{"paper_id":"400d9097-2b91-457d-b108-e791ceba9712","body_text":"Rapid and sensitive on-site detection of Clothianidin in surface waters with a reusable fiber-embedded optofluidic biochip | 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 Research Article Rapid and sensitive on-site detection of Clothianidin in surface waters with a reusable fiber-embedded optofluidic biochip Laiya Lu, Min Wang, Tianxiang Ji, Siyan Liu, Yuxin Zhuo, Feng Long This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6323851/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 May, 2025 Read the published version in Microchimica Acta → Version 1 posted 9 You are reading this latest preprint version Abstract Clothianidin (CLO), a highly effective neonicotinoid insecticide, is globally utilized to combat both sucking and chewing pests. There is an increasing demand for rapid and high-frequency on-site detection of CLO in water and food sources due to its high toxicity to non-target organisms. To address this, we introduce an innovative and decentralized testing method for CLO employing a reusable fiber-embedded optofluidic biochip. This biochip leverages evanescent wave fluorescence, indirect competitive immunoassay, and optofluidic technology to provide reliable, rapid, straightforward, and cost-effective CLO measurements. The integration of an all-fiber optical structure with a tapered fiber nano-biosensor significantly enhances fluorescence excitation and collection efficiency, bolstering the biochip's on-site detection capabilities and scalability. This biochip demonstrated high sensitivity and specificity in detecting CLO, achieving a satisfactory limit of detection of 1.0 µg/L within 12 min. It was successfully applied for rapid on-site screening of CLO in surface waters and food in the Beijing-Tianjin region, offering timely and decentralized feedback. The biochip detected spiked surface waters and food with satisfactory recovery rates. These confirm the biochip's potential as a robust tool for rapid and high-frequency on-site CLO screening in water and food, particularly in settings with limited resources. This biochip is highly adaptable and can be easily expanded to detect other trace pollutants by utilizing the appropriate functionalized fiber biosensors and antibodies. Fiber-embedded optofluidic biochip Clothianidin Evanescent wave fluorescence Immunoassay On-site detection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Induction Clothianidin (CLO) is a novel neonicotinoid insecticide employed to control sucking and chewing types of pests globally [ 1 , 2 ]. This insecticide has broad-spectrum insecticidal activity, acting on the nicotinic acetylcholine esterase receptors in the insect nervous system, blocking the normal conduction of signals in the insect central nervous system, and leading to their paralysis and death [ 3 , 2 ]. Although CLO is characterized by low toxicity, long-lasting effectiveness, and no cross-resistance compared with conventional pesticides [ 4 ], it is highly toxic to non-target organisms such as bees and birds [ 5 ]. In nature, various nontarget organisms can be exposed to this insecticide through contaminated food, water, and air [ 6 ]. Even the ingestion of a small number of treated seeds by birds can lead to death [ 7 ]. The excessive use of CLO may lead to residue levels exceeding safety standards in agricultural products [ 8 ], posing a threat to human health [ 9 , 10 ]. It may result in oxidative stress, neurological symptoms, metabolic changes, osteoporosis, and liver cancer [ 11 , 12 ]. Studies demonstrated that only 5% of the active ingredients [ 13 ] are absorbed by crops, with the majority entering the environment [ 14 , 15 ]. CLO has become among the most readily detectable neonicotinoid compounds in urban [ 16 ] and agricultural watersheds [ 8 ]. According to the European Food Safety Authority (EFSA) risk assessment in 2018, the European Union has prohibited all outdoor uses of CLO [ 17 , 5 ]. Nevertheless, CLO remains a high-quality insecticide for which there are currently no immediate alternatives [ 18 ]. In China, there are 374 registrations of CLO, mainly for the control of aphids, thrips, whiteflies, and other pests. Inspection data from 2023 show an increase in the number of batches of products that exceed the standard, including chili peppers, cowpeas, bananas, celery, and mangoes. To ensure the safety of agricultural products, China has established maximum residue limits (MRLs) for CLO in food, such as 0.05 mg/kg for chili peppers, 0.2 mg/kg for ginger, 0.01 mg/kg for cowpea, 0.02 mg/kg for raw milk and 2 mg/kg for leafy vegetables. Various centralized testing technologies, including gas chromatography (GC), high-performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS), detect CLO with high sensitivity and accuracy [ 19 , 20 ]. However, these methods require complex pre-treatment procedures, detailed personnel training, and expensive and sophisticated instruments [ 21 ], thus lacking capacity for rapid, high-frequency, and point-of-need testing [ 22 ]. Biological molecules such as antibodies, due to their high specificity and affinity, have been widely used to establish sensitive and rapid detection methods for CLO. Various immunoassay methods, such as enzyme-linked immunosorbent assay (ELISA) [ 23 ], chemiluminescent enzyme immunoassay (CLEIA) [ 24 ], fluorescence immunoassay [ 25 ], and lateral flow immunoassay (FLIA) [ 26 ], have been developed for rapid CLO detection. However, several factors limit their practical application, including the inability to perform quantitative assays, low sensitivity, and susceptibility to matrix interference [ 27 ]. In response to these challenges, a portable and reusable fiber-embedded optofluidic biochip has been constructed for the rapid and sensitive on-site detection of CLO [ 28 ]. Optofluidic biochips that combines the outstanding features of optics with those of microfluidic biochips, have emerged as a versatile and potent assay platform for detecting trace toxic substance [ 29 , 30 ]. Traditional optofluidic biochips, however, have been hindered by their reliance on bulky, intricate, and costly detection equipment, such as confocal fluorescence microscopes [ 31 ]. These setups necessitate numerous free-space optical components, meticulous optical alignment, and burdensome maintenance. These factors have constrained their applicability in point-of-need testing scenarios [ 32 ]. In our system, the breakthrough lies in the integration of optical fibers into optofluidic chips, which has opened up new analytical possibilities [ 33 , 34 ]. The advantages of optical fibers include their compact size, large surface-area-to-volume ratio, ease of surface modification, excellent biocompatibility, and resistance to electromagnetic interference [ 35 , 36 ]. The portable fiber-embedded optofluidic biochip utilizes a compact all-fiber optical system for the transmission of excitation light and fluorescence. This design eliminates the need for multiple optically separate components and precise optical alignment, resulting in a sensitive, miniaturized, and portable system. For precise CLO detection, we have embedded a highly reusable CLO-OVA functionalized fiber biosensor into the optofluidic chip, which serves dual roles as both biorecognition element and optical transmission component. By employing the fiber-embedded optofluidic biochip, we have established an indirect competitive immunoassay method for the rapid on-site detection of CLO in a variety of food and environmental samples. This biochip boasts several unique features, including enhanced sensitivity and stability, and a compact optical construction that eliminates the need for optical alignment. These attributes position it as a promising contender for food and environmental monitoring applications. 2. Materials and Methods 2.1 Chemicals and materials CLO, Chlorbenzuron, Bifenthrin, Isoprocarb, Buprofezin, and Thiamethoxam were purchased from Beijing zhongkezhijian Biotechnology (Beijing, China). Bovine serum albumin (BSA), 3-Mercaptopropyltrimethoxysilane (MTS), 4-maleimidobutyric acid N-hydroxysuccinimide ester (GMBS), and hydrofluoric acid (HF) were purchased from Sigma-Aldrich (Steinheim, Germany). All other chemical reagents were purchased from Beijing Chemical Reagent Co. (Beijing, China). Monoclonal anti-CLO antibodies were obtained from Shandong Landu Bio-sciences & technology Co. (Shandong, China). Sodium dodecyl sulfate (SDS, 0.5%, pH = 1.9), phosphate buffer (PBS, pH = 7.4), antibody dilution solution, and Cy5.5 labeled anti-CLO antibody (Cy5.5-anti-CLO antibody) were prepared by our group (Note S1 in Supporting information). Standard stock solution (1.0 mg/mL) of CLO was prepared by dissolving an appropriate amount of CLO in 0.01 mol/L PBS and kept at 4 ºC until use. 2.2 Preparation of reusable Tapered functionalized fiber nano-biosensor To ensure accurate detection of CLO, a reusable functionalized fiber nano-biosensor was prepared. The functionalized tapered fiber nano-biosensor was prepared using a 5.5-cm long quartz fiber (ϕ 600 µm, NA = 0.22) with a 3.0 cm of coating layer stripped to form the biosensing section, which was in-situ etched using hydrofluoric acid (HF) tube-etching method. The combination tapered fiber nano-biosensor with a nanopore layer was fabricated by controlling etching conditions and functionalized with CLO-ovalbumin (CLO-OVA) according to the previous described methods (Note S2 in Supporting Information) [ 37 , 38 ]. 2.3 Fiber-embedded optofluidic biochip The reusable fiber-embedded optofluidic biochip is ingeniously crafted by integrating a functionalized fiber nano-biosensor into an optofluidic chip. Crafted through advanced 3D printing technology, the biochip is endowed with a micro-channel with 3.0 cm in length and 600 µm in diameter, having an effective volume of 20 µL. The 3D architecture of the biochip revolutionizes the fabrication and assembly process, enhancing mass transfer rates and reaction efficiency, which translates to faster and more sensitive detection capabilities. To achieve the fluorescence signal detection, a portable all-fiber evanescent wave fluorescence detection device was used (Fig. 1 D and 1 E). This portable detection device is equipped with a 635 nm laser (8 mW) that provides monochromatic light source, and a single-multimode fiber coupler (SMFC) that adeptly handles the transmission of the excitation and fluorescence light (Fig. 1 D). The excitation light enters the fiber nano-biosensor and propagates via total internal reflection, generating an evanescent wave on the biosensor's surface with an effective penetration depth of less than 100 nm. The Cy5.5-anti-CLO antibody, which binds to the CLO-OVA immobilized on the biosensor's surface, is excited by the evanescent wave. A portion of fluorescence is coupled back into the fiber nano-biosensor. The collected fluorescence is subsequently detected by an ultrasensitive mini-photodetector, the PD-1000. Various solutions, such as buffer, sample, and regeneration solutions, are delivered to the fiber-embedded optofluidic biochip in a controlled manner by a peristaltic pump and a six-position valve. The integrated signal and data processing system analyzed the fluorescence signals. The resulting fluorescence signal trace was clearly displayed on the user interface. 2.4 Detection of CLO in surface waters in Beijing-Tianjin area To assess the environmental monitoring capabilities of the fiber-embedded optofluidic biochip, we gathered surface water specimens from two significant sites in Beijing, China: the Beijing Botanical Garden and The Summer Palace, on December 15, 2023. To broaden the scope of our investigation, we collected water samples from ten varied locations across the Haihe River basin in Tianjin, China, on March 22, April 20, and May 26, 2024. These samples with and without spiked CLO were directly on-site detected by the fiber-embedded optofluidic biochip without any pre-treatment. For a thorough comparative analysis and precise detection of CLO in the collected surface water samples via HPLC, a thorough pre-treatment procedure was indispensable. Initially, each 500 mL water sample was spiked with various concentrations of CLO and was filtered through a glass fiber filter (0.7 µm, Whatman GF/F 47 mm, UK) to eliminate particulate matter. Subsequently, the samples were enriched using solid-phase extraction with an Oasis HLB column (6 cm³, 200 mg, Waters, USA). Following this, the HLB columns were rinsed with deionized water and eluted with methanol. The eluates were then concentrated to a final volume of 1 mL under a nitrogen stream and subsequently filtered through nylon membrane filters (0.45 µm, Whatman, UK). The prepared extracts were analyzed by HPLC (Note S3 in Supporting Information). 2.5 Detection of CLO in foodstuffs To evaluate the performance of the fiber-embedded optofluidic biochip in food analysis, we performed CLO detection in milk and baby Chinese cabbage purchased from a local supermarket. For the milk samples, they were spiked various concentrations of CLO, and blended with a mixture of acetonitrile and 0.2% trifluoroacetic acid. Followed by centrifugation, the supernatant was decanted and passed through a 0.22 µm hydrophobic polytetrafluoroethylene (PTFE) filter. The treated milk samples were analyzed using the fiber-embedded optofluidic biochip. In the case of the baby Chinese cabbage, its juice was extracted using a manual squeezing technique and spiked with various concentrations of CLO. The resulting juice was then filtered through the same 0.22 µm PTFE filter to ensure cleanliness. The filtered samples were subjected to analysis via the biochip. After pre-treatment as water samples, these samples were also analyzed using HPLC. 3. Results and discussion 3.1 Indirect competitive immunoassay mechanism of CLO Figure 2 A illustrates the indirect competitive immunoassay mechanism of CLO and outlines a typical detection cycle using this advanced biochip. Initially, 30 µL of sample containing varying concentrations of CLO and 30 µL of Cy5.5-anti-CLO antibody solution at a predetermined concentration was mixed and incubated for a set period. During this incubation, some of the Cy5.5-anti-CLO antibodies bind with CLO. Concurrently, a 10 mM PBS solution is introduced into the optofluidic biochip to establish a baseline signal (Phase I in Fig. 2 B). Subsequently, the mixture of CLO and Cy5.5-anti-CLO antibody is added into the biochip for initiating an indirect competitive immunoreaction. Antibodies with available binding sites in the mixture bind to the immobilized CLO-OVA on the fiber nano-biosensor's surface. The Cy5.5-anti-CLO antibodies are then excited by the evanescent wave field generated at the sensing surface, and the resulting fluorescence signal is recorded in real-time (Phase II in Fig. 2 B). The fluorescence intensity, which is indicative of the number of antibodies bound to the fiber biosensor's surface, is inversely proportional to the CLO concentration in the samples. To reuse the fiber biosensor, a 0.5% SDS solution at pH = 1.9 is employed to elute the Cy5.5-anti-CLO antibody from the biosensing surface (Phase III in Fig. 2 B). Finally, a 10 mM PBS solution is introduced to the sample cell, flushing away any residual antibody and SDS solution (Phase IV in Fig. 2 B), thus regenerating the fiber biosensor for subsequent tests. The entire detection cycle is completed in less than 10 min. 3.2 Characteristics of the reusable functionalized fiber-embedded optofluidic biochip In the reusable functionalized fiber-embedded optofluidic biochip (Fig. 2 A), the CLO-OVA functionalized fiber nano-biosensor was used as a specific biorecognition element for CLO and a transducer for fluorescence detection. The SEM image showed the formation of nano-pore and the successful modification of CLO-OVA on the fiber nano-biosensor (Fig. 1 B, bottom left). The fluorescence microscopy image demonstrated the significant fluorescence signal when the Cy5.5-anti-CLO antibody was introduced and bound with CLO-OVA immobilized on fiber nano-biosensor (Fig. 1 B, bottom right). To evaluate its performance, titration experiments were conducted using the Cy5.5-anti-CLO antibody. First, when 0.125 µg/mL Cy5.5-anti-CLO antibody was introduced into the optofluidic biochip, the fluorescence intensity increased over the time (Fig. 2 B). The highest fluorescence intensity was obtained due to the most Cy5.5-anti-CLO antibody specifically bound with the CLO-OVA immobilized on the fiber nano-biosensor surface. Second, when the mixture of CLO and Cy5.5-anti-CLO antibody was added, the fluorescence intensity also increased but a lower light intensity was obtained compared with the above results. Third, when the mixture of a large excess of potential interferences (100.0 µg/L Chlorbenzuron, Bifenthrin, Isoprocarb, Buprofezin, Thiamethoxam) and Cy5.5-anti-CLO antibody was added, no significant decrease of fluorescence intensity was observed. These results demonstrated that the CLO-OVA were successfully modified onto the fiber nano-biosensor, and the observed signal decrease originated from the CLO in solution competitively bound with Cy5.5-anti-CLO antibody. The Cy5.5-anti-CLO antibody was highly selective towards CLO and had few cross-reactivity for other interferences. Based on indirect competition immunoassay principle, the biochip could be applied for the specific detection of CLO. The biosensor's surface must possess excellent regeneration capabilities, which are essential for maintaining the precision of detection results, reducing costs, expediting the detection process, and streamlining operational procedures. The reusability, stability, and activity of the CLO-OVA functionalized biosensor were rigorously assessed through a series of bioassays. The fiber nano-biosensor could be effectively regenerated by employing a 0.5% SDS solution at pH 1.9 to detach the bound Cy5.5-anti-CLO antibody from its surface (Fig. 2 B). As depicted in Fig. 3 B, the fiber nano-biosensor's performance remained consistent after 200 cycles of regeneration, with the coefficient of variation (CV) for the fluorescence signal consistently below 1.9% across all tests. This demonstrates the biosensor's remarkable reusability and stability. Subsequent to these evaluations, the biochip's shelf-life was also investigated. The results revealed that the biochip exhibited high storage stability when kept at 4ºC, retaining 92% of its initial response even after 60 days, as illustrated in Fig. 3 C. 3.3 Optimization of detection conditions To enhance the detection performance, a series of conditions were meticulously optimized, including the pre-reaction time, incubation duration, and antibody concentration. The optimization began with the pre-reaction time between the Cy5.5-anti-CLO antibody and CLO, as illustrated in Fig. 3 A. It was observed that an extended pre-reaction of CLO with the Cy5.5-anti-CLO antibodies led to a reduction in the fluorescence signal, with a plateau being reached after 2 min. Consequently, a 2-minute pre-reaction time was adopted for all subsequent experiments. Figure 3 B demonstrates the impact of incubation time on fluorescence intensity. When the biochip was exposed to an anti-CLO antibody solution without CLO or with a low concentration of CLO (10.0 µg/L), the fluorescence intensity increased rapidly at first and then plateaued after 20 min. In contrast, when a mixture containing a high concentration of CLO (1000.0 µg/L) along with the anti-CLO antibody, the fluorescence intensity quickly reached a plateau within just 5 min. Taking into account the necessity for rapid on-site screening of CLO, an incubation time of 10 min was determined to be optimal for CLO detection. The fiber-embedded optofluidic biochip functions on the principle of an indirect competition immunoassay, where the detection sensitivity for CLO is significantly influenced by the concentration of the antibodies used. To ascertain the optimal concentration of Cy5.5-anti-CLO antibody, a sensitivity index ( ε A ) was introduced, as defined by Eq. ( 1 ). $$\\:{\\varepsilon\\:}_{\\text{A}}=\\frac{{I}_{\\text{A}0}-{I}_{\\text{A}1}}{{I}_{\\text{A}0}}$$ 1 where, \\(\\:{I}_{\\text{A}0}\\) and \\(\\:{I}_{\\text{A}1}\\) represent the net fluorescent intensities of the samples in the absence and presence of the target analyte, respectively. The optimal antibody concentration was chosen based on a balance between suitable fluorescence intensity and a high sensitivity index. As depicted in Fig. 3 C, the fluorescence signal increased with the rising concentration of Cy5.5-anti-CLO antibody, as this allowed for more antibodies to bind with CLO-OVA that was immobilized on the fiber biosensor. Conversely, the sensitivity index ε A increased with decreasing antibody concentration, as the lower concentration favored the competitive binding of free CLO in solution, leading to fewer antibodies binding with CLO-OVA. After considering both the sensitivity index and the appropriate fluorescence intensity, a Cy5.5-anti-CLO antibody concentration of 0.125 µg/mL was identified as the optimal concentration for use in subsequent experiments. 3.4 Dose-response curve of CLO immunoassay After determining the optimal detection parameters, quantitative analyses of CLO were conducted using the fiber-embedded optofluidic biochip. The Cy5.5-anti-CLO antibody was premixed with a range of CLO concentrations and incubated for 2 min. This mixture was then introduced into the optofluidic biochip for fluorescent detection. Figure 4 A displays the typical real-time fluorescence signal traces recorded for various CLO concentrations. As anticipated, an increase in CLO concentration led to a corresponding decrease in fluorescence intensity. This reduction is attributed to the competition between free CLO and CLO-OVA on the fiber biosensor for binding sites on the Cy5.5-anti-CLO antibody, with free CLO occupying the active sites and thereby reducing the binding of the antibody to CLO-OVA. For each assay, the effective fluorescence signal of the samples ( I s ), with the baseline signal subtracted, was normalized against that of a blank sample using the following formula: ΔI s = I s / I b (2) where I b represents the fluorescent intensity of the blank sample, while I s denotes the fluorescent intensity of the sample under test. These normalized results are then employed to establish a dose-response relationship, which is modeled using a four-parameter logistic equation (Note S4 in Supporting Information). The standard deviation of less than 3.1% for each CLO concentration, with three replicates (n = 3), underscores the high stability of the fiber-embedded optofluidic biochip for CLO detection, as shown in Fig. 4 B. The limit of detection (LOD) for CLO, calculated to be 1.0 µg/L based on three times the standard deviation (3σ), is notably lower than the maximum residue limits (MRLs) established by various countries and organizations, and is also on par with the LODs of other methods, as detailed in Table S1 . This achievement is attributed to the high affinity of the anti-CLO antibody and the exceptional sensitivity of the fiber-embedded optofluidic biochip. The biochip demonstrated a linear response to CLO concentrations ranging from 5.0 to 500.0 µg/L. The fiber-embedded optofluidic biochip stands out against traditional detection methods with several remarkable characteristics. Firstly, its all-fiber optical architecture enhances the efficiency of fluorescence excitation and collection, bolstering its capabilities for on-site detection and scalability. Additionally, the nanopore structure of the fiber nano-biosensor increases the immobilization of biorecognition molecules on its surface and intensifies the interaction between the enhanced evanescent field and the dye, thereby significantly boosting detection sensitivity. Secondly, the fiber-embedded optofluidic biochip offers a plug-and-play simplicity that is easy to operate, making it resilient to stringent operating conditions and capable of providing rapid, decentralized feedback. Thirdly, this innovative biosensing platform is highly versatile, easily adaptable for detecting other trace pollutants by utilizing the appropriate functionalized fiber nano-biosensors and antibodies. Furthermore, this biosensing platform is endowed with numerous additional advantages, including its compact size, portability, reusability, user-friendliness, and cost-effectiveness. These features render it an exemplary solution for the rapid on-site screening of CLO, which is crucial for improving symptom management and enhancing health-related quality of life, especially in settings with limited resources. 3.5 Detection of CLO in water environments To assess the practical application of the fiber-embedded optofluidic biochip, a series of surface water samples were collected from various locations, including the Beijing Botanical Garden, the Summer Palace, and the Haihe River basin, for detection of CLO. Specifically, thirty water samples were gathered from ten different sites within the Haihe River basin (Figure S1 ) over a three-month period from March to May 2024. These samples were tested directly using the fiber-embedded optofluidic biochip without any prior treatment, and no CLO was detected in any of the samples (Table 1 and Table S2). To corroborate these findings, the same water samples underwent rigorous pretreatment and were analyzed using HPLC; no CLO was detected (Table S2). These outcomes suggest that the risk of CLO contamination in the Beijing-Tianjin area is minimal. To further assess the accuracy of the fiber-embedded optofluidic biochip, the water samples from the Beijing Botanical Garden and the Summer Palace were spiked with different concentrations of CLO (20, 40, and 60 µg/L CLO) and retested. The recovery rates of CLO in these spiked samples, as detailed in Table 1 , varied from 80 to 110%. These were verified via HPLC (Table 1 ), with a strong relationship observed between the measured results obtained using the biochip and HPLC (Fig. 5 ). These results indicate that the fiber-embedded optofluidic biochip provides satisfactory accuracy and reliability for detecting CLO in real-world water samples. In conclusion, the fiber-embedded optofluidic biochip emerges as a robust tool for the rapid on-site decentralized screening of CLO in aquatic environments, offering a valuable contribution to environmental monitoring and public health. Table 1 Determination of CLO in environment water and food samples by fiber-embedded optofluidic biochip and HPLC Samples Original water (µg/L) Spiked Conc. (µg/L) Detection Conc. (µg/L) RSD (%) Recovery (%) HPLC(µg/L) Recovery (%) The Summer Palace < 1.0 20 20.25 9.1 101.25% 19.286 96.43% < 1.0 40 33.70 2.3 84.25% 39.26 98.15% < 1.0 60 50.05 2.2 83.42% 53.994 89.99% Beijing Botanical Garden < 1.0 20 19.85 0.2 99.25% 18.518 92.59% < 1.0 40 33.85 8.7 84.63% 44.28 110.70% < 1.0 60 52.40 4.5 87.33% 64.554 107.59% Milk < 1.0 20 19.66 16.6 98.30% 19.286 96.43% < 1.0 40 46.81 7.0 117.04% 40.06 100.15% < 1.0 60 60.56 14.7 100.94% 62.682 104.47% Baby Chinese cabbage < 1.0 20 21.80 16.1 109.00% 20.264 101.32% < 1.0 40 36.50 5.8 91.25% 36.384 90.96% < 1.0 60 50.46 5.1 84.11% 57.318 95.53% 3.6 Towards the universal immunoassay platform for CLO detection To showcase its versatility, the fiber-embedded optofluidic biochip was deployed to detect CLO in various food items, specifically milk and Chinese cabbage. The performance of the biochip in food detection was evaluated under these real-world conditions. Given the complexity of milk's composition, which could potentially interfere with antigen-antibody interactions, the milk samples were extracted using a solution containing acetonitrile and trifluoroacetic acid (0.2%). After filtration, the supernatant was analyzed using the fiber-embedded optofluidic biochip, and no CLO was detected in these samples (Table 1 ). For the detection of CLO in Chinese cabbage, the cabbage was squeezed to extract its juice, which was then filtered through a 0.22 µm PTFE filter film. The filtered solution was subsequently tested with the biochip, and again, no CLO was also detected (Table 1 ). To further assess the biochip's accuracy, two types of food samples were spiked with various concentrations of CLO (20, 40, and 60 µg/L). As indicated in Table 1 , the recovery rates of CLO in these spiked food samples ranged from 80 to 120%, which were also verified via HPLC (Table 1 ) with a strong relationship observed between the measured results obtained using the biochip and HPLC (Fig. 5 ). These results indicate that the fiber-embedded optofluidic biochip offers satisfactory accuracy and reliability for detecting CLO in actual food samples. Thus, the fiber-embedded optofluidic biochip proves to be a viable tool for the rapid decentralized screening of CLO in food products, extending its utility beyond environmental samples to include food safety applications. 4. Conclusion In this study, an innovative and decentralized testing method for CLO employing a reusable fiber-embedded optofluidic biochip is described through integrating evanescent wave fluorescence, indirect competition immunoassay, and optofluidic biochip technology. The combination of all-fiber optical structure and tapered fiber nano-biosensor significantly improves the fluorescence excitation and collection efficiency, as well as its on-site detection ability and scalability. The nanopore structure of the fiber nano-biosensor enhanced the number of biorecognition molecules immobilized on its surface and the interaction between the enhanced evanescent field and dye, allowing a further increase in the detection sensitivity. The all-fiber optical structure and the plug-and-play biochip allows it to achieve rapid on-site screening of target pollutants, and unaffected by stringent operating conditions, thus providing timely feedback in a decentralized and rapid fashion. The fiber-embedded optofluidic biochip was successfully used for rapid on-site screening of the CLO in the surface waters and food in Beijing-Tianjin area. Although no positive results that was validated by HPLC were obtained, the spiked surface waters and food samples were detected with satisfactory recovery rates. Several outstanding features of the fiber-embedded optofluidic biochip, such as rapidity, compact size, portability, reusability, user-friendliness, and cost-efficiency, allow it an ideal solution for rapid on-site screening of CLO, towards improving symptom management and enhancing health-related quality of life, particularly in resource-limited environments. This new biosensing platform is highly versatile and easily extended to detect other trace pollutants using the corresponding functionalized fiber nano-biosensors and antibodies. Declarations CRediT authorship contribution statement Laiya Lu: Conceptualization, Methodology, Validation, Writing-original draft, Funding acquisition. Tianxiang Ji and Min Wang: Methodology, Investigation. Siyan Liu and Yuxin Zhuo: Investigation, Data curation. Feng Long: Supervision, Funding acquisition, Methodology, Writing-review&editing. Declaration of competing interest The authors declare no competing interests. Acknowledgments This work was financially supported by Beijing Science and Technology Planning Project (Z221100007122001), and the Natural Science Foundation of Beijing (8242031). References Nanda, S., Ganguly, A., Mandi, M., et al., 2024. Chronic sub-lethal exposure to clothianidin triggers organismal and sub-organismal-level health hazards in a non-target organism, Drosophila melanogaster. Sci. Total Environ. 932, 172783. Sales-Alba, A., Cruz-Alcalde, A., López-Vinent, N., et al., 2023. Removal of neonicotinoid insecticide clothianidin from water by ozone-based oxidation: Kinetics and transformation products. Sep. Purif. Technol. 316, 123735. Hirano, T., Ohno, S., Ikenaka, Y., et al., 2024. Quantification of the tissue distribution and accumulation of the neonicotinoid pesticide clothianidin and its metabolites in maternal and fetal mice. Toxicology and Applied Pharmacology 484, 116847. Naumann, T., Bento, C.P.M., Wittmann, A., et al., 2022. Occurrence and ecological risk assessment of neonicotinoids and related insecticides in the Bohai Sea and its surrounding rivers, China. Water Res. 209, 117912. Schaafsma, A., Limay-Rios, V., Baute, T., et al., 2015. Neonicotinoid Insecticide Residues in Surface Water and Soil Associated with Commercial Maize (Corn) Fields in Southwestern Ontario. PLoS One 10, e0118139. Addy-Orduna, L.M., Brodeur, J.C., Mateo, R., 2019. Oral acute toxicity of imidacloprid, thiamethoxam and clothianidin in eared doves: A contribution for the risk assessment of neonicotinoids in birds. Sci. Total Environ. 650, 1216-1223. Gibbons, D., Morrissey, C., Mineau, P., 2015. A review of the direct and indirect effects of neonicotinoids and fipronil on vertebrate wildlife. Environmental Science and Pollution Research 22, 103-118. Wang, Y., Shen, J., Lang, H., et al., 2024. Elevated temperature magnifies the acute and chronic toxicity of clothianidin to Eisenia fetida. Environ. Pollut. 355, 124210. Wang, X., Anadón, A., Wu, Q., et al., 2018. Mechanism of Neonicotinoid Toxicity: Impact on Oxidative Stress and Metabolism. Annual Review of Pharmacology and Toxicology 58, 471-507. Zhou, Y., Zhang, Z., Jing, J., et al., 2023. Integrating environmental carry capacity based on pesticide risk assessment in soil management: A case study for China. J. Hazard. Mater. 460, 132341. Mahai, G., Wan, Y., Wang, A., et al., 2023. Exposure to multiple neonicotinoid insecticides, oxidative stress, and gestational diabetes mellitus: Association and potential mediation analyses. Environ. Int. 179, 108173. Zhang, D., Lu, S., 2022. Human exposure to neonicotinoids and the associated health risks: A review. Environ. Int. 163, 107201. Wood, T.J., Goulson, D., 2017. The environmental risks of neonicotinoid pesticides: a review of the evidence post 2013. Environmental Science and Pollution Research 24, 17285-17325. Alford, A.M., Krupke, C.H., 2019. Movement of the Neonicotinoid Seed Treatment Clothianidin into Groundwater, Aquatic Plants, and Insect Herbivores. Environ. Sci. Technol. 53, 14368-14376. Zhao, R., Zhang, Z., Yu, M., et al., 2024. New strategy for improving the rainfastness and control effect on Monochamus alternatus of clothianidin via a castor oil-based polyurethane nanoemulsion. Environmental Technology & Innovation 34, 103564. Woodward, E.E., Hladik, M.L., Main, A.R., et al., 2022. Comparing imidacloprid, clothianidin, and azoxystrobin runoff from lettuce fields using a soil drench or treated seeds in the Salinas Valley, California. Environ. Pollut. 315, 120325. Pook, C., Gritcan, I., 2019. Validation and application of a modified QuEChERS method for extracting neonicotinoid residues from New Zealand maize field soil reveals their persistence at nominally hazardous concentrations. Environ. Pollut. 255, 113075. Jeschke, P., Nauen, R., Schindler, M., et al., 2011. Overview of the Status and Global Strategy for Neonicotinoids. Journal of Agricultural and Food Chemistry 59, 2897-2908. Kim, B.M., Park, J.-S., Choi, J.-H., et al., 2012. Residual determination of clothianidin and its metabolites in three minor crops via tandem mass spectrometry. Food Chem. 131, 1546-1551. Liu, L., Peng, M., Xu, K., et al., 2024. Fluorescence Quenching Detection of Clothianidin in Fruit and Vegetable Samples Using MAPbBr3 Perovskite Quantum Dots. ACS Applied Nano Materials 7, 9176-9183. Liang, Z., Mahmoud Abdelshafy, A., Luo, Z., et al., 2022. Occurrence, detection, and dissipation of pesticide residue in plant-derived foodstuff: A state-of-the-art review. Food Chem. 384, 132494. Yang, H., Xia, L., Zheng, J., et al., 2023. Screening and identification of a DNA aptamer to construct the label-free fluorescent aptasensor for ultrasensitive and selective detection of clothianidin residue in agricultural products. Talanta 262, 124712. Li, M., Hua, X., Ma, M., et al., 2014. Detecting clothianidin residues in environmental and agricultural samples using rapid, sensitive enzyme-linked immunosorbent assay and gold immunochromatographic assay. Sci. Total Environ. 499, 1-6. Xu, Z.-L., Sun, W.-J., Yang, J.-Y., et al., 2012. Development of a Solid-Phase Extraction Coupling Chemiluminescent Enzyme Immunoassay for Determination of Organophosphorus Pesticides in Environmental Water Samples. Journal of Agricultural and Food Chemistry 60, 2069-2075. Sheng, E., Shi, H., Zhou, L., et al., 2016. Dual-labeled time-resolved fluoroimmunoassay for simultaneous detection of clothianidin and diniconazole in agricultural samples. Food Chem. 192, 525-530. Lai, X., Cao, W., Zhang, G., et al., 2024. Traffic signal-inspired fluorescence lateral flow immunoassay utilizing self-assembled AIENP@Ni/EC for simultaneous multi-pesticide residue detection. Chem. Eng. J. 501, 157565. Borah, P., Biswas, R., 2023. Impactful analytical schemes for assessing pesticides in tea: A comprehensive review. Measurement 221, 113505. Taitt, Chris R., Anderson, G.P., Ligler, F.S., 2016. Evanescent wave fluorescence biosensors: Advances of the last decade. Biosensors and Bioelectronics 76, 103-112. Fernandez-Cuesta, I., Llobera, A., Ramos-Payán, M., 2022. Optofluidic systems enabling detection in real samples: A review. Anal. Chim. Acta 1192, 339307. Li, W., Wang, H., Yang, R., et al., 2018. Integrated multichannel all-fiber optofluidic biosensing platform for sensitive and simultaneous detection of trace analytes. Anal. Chim. Acta 1040, 112-119. Chen, L., Yu, L., Liu, Y., et al., 2023. Valve-Adjustable Optofluidic Bio-Imaging Platform for Progressive Stenosis Investigation. ACS Sensors 8, 3104-3115. Wang, Y., Gao, R., Zhan, C., et al., 2025. SERS-based microfluidic sensor for sensitive detection of circulating tumor markers: A critical review. Coord. Chem. Rev. 523, 216289. Pidenko, S.A., Burmistrova, N.A., Shuvalov, A.A., et al., 2018. Microstructured optical fiber-based luminescent biosensing: Is there any light at the end of the tunnel? - A review. Anal. Chim. Acta 1019, 14-24. Zhao, Y., Hu, X.-g., Hu, S., et al., 2020. Applications of fiber-optic biochemical sensor in microfluidic chips: A review. Biosensors and Bioelectronics 166, 112447. Aralekallu, S., Boddula, R., Singh, V., 2023. Development of glass-based microfluidic devices: A review on its fabrication and biologic applications. Materials & Design 225, 111517. Yin, M.-j., Gu, B., An, Q.-F., et al., 2018. Recent development of fiber-optic chemical sensors and biosensors: Mechanisms, materials, micro/nano-fabrications and applications. Coord. Chem. Rev. 376, 348-392. Chen, D., Xu, W., Lu, Y., et al., 2024. Rapid and sensitive parallel on-site detection of antibiotics and resistance genes in aquatic environments using evanescent wave dual-color fluorescence fiber-embedded optofluidic nanochip. Biosensors and Bioelectronics 257, 116281. Long, F., He, M., Zhu, A.N., et al., 2009. Portable optical immunosensor for highly sensitive detection of microcystin-LR in water samples. Biosensors and Bioelectronics 24, 2346-2351. Additional Declarations No competing interests reported. Supplementary Files Supportinginformation.docx floatimage1.jpeg Graphical abstract Cite Share Download PDF Status: Published Journal Publication published 23 May, 2025 Read the published version in Microchimica Acta → Version 1 posted Editorial decision: Revision requested 18 Apr, 2025 Reviews received at journal 17 Apr, 2025 Reviewers agreed at journal 14 Apr, 2025 Reviews received at journal 10 Apr, 2025 Reviewers agreed at journal 01 Apr, 2025 Reviewers invited by journal 01 Apr, 2025 Editor assigned by journal 30 Mar, 2025 Submission checks completed at journal 30 Mar, 2025 First submitted to journal 27 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6323851\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":444922866,\"identity\":\"0b0937b8-bb7d-4d71-8a6e-dd5e65d42612\",\"order_by\":0,\"name\":\"Laiya Lu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Renmin University of China\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Laiya\",\"middleName\":\"\",\"lastName\":\"Lu\",\"suffix\":\"\"},{\"id\":444922867,\"identity\":\"c947e057-a9cd-4025-bfe8-d96b242c2eaa\",\"order_by\":1,\"name\":\"Min Wang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Renmin University of China\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Min\",\"middleName\":\"\",\"lastName\":\"Wang\",\"suffix\":\"\"},{\"id\":444922868,\"identity\":\"a212d057-c73e-4c42-84a1-5deef2b91147\",\"order_by\":2,\"name\":\"Tianxiang Ji\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Renmin University of China\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Tianxiang\",\"middleName\":\"\",\"lastName\":\"Ji\",\"suffix\":\"\"},{\"id\":444922869,\"identity\":\"2c8bedad-a323-4a4f-9c43-ae451eaddbd1\",\"order_by\":3,\"name\":\"Siyan Liu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Renmin University of China\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Siyan\",\"middleName\":\"\",\"lastName\":\"Liu\",\"suffix\":\"\"},{\"id\":444922870,\"identity\":\"fc385e98-f475-496a-a948-5ff8bdfa025e\",\"order_by\":4,\"name\":\"Yuxin Zhuo\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Renmin University of China\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yuxin\",\"middleName\":\"\",\"lastName\":\"Zhuo\",\"suffix\":\"\"},{\"id\":444922871,\"identity\":\"7cc653a3-3648-4b52-9137-ce9729e98cc1\",\"order_by\":5,\"name\":\"Feng Long\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYBACPhBRYcAmB+GyEaEFrOZMBZ8xqVrOyCU2EK+Fvffwi4NtZunbZyQ/YPhQdpiBf3YDAS0859IsDral5c65kWbAOOPcYQaJOwcIaJHIMTP+2HYsd4ZEDgMzb9thBgOJBMJaDA62/U+XAGn5S6QW4wcHzrAlgLUwEqWF54wZw4EKNsMZPM8MDvacS+eRuEFACz97j/GHAwZs8hLsyQ8f/CizluOfQUAL2G0w1gEg5iGoHgiYPxCjahSMglEwCkYwAABq6j6K2e/XkwAAAABJRU5ErkJggg==\",\"orcid\":\"\",\"institution\":\"Renmin University of China\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Feng\",\"middleName\":\"\",\"lastName\":\"Long\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-03-28 01:23:14\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-6323851/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-6323851/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1007/s00604-025-07220-y\",\"type\":\"published\",\"date\":\"2025-05-23T15:58:34+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":81021023,\"identity\":\"69662960-6bf4-4a4b-a553-650625fffbbe\",\"added_by\":\"auto\",\"created_at\":\"2025-04-21 09:41:09\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":768404,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eTapered functionalized fiber-embedded optofluidic biochip. (A) Tapered fiber nano-biosensor using HF acid-etched method. (B) CLO-functionalized fiber nano-biosensor. Bottom-left: SEM image; Bottom-right: Image of fiber nano-biosensor modified with CLO-OVA after addition of Cy5.5-anti-CLO antibody. (C) CLO-functionalized fiber-embedded optofluidic biochip. (D) Scheme of all-fiber fluorescence detection device. (E) On-site detection of CLO using fiber-embedded optofluidic biochip.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6323851/v1/a266a37cd95faee400722541.png\"},{\"id\":81022417,\"identity\":\"a31a4e0f-9e6e-40a6-9518-23c4b473a1bb\",\"added_by\":\"auto\",\"created_at\":\"2025-04-21 09:57:09\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":351374,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eIndirect competitive immunoassay mechanism of CLO. (A) Scheme of CLO immunoassay principle. (B) Typical detection curves of CLO using the fiber-embedded optofluidic biochip. The concentration of Cy5.5-anti-CLO antibody is 0.1 µg/mL.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6323851/v1/c30f1c91a7d62a4abcde78b9.png\"},{\"id\":81021026,\"identity\":\"19e9f8bd-81e1-4090-9386-fe2baa6d5631\",\"added_by\":\"auto\",\"created_at\":\"2025-04-21 09:41:09\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":82041,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eCharacteristics of the reusable functionalized fiber-embedded optofluidic biochip. (A) Specificity. The concentration of CLO, Chlorbenzuron, Bifenthrin, Isoprocarb, Buprofezin, and Thiamethoxam is 100.0 μg/L. (B) Reusability. (C) Storage stability. Detection condition: the Cy5.5-anti-CLO antibody concentration is 0.125 µg/mL.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"33.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6323851/v1/c0ffcfa3dba8b46ff348fed7.png\"},{\"id\":81021028,\"identity\":\"1d2b7c16-fb42-49c8-a0f3-e1d65a9202b3\",\"added_by\":\"auto\",\"created_at\":\"2025-04-21 09:41:09\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":180097,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eFigure 3. \\u003cstrong\\u003eOptimization of detection conditions. \\u003c/strong\\u003e(A) Optimization of pre-reaction time. Detection conditions: 0.125 µg/mL anti-CLO antibody, 50.0 µg/L CLO. (B) Optimization of incubation time. Detection conditions: 0.125 µg/mL anti-CLO antibody, 0, 10, or 500.0 µg/L CLO, 2 min pre-reaction. (C) Optimization of anti-CLO antibody concentration. Detection conditions: 0.05, 0.125, 0.25, or 0.5 µg/mL anti-CLO antibody concentration; 0 or 30 µg/L CLO; 2 min pre-reaction, 6 min incubation time. Error bars correspond to the standard deviation (n = 3).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6323851/v1/993f5548152f184aa5921527.png\"},{\"id\":81021027,\"identity\":\"04b38764-95db-4a54-ab90-d0a12f50deeb\",\"added_by\":\"auto\",\"created_at\":\"2025-04-21 09:41:09\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":180802,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eFigure 4. (A) Real-time curves of various concentrations of CLO. (B) Dose-response curve of CLO using the reusable fiber-embedded microfluidic biochip.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6323851/v1/e57ef81e9010377157b51197.png\"},{\"id\":81021034,\"identity\":\"959c963b-cc94-4462-b348-311ba8deea8e\",\"added_by\":\"auto\",\"created_at\":\"2025-04-21 09:41:09\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":362303,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eFigure 5\\u003c/strong\\u003e. Comparison between the fiber-embedded microfluidic biochip and HPLC (S1, S2, S3, S4 are the Summer Palace, Beijing Botanical Garden, Milk, Baby Chinese cabbage, respectively, ***P\\u0026lt;0.001, **P\\u0026lt;0.01, *P\\u0026lt;0.05). The error bars correspond to the standard deviations of the data points from five repeated experiments (n = 5).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6323851/v1/43a654f598a4695858899b64.png\"},{\"id\":83460485,\"identity\":\"2068fba9-5977-4da7-88c4-317d01616dd9\",\"added_by\":\"auto\",\"created_at\":\"2025-05-26 16:12:38\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2747416,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6323851/v1/093bd398-e4a3-4946-a63f-9b1a4a5207f4.pdf\"},{\"id\":81021053,\"identity\":\"ad6296fd-8303-4e1f-8a79-61eb74c2be59\",\"added_by\":\"auto\",\"created_at\":\"2025-04-21 09:41:10\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":19982772,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Supportinginformation.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6323851/v1/93f5f9add7fb4668258c68a8.docx\"},{\"id\":81022418,\"identity\":\"986f4878-60fd-4900-81bc-9823cded884e\",\"added_by\":\"auto\",\"created_at\":\"2025-04-21 09:57:09\",\"extension\":\"jpeg\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":751379,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eGraphical abstract\\u003c/strong\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage1.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6323851/v1/288f1d1cfc7e6a8a0ea43940.jpeg\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Rapid and sensitive on-site detection of Clothianidin in surface waters with a reusable fiber-embedded optofluidic biochip\",\"fulltext\":[{\"header\":\"1. Induction\",\"content\":\"\\u003cp\\u003eClothianidin (CLO) is a novel neonicotinoid insecticide employed to control sucking and chewing types of pests globally [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. This insecticide has broad-spectrum insecticidal activity, acting on the nicotinic acetylcholine esterase receptors in the insect nervous system, blocking the normal conduction of signals in the insect central nervous system, and leading to their paralysis and death [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. Although CLO is characterized by low toxicity, long-lasting effectiveness, and no cross-resistance compared with conventional pesticides [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e], it is highly toxic to non-target organisms such as bees and birds [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. In nature, various nontarget organisms can be exposed to this insecticide through contaminated food, water, and air [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e]. Even the ingestion of a small number of treated seeds by birds can lead to death [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. The excessive use of CLO may lead to residue levels exceeding safety standards in agricultural products [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e], posing a threat to human health [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. It may result in oxidative stress, neurological symptoms, metabolic changes, osteoporosis, and liver cancer [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e]. Studies demonstrated that only 5% of the active ingredients [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e] are absorbed by crops, with the majority entering the environment [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e]. CLO has become among the most readily detectable neonicotinoid compounds in urban [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e] and agricultural watersheds [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. According to the European Food Safety Authority (EFSA) risk assessment in 2018, the European Union has prohibited all outdoor uses of CLO [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. Nevertheless, CLO remains a high-quality insecticide for which there are currently no immediate alternatives [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e]. In China, there are 374 registrations of CLO, mainly for the control of aphids, thrips, whiteflies, and other pests. Inspection data from 2023 show an increase in the number of batches of products that exceed the standard, including chili peppers, cowpeas, bananas, celery, and mangoes. To ensure the safety of agricultural products, China has established maximum residue limits (MRLs) for CLO in food, such as 0.05 mg/kg for chili peppers, 0.2 mg/kg for ginger, 0.01 mg/kg for cowpea, 0.02 mg/kg for raw milk and 2 mg/kg for leafy vegetables.\\u003c/p\\u003e \\u003cp\\u003eVarious centralized testing technologies, including gas chromatography (GC), high-performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS), detect CLO with high sensitivity and accuracy [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]. However, these methods require complex pre-treatment procedures, detailed personnel training, and expensive and sophisticated instruments [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e], thus lacking capacity for rapid, high-frequency, and point-of-need testing [\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e]. Biological molecules such as antibodies, due to their high specificity and affinity, have been widely used to establish sensitive and rapid detection methods for CLO. Various immunoassay methods, such as enzyme-linked immunosorbent assay (ELISA) [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e], chemiluminescent enzyme immunoassay (CLEIA) [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e], fluorescence immunoassay [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e], and lateral flow immunoassay (FLIA) [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e], have been developed for rapid CLO detection. However, several factors limit their practical application, including the inability to perform quantitative assays, low sensitivity, and susceptibility to matrix interference [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eIn response to these challenges, a portable and reusable fiber-embedded optofluidic biochip has been constructed for the rapid and sensitive on-site detection of CLO [\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e]. Optofluidic biochips that combines the outstanding features of optics with those of microfluidic biochips, have emerged as a versatile and potent assay platform for detecting trace toxic substance [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e]. Traditional optofluidic biochips, however, have been hindered by their reliance on bulky, intricate, and costly detection equipment, such as confocal fluorescence microscopes [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e]. These setups necessitate numerous free-space optical components, meticulous optical alignment, and burdensome maintenance. These factors have constrained their applicability in point-of-need testing scenarios [\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e]. In our system, the breakthrough lies in the integration of optical fibers into optofluidic chips, which has opened up new analytical possibilities [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e]. The advantages of optical fibers include their compact size, large surface-area-to-volume ratio, ease of surface modification, excellent biocompatibility, and resistance to electromagnetic interference [\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e]. The portable fiber-embedded optofluidic biochip utilizes a compact all-fiber optical system for the transmission of excitation light and fluorescence. This design eliminates the need for multiple optically separate components and precise optical alignment, resulting in a sensitive, miniaturized, and portable system. For precise CLO detection, we have embedded a highly reusable CLO-OVA functionalized fiber biosensor into the optofluidic chip, which serves dual roles as both biorecognition element and optical transmission component. By employing the fiber-embedded optofluidic biochip, we have established an indirect competitive immunoassay method for the rapid on-site detection of CLO in a variety of food and environmental samples. This biochip boasts several unique features, including enhanced sensitivity and stability, and a compact optical construction that eliminates the need for optical alignment. These attributes position it as a promising contender for food and environmental monitoring applications.\\u003c/p\\u003e \"},{\"header\":\"2. Materials and Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.1 Chemicals and materials\\u003c/h2\\u003e \\u003cp\\u003eCLO, Chlorbenzuron, Bifenthrin, Isoprocarb, Buprofezin, and Thiamethoxam were purchased from Beijing zhongkezhijian Biotechnology (Beijing, China). Bovine serum albumin (BSA), 3-Mercaptopropyltrimethoxysilane (MTS), 4-maleimidobutyric acid N-hydroxysuccinimide ester (GMBS), and hydrofluoric acid (HF) were purchased from Sigma-Aldrich (Steinheim, Germany). All other chemical reagents were purchased from Beijing Chemical Reagent Co. (Beijing, China).\\u003c/p\\u003e \\u003cp\\u003eMonoclonal anti-CLO antibodies were obtained from Shandong Landu Bio-sciences \\u0026amp; technology Co. (Shandong, China). Sodium dodecyl sulfate (SDS, 0.5%, pH\\u0026thinsp;=\\u0026thinsp;1.9), phosphate buffer (PBS, pH\\u0026thinsp;=\\u0026thinsp;7.4), antibody dilution solution, and Cy5.5 labeled anti-CLO antibody (Cy5.5-anti-CLO antibody) were prepared by our group (Note S1 in Supporting information). Standard stock solution (1.0 mg/mL) of CLO was prepared by dissolving an appropriate amount of CLO in 0.01 mol/L PBS and kept at 4 \\u0026ordm;C until use.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.2 Preparation of reusable Tapered functionalized fiber nano-biosensor\\u003c/h2\\u003e \\u003cp\\u003eTo ensure accurate detection of CLO, a reusable functionalized fiber nano-biosensor was prepared. The functionalized tapered fiber nano-biosensor was prepared using a 5.5-cm long quartz fiber (ϕ 600 \\u0026micro;m, NA\\u0026thinsp;=\\u0026thinsp;0.22) with a 3.0 cm of coating layer stripped to form the biosensing section, which was \\u003cem\\u003ein-situ\\u003c/em\\u003e etched using hydrofluoric acid (HF) tube-etching method. The combination tapered fiber nano-biosensor with a nanopore layer was fabricated by controlling etching conditions and functionalized with CLO-ovalbumin (CLO-OVA) according to the previous described methods (Note S2 in Supporting Information) [\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.3 Fiber-embedded optofluidic biochip\\u003c/h2\\u003e \\u003cp\\u003eThe reusable fiber-embedded optofluidic biochip is ingeniously crafted by integrating a functionalized fiber nano-biosensor into an optofluidic chip. Crafted through advanced 3D printing technology, the biochip is endowed with a micro-channel with 3.0 cm in length and 600 \\u0026micro;m in diameter, having an effective volume of 20 \\u0026micro;L. The 3D architecture of the biochip revolutionizes the fabrication and assembly process, enhancing mass transfer rates and reaction efficiency, which translates to faster and more sensitive detection capabilities. To achieve the fluorescence signal detection, a portable all-fiber evanescent wave fluorescence detection device was used (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD and \\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eE). This portable detection device is equipped with a 635 nm laser (8 mW) that provides monochromatic light source, and a single-multimode fiber coupler (SMFC) that adeptly handles the transmission of the excitation and fluorescence light (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD). The excitation light enters the fiber nano-biosensor and propagates via total internal reflection, generating an evanescent wave on the biosensor's surface with an effective penetration depth of less than 100 nm. The Cy5.5-anti-CLO antibody, which binds to the CLO-OVA immobilized on the biosensor's surface, is excited by the evanescent wave. A portion of fluorescence is coupled back into the fiber nano-biosensor. The collected fluorescence is subsequently detected by an ultrasensitive mini-photodetector, the PD-1000.\\u003c/p\\u003e \\u003cp\\u003eVarious solutions, such as buffer, sample, and regeneration solutions, are delivered to the fiber-embedded optofluidic biochip in a controlled manner by a peristaltic pump and a six-position valve. The integrated signal and data processing system analyzed the fluorescence signals. The resulting fluorescence signal trace was clearly displayed on the user interface.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.4 Detection of CLO in surface waters in Beijing-Tianjin area\\u003c/h2\\u003e \\u003cp\\u003eTo assess the environmental monitoring capabilities of the fiber-embedded optofluidic biochip, we gathered surface water specimens from two significant sites in Beijing, China: the Beijing Botanical Garden and The Summer Palace, on December 15, 2023. To broaden the scope of our investigation, we collected water samples from ten varied locations across the Haihe River basin in Tianjin, China, on March 22, April 20, and May 26, 2024. These samples with and without spiked CLO were directly on-site detected by the fiber-embedded optofluidic biochip without any pre-treatment.\\u003c/p\\u003e \\u003cp\\u003eFor a thorough comparative analysis and precise detection of CLO in the collected surface water samples via HPLC, a thorough pre-treatment procedure was indispensable. Initially, each 500 mL water sample was spiked with various concentrations of CLO and was filtered through a glass fiber filter (0.7 \\u0026micro;m, Whatman GF/F 47 mm, UK) to eliminate particulate matter. Subsequently, the samples were enriched using solid-phase extraction with an Oasis HLB column (6 cm\\u0026sup3;, 200 mg, Waters, USA). Following this, the HLB columns were rinsed with deionized water and eluted with methanol. The eluates were then concentrated to a final volume of 1 mL under a nitrogen stream and subsequently filtered through nylon membrane filters (0.45 \\u0026micro;m, Whatman, UK). The prepared extracts were analyzed by HPLC (Note S3 in Supporting Information).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.5 Detection of CLO in foodstuffs\\u003c/h2\\u003e \\u003cp\\u003eTo evaluate the performance of the fiber-embedded optofluidic biochip in food analysis, we performed CLO detection in milk and baby Chinese cabbage purchased from a local supermarket. For the milk samples, they were spiked various concentrations of CLO, and blended with a mixture of acetonitrile and 0.2% trifluoroacetic acid. Followed by centrifugation, the supernatant was decanted and passed through a 0.22 \\u0026micro;m hydrophobic polytetrafluoroethylene (PTFE) filter. The treated milk samples were analyzed using the fiber-embedded optofluidic biochip. In the case of the baby Chinese cabbage, its juice was extracted using a manual squeezing technique and spiked with various concentrations of CLO. The resulting juice was then filtered through the same 0.22 \\u0026micro;m PTFE filter to ensure cleanliness. The filtered samples were subjected to analysis via the biochip. After pre-treatment as water samples, these samples were also analyzed using HPLC.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"3. Results and discussion\",\"content\":\"\\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.1 Indirect competitive immunoassay mechanism of CLO\\u003c/h2\\u003e \\u003cp\\u003eFigure \\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA illustrates the indirect competitive immunoassay mechanism of CLO and outlines a typical detection cycle using this advanced biochip. Initially, 30 \\u0026micro;L of sample containing varying concentrations of CLO and 30 \\u0026micro;L of Cy5.5-anti-CLO antibody solution at a predetermined concentration was mixed and incubated for a set period. During this incubation, some of the Cy5.5-anti-CLO antibodies bind with CLO. Concurrently, a 10 mM PBS solution is introduced into the optofluidic biochip to establish a baseline signal (Phase I in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). Subsequently, the mixture of CLO and Cy5.5-anti-CLO antibody is added into the biochip for initiating an indirect competitive immunoreaction. Antibodies with available binding sites in the mixture bind to the immobilized CLO-OVA on the fiber nano-biosensor's surface. The Cy5.5-anti-CLO antibodies are then excited by the evanescent wave field generated at the sensing surface, and the resulting fluorescence signal is recorded in real-time (Phase II in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). The fluorescence intensity, which is indicative of the number of antibodies bound to the fiber biosensor's surface, is inversely proportional to the CLO concentration in the samples. To reuse the fiber biosensor, a 0.5% SDS solution at pH\\u0026thinsp;=\\u0026thinsp;1.9 is employed to elute the Cy5.5-anti-CLO antibody from the biosensing surface (Phase III in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). Finally, a 10 mM PBS solution is introduced to the sample cell, flushing away any residual antibody and SDS solution (Phase IV in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB), thus regenerating the fiber biosensor for subsequent tests. The entire detection cycle is completed in less than 10 min.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.2 Characteristics of the reusable functionalized fiber-embedded optofluidic biochip\\u003c/h2\\u003e \\u003cp\\u003eIn the reusable functionalized fiber-embedded optofluidic biochip (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA), the CLO-OVA functionalized fiber nano-biosensor was used as a specific biorecognition element for CLO and a transducer for fluorescence detection. The SEM image showed the formation of nano-pore and the successful modification of CLO-OVA on the fiber nano-biosensor (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB, bottom left). The fluorescence microscopy image demonstrated the significant fluorescence signal when the Cy5.5-anti-CLO antibody was introduced and bound with CLO-OVA immobilized on fiber nano-biosensor (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB, bottom right).\\u003c/p\\u003e \\u003cp\\u003eTo evaluate its performance, titration experiments were conducted using the Cy5.5-anti-CLO antibody. First, when 0.125 \\u0026micro;g/mL Cy5.5-anti-CLO antibody was introduced into the optofluidic biochip, the fluorescence intensity increased over the time (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). The highest fluorescence intensity was obtained due to the most Cy5.5-anti-CLO antibody specifically bound with the CLO-OVA immobilized on the fiber nano-biosensor surface. Second, when the mixture of CLO and Cy5.5-anti-CLO antibody was added, the fluorescence intensity also increased but a lower light intensity was obtained compared with the above results. Third, when the mixture of a large excess of potential interferences (100.0 \\u0026micro;g/L Chlorbenzuron, Bifenthrin, Isoprocarb, Buprofezin, Thiamethoxam) and Cy5.5-anti-CLO antibody was added, no significant decrease of fluorescence intensity was observed. These results demonstrated that the CLO-OVA were successfully modified onto the fiber nano-biosensor, and the observed signal decrease originated from the CLO in solution competitively bound with Cy5.5-anti-CLO antibody. The Cy5.5-anti-CLO antibody was highly selective towards CLO and had few cross-reactivity for other interferences. Based on indirect competition immunoassay principle, the biochip could be applied for the specific detection of CLO.\\u003c/p\\u003e \\u003cp\\u003eThe biosensor's surface must possess excellent regeneration capabilities, which are essential for maintaining the precision of detection results, reducing costs, expediting the detection process, and streamlining operational procedures. The reusability, stability, and activity of the CLO-OVA functionalized biosensor were rigorously assessed through a series of bioassays. The fiber nano-biosensor could be effectively regenerated by employing a 0.5% SDS solution at pH 1.9 to detach the bound Cy5.5-anti-CLO antibody from its surface (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). As depicted in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB, the fiber nano-biosensor's performance remained consistent after 200 cycles of regeneration, with the coefficient of variation (CV) for the fluorescence signal consistently below 1.9% across all tests. This demonstrates the biosensor's remarkable reusability and stability. Subsequent to these evaluations, the biochip's shelf-life was also investigated. The results revealed that the biochip exhibited high storage stability when kept at 4\\u0026ordm;C, retaining 92% of its initial response even after 60 days, as illustrated in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.3 Optimization of detection conditions\\u003c/h2\\u003e \\u003cp\\u003eTo enhance the detection performance, a series of conditions were meticulously optimized, including the pre-reaction time, incubation duration, and antibody concentration. The optimization began with the pre-reaction time between the Cy5.5-anti-CLO antibody and CLO, as illustrated in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA. It was observed that an extended pre-reaction of CLO with the Cy5.5-anti-CLO antibodies led to a reduction in the fluorescence signal, with a plateau being reached after 2 min. Consequently, a 2-minute pre-reaction time was adopted for all subsequent experiments.\\u003c/p\\u003e \\u003cp\\u003eFigure \\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB demonstrates the impact of incubation time on fluorescence intensity. When the biochip was exposed to an anti-CLO antibody solution without CLO or with a low concentration of CLO (10.0 \\u0026micro;g/L), the fluorescence intensity increased rapidly at first and then plateaued after 20 min. In contrast, when a mixture containing a high concentration of CLO (1000.0 \\u0026micro;g/L) along with the anti-CLO antibody, the fluorescence intensity quickly reached a plateau within just 5 min. Taking into account the necessity for rapid on-site screening of CLO, an incubation time of 10 min was determined to be optimal for CLO detection.\\u003c/p\\u003e \\u003cp\\u003eThe fiber-embedded optofluidic biochip functions on the principle of an indirect competition immunoassay, where the detection sensitivity for CLO is significantly influenced by the concentration of the antibodies used. To ascertain the optimal concentration of Cy5.5-anti-CLO antibody, a sensitivity index (\\u003cem\\u003eε\\u003c/em\\u003e\\u003csub\\u003eA\\u003c/sub\\u003e) was introduced, as defined by Eq.\\u0026nbsp;(\\u003cspan refid=\\\"Equ1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e).\\u003cdiv id=\\\"Equ1\\\" class=\\\"Equation\\\"\\u003e\\u003cdiv format=\\\"TEX\\\" class=\\\"mathdisplay\\\" id=\\\"FileID_Equ1\\\" name=\\\"EquationSource\\\"\\u003e\\n$$\\\\:{\\\\varepsilon\\\\:}_{\\\\text{A}}=\\\\frac{{I}_{\\\\text{A}0}-{I}_{\\\\text{A}1}}{{I}_{\\\\text{A}0}}$$\\u003c/div\\u003e\\u003cdiv class=\\\"EquationNumber\\\"\\u003e1\\u003c/div\\u003e\\u003c/div\\u003e\\u003c/p\\u003e \\u003cp\\u003ewhere, \\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{I}_{\\\\text{A}0}\\\\)\\u003c/span\\u003e\\u003c/span\\u003eand \\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:{I}_{\\\\text{A}1}\\\\)\\u003c/span\\u003e\\u003c/span\\u003e represent the net fluorescent intensities of the samples in the absence and presence of the target analyte, respectively. The optimal antibody concentration was chosen based on a balance between suitable fluorescence intensity and a high sensitivity index. As depicted in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC, the fluorescence signal increased with the rising concentration of Cy5.5-anti-CLO antibody, as this allowed for more antibodies to bind with CLO-OVA that was immobilized on the fiber biosensor. Conversely, the sensitivity index \\u003cem\\u003eε\\u003c/em\\u003e\\u003csub\\u003eA\\u003c/sub\\u003e increased with decreasing antibody concentration, as the lower concentration favored the competitive binding of free CLO in solution, leading to fewer antibodies binding with CLO-OVA. After considering both the sensitivity index and the appropriate fluorescence intensity, a Cy5.5-anti-CLO antibody concentration of 0.125 \\u0026micro;g/mL was identified as the optimal concentration for use in subsequent experiments.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.4 Dose-response curve of CLO immunoassay\\u003c/h2\\u003e \\u003cp\\u003eAfter determining the optimal detection parameters, quantitative analyses of CLO were conducted using the fiber-embedded optofluidic biochip. The Cy5.5-anti-CLO antibody was premixed with a range of CLO concentrations and incubated for 2 min. This mixture was then introduced into the optofluidic biochip for fluorescent detection. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA displays the typical real-time fluorescence signal traces recorded for various CLO concentrations. As anticipated, an increase in CLO concentration led to a corresponding decrease in fluorescence intensity. This reduction is attributed to the competition between free CLO and CLO-OVA on the fiber biosensor for binding sites on the Cy5.5-anti-CLO antibody, with free CLO occupying the active sites and thereby reducing the binding of the antibody to CLO-OVA.\\u003c/p\\u003e \\u003cp\\u003eFor each assay, the effective fluorescence signal of the samples (\\u003cem\\u003eI\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e), with the baseline signal subtracted, was normalized against that of a blank sample using the following formula:\\u003c/p\\u003e \\u003cp\\u003e \\u003cem\\u003eΔI\\u003c/em\\u003e \\u003csub\\u003es\\u003c/sub\\u003e=\\u003cem\\u003eI\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e/\\u003cem\\u003eI\\u003c/em\\u003e\\u003csub\\u003eb\\u003c/sub\\u003e (2)\\u003c/p\\u003e \\u003cp\\u003ewhere \\u003cem\\u003eI\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003eb\\u003c/em\\u003e\\u003c/sub\\u003e represents the fluorescent intensity of the blank sample, while \\u003cem\\u003eI\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003es\\u003c/em\\u003e\\u003c/sub\\u003e denotes the fluorescent intensity of the sample under test. These normalized results are then employed to establish a dose-response relationship, which is modeled using a four-parameter logistic equation (Note S4 in Supporting Information). The standard deviation of less than 3.1% for each CLO concentration, with three replicates (n\\u0026thinsp;=\\u0026thinsp;3), underscores the high stability of the fiber-embedded optofluidic biochip for CLO detection, as shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB.\\u003c/p\\u003e \\u003cp\\u003eThe limit of detection (LOD) for CLO, calculated to be 1.0 \\u0026micro;g/L based on three times the standard deviation (3σ), is notably lower than the maximum residue limits (MRLs) established by various countries and organizations, and is also on par with the LODs of other methods, as detailed in Table \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e. This achievement is attributed to the high affinity of the anti-CLO antibody and the exceptional sensitivity of the fiber-embedded optofluidic biochip. The biochip demonstrated a linear response to CLO concentrations ranging from 5.0 to 500.0 \\u0026micro;g/L.\\u003c/p\\u003e \\u003cp\\u003eThe fiber-embedded optofluidic biochip stands out against traditional detection methods with several remarkable characteristics. Firstly, its all-fiber optical architecture enhances the efficiency of fluorescence excitation and collection, bolstering its capabilities for on-site detection and scalability. Additionally, the nanopore structure of the fiber nano-biosensor increases the immobilization of biorecognition molecules on its surface and intensifies the interaction between the enhanced evanescent field and the dye, thereby significantly boosting detection sensitivity. Secondly, the fiber-embedded optofluidic biochip offers a plug-and-play simplicity that is easy to operate, making it resilient to stringent operating conditions and capable of providing rapid, decentralized feedback. Thirdly, this innovative biosensing platform is highly versatile, easily adaptable for detecting other trace pollutants by utilizing the appropriate functionalized fiber nano-biosensors and antibodies. Furthermore, this biosensing platform is endowed with numerous additional advantages, including its compact size, portability, reusability, user-friendliness, and cost-effectiveness. These features render it an exemplary solution for the rapid on-site screening of CLO, which is crucial for improving symptom management and enhancing health-related quality of life, especially in settings with limited resources.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.5 Detection of CLO in water environments\\u003c/h2\\u003e \\u003cp\\u003eTo assess the practical application of the fiber-embedded optofluidic biochip, a series of surface water samples were collected from various locations, including the Beijing Botanical Garden, the Summer Palace, and the Haihe River basin, for detection of CLO. Specifically, thirty water samples were gathered from ten different sites within the Haihe River basin (Figure \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e) over a three-month period from March to May 2024. These samples were tested directly using the fiber-embedded optofluidic biochip without any prior treatment, and no CLO was detected in any of the samples (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e and Table S2). To corroborate these findings, the same water samples underwent rigorous pretreatment and were analyzed using HPLC; no CLO was detected (Table S2). These outcomes suggest that the risk of CLO contamination in the Beijing-Tianjin area is minimal.\\u003c/p\\u003e \\u003cp\\u003eTo further assess the accuracy of the fiber-embedded optofluidic biochip, the water samples from the Beijing Botanical Garden and the Summer Palace were spiked with different concentrations of CLO (20, 40, and 60 \\u0026micro;g/L CLO) and retested. The recovery rates of CLO in these spiked samples, as detailed in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e, varied from 80 to 110%. These were verified via HPLC (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e), with a strong relationship observed between the measured results obtained using the biochip and HPLC (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). These results indicate that the fiber-embedded optofluidic biochip provides satisfactory accuracy and reliability for detecting CLO in real-world water samples. In conclusion, the fiber-embedded optofluidic biochip emerges as a robust tool for the rapid on-site decentralized screening of CLO in aquatic environments, offering a valuable contribution to environmental monitoring and public health.\\u003c/p\\u003e \\u003cp\\u003e \\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\\u003eDetermination of CLO in environment water and food samples by fiber-embedded optofluidic biochip and HPLC\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"8\\\"\\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=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c7\\\" colnum=\\\"7\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c8\\\" colnum=\\\"8\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eSamples\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eOriginal water\\u003c/p\\u003e \\u003cp\\u003e(\\u0026micro;g/L)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eSpiked Conc. (\\u0026micro;g/L)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eDetection Conc.\\u003c/p\\u003e \\u003cp\\u003e(\\u0026micro;g/L)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eRSD\\u003c/p\\u003e \\u003cp\\u003e(%)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eRecovery\\u003c/p\\u003e \\u003cp\\u003e(%)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eHPLC(\\u0026micro;g/L)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003eRecovery\\u003c/p\\u003e \\u003cp\\u003e(%)\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"2\\\" rowspan=\\\"3\\\"\\u003e \\u003cp\\u003eThe Summer Palace\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u0026lt;\\u0026thinsp;1.0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e20\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e20.25\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e9.1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e101.25%\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e19.286\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e96.43%\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u0026lt;\\u0026thinsp;1.0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e40\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e33.70\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e2.3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e84.25%\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e39.26\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e98.15%\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u0026lt;\\u0026thinsp;1.0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e60\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e50.05\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e2.2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e83.42%\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e53.994\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e89.99%\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"2\\\" rowspan=\\\"3\\\"\\u003e \\u003cp\\u003eBeijing Botanical Garden\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u0026lt;\\u0026thinsp;1.0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e20\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e19.85\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0.2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e99.25%\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e18.518\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e92.59%\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u0026lt;\\u0026thinsp;1.0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e40\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e33.85\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e8.7\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e84.63%\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e44.28\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e110.70%\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u0026lt;\\u0026thinsp;1.0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e60\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e52.40\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e4.5\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e87.33%\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e64.554\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e107.59%\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"2\\\" rowspan=\\\"3\\\"\\u003e \\u003cp\\u003eMilk\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u0026lt;\\u0026thinsp;1.0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e20\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e19.66\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e16.6\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e98.30%\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e19.286\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e96.43%\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u0026lt;\\u0026thinsp