in the sensor system were measured at varying concentrations of carbendazim standard solution (1, 2, 3, 4, 5, 6, and 7 \u0026micro;g/mL). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the fluorescence intensity of the system increased with increasing carbendazim concentrations. Within the concentration range of 0.5\u0026ndash;7 \u0026micro;g/mL, the relationship between carbendazim concentration (C) and (F\u0026thinsp;\u0026minus;\u0026thinsp;F\u003csub\u003e0\u003c/sub\u003e)/F\u003csub\u003e0\u003c/sub\u003e (where F indicates the fluorescence intensity after carbendazim addition) was linear. The equation derived is (F\u0026thinsp;\u0026minus;\u0026thinsp;F\u003csub\u003e0\u003c/sub\u003e)/F\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.2099C\u003csub\u003ecarbendazim\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;0.3349, with a correlation coefficient of 0.9913. Using the 3σ/s principle, the calculated limit of detection (LOD) is 0.44 \u0026micro;g/mL. These results indicate that the sensor exhibits high sensitivity for carbendazim detection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.5.2. Method specificity\u003c/h2\u003e \u003cp\u003eSpecificity is a crucial indicator for assessing whether the test system can reliably detect carbendazim without interference from other substances (such as similar pesticides) that may be present in actual sample tests. Under optimized conditions, the biosensor was used to detect 100 \u0026micro;g/mL of carbendazim and 50 \u0026micro;g/mL of seven common pesticide residues (dinotefuran, ethiprole, acetamiprid, fipronil, pymetrozine, benomyl and thiophanate), and the corresponding fluorescence intensities were recorded. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e, the fluorescence intensity increased significantly when only carbendazim was present in the system. Conversely, the fluorescence intensity of the other substances remained minimal, even when present at concentrations 10 times higher than carbendazim. These results confirm that the fluorescent Apt sensor has good selectivity for detecting carbendazim in Chinese herbal slices.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Repeatability and stability of the method\u003c/h2\u003e \u003cp\u003eTo evaluate the repeatability and stability of the method, the carbendazim standard solution (100 \u0026micro;g/mL) was measured in triplicate under optimized conditions, and the corresponding fluorescence intensities were recorded. The (F\u0026thinsp;\u0026minus;\u0026thinsp;F\u003csub\u003e0\u003c/sub\u003e)/F\u003csub\u003e0\u003c/sub\u003e values were calculated, where F represents the fluorescence intensity after adding carbendazim, and F\u003csub\u003e0\u003c/sub\u003e is the fluorescence intensity without carbendazim. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e, after 14 d, the fluorescence intensity of the sensor remained at 95%, demonstrating that the constructed fluorescence Apt sensor exhibits good repeatability and stability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Chinese medicine actual sample test\u003c/h2\u003e \u003cp\u003eTo assess the practical application of the proposed method, the sensor system was used to detect carbendazim concentrations in herbal slices of Sophora flavescens and yam. First, the sensor was used to detect carbendazim in the Chinese yam and Sophora flavescens, but no detectable carbendazim residues were found in the samples. Then, three different concentrations of carbendazim (5, 6, and 7 \u0026micro;g/mL) were added to the diluted reserve solution in item 2.7. The results are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The recoveries of carbendazim in the two sample types ranged from 90.68\u0026ndash;106.29%, with RSDs between 0.52% and 1.60%. These results indicate that the method has good practicability and can be extensively used for the rapid detection of carbendazim in traditional Chinese medicine decoction pieces, offering a new approach to determining pesticide residues.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSpiked recoveries in real samples.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAdded (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFound (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRecovery (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRSD (%, n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eSophora\u003c/p\u003e \u003cp\u003eflavescens\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e106.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e101.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eYam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this study, we developed a biosensor utilizing FRET between AuNPs and GQDs for the detection of carbendazim pesticide residues in traditional Chinese medicine decoction pieces. Under optimized conditions, the linear range for the fluorescence Apt sensor was 0.5\u0026ndash;7 \u0026micro;g/mL, with an LOD of 0.44 \u0026micro;g/mL. The results demonstrate that the biosensor exhibits a broad linear detection range, a low detection limit, and the potential to rapidly detect carbendazim in actual samples of traditional Chinese medicine. This method offers simple operation, a short detection time, and minimal equipment requirements. In addition, this fluorescent Apt sensor could be adapted to detect other chemicals that have corresponding Apts.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eMethodology, Z.W; Writing\u0026mdash;original draft, X.L and Y.H.; Writing\u0026mdash;review \u0026amp; editing, T.F.; Funding acquisition, T.F. and K.P. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by the Special Fund for Science and Technology Innovation Teams of Shanxi Province (No. 202304051001044) and the Natural Science Foundation Project of Shanxi Province (No. 202303021221158).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAll data generated or analysed during this study are included in this published article\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eDong, J.; Yang, H.; Li, Y.; Liu, A.; Wei, W.; \u0026amp; Liu, S. 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Electrochemical Biosensor Based on Well-Dispersed Boron Nitride Colloidal Nanoparticles and DNA Aptamers for Ultrasensitive Detection of Carbendazim. \u003cem\u003eACS Omega\u003c/em\u003e 2021, 6(41):27405-27411.\u003c/li\u003e\n \u003cli\u003eXiao, S.; Lu, J.; Sun, L.; An, S. A simple and sensitive AuNPs-based colorimetric aptasensor for specific detection of azlocillin. \u003cem\u003eSpectrochim Acta A Mol Biomol Spectrosc\u003c/em\u003e 2022, 271:120924. Doi: 10.1016/j.saa.2022.120924.\u003c/li\u003e\n \u003cli\u003eSu, L.; Wang, S.; Wang, L.; Yan, Z.; Yi, H.; Zhang, D.; Shen, G.; Ma, Y. Fluorescent aptasensor for carbendazim detection in aqueous samples based on gold nanoparticles quenching Rhodamine B. \u003cem\u003eSpectrochim Acta A Mol Biomol Spectrosc\u003c/em\u003e 2020, 225:117511. Doi: 10.1016/j.saa.2019.117511.\u003c/li\u003e\n \u003cli\u003eSun, Y.; Qi, T.; Jin, Y.; Liang, L.; Zhao, J. A signal-on fluorescent aptasensor based on gold nanoparticles for kanamycin detection. \u003cem\u003eRSC Adv\u003c/em\u003e 2021, 11(17), 10054-10060.\u003c/li\u003e\n \u003cli\u003eChen, X. X.; Lin, Z. Z.; Hong, C. Y.; Zhong, H. P.; Yao, Q. H.; Huang, Z. Y. Label-Free Fluorescence-Based Aptasensor for the Detection of Sulfadimethoxine in Water and Fish. \u003cem\u003eAppl Spectrosc\u003c/em\u003e 2019, 73(3):294-303.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Biosensor, fluorescence, gold nanoparticles, graphene quantum dots, carbendazim, aptamer","lastPublishedDoi":"10.21203/rs.3.rs-6225961/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6225961/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA fluorescence biosensing method based on fluorescence resonance energy transfer between gold nanoparticles (AuNPs) and graphene quantum dots (GQDs) was developed for the rapid detection of carbendazim. Upon adding the aptamer to the AuNPs solution, the aptamer binds to the AuNPs, preventing their aggregation in a high-concentration NaCl solution. Therefore, the AuNPs retain their optical properties and can quench the fluorescence of GQDs. When carbendazim is introduced, it specifically binds to the aptamer on the AuNPs, forming a complex. This interaction causes the AuNPs to lose the protective effect of the aptamer and aggregate in the high-concentration NaCl solution, leading to a loss in their ability to quench GQDs fluorescence, which increases the fluorescence intensity of the solution. This method was applied to recover carbendazim from the leaves of Chinese herbal medicines, achieving recoveries of 90.68%-106.29%. The results demonstrate that the sensor is simple, convenient, and effective.\u003c/p\u003e","manuscriptTitle":"Signal-on fluorescent strategy for label-free detection of carbendazim based on gold nanoparticles quenching graphene quantum dots","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-21 12:03:32","doi":"10.21203/rs.3.rs-6225961/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7737dee1-2a7c-411a-a33c-6f1efe3ea8eb","owner":[],"postedDate":"April 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":47403962,"name":"Biological sciences/Biochemistry"},{"id":47403963,"name":"Physical sciences/Nanoscience and technology"}],"tags":[],"updatedAt":"2025-11-19T10:38:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-21 12:03:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6225961","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6225961","identity":"rs-6225961","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}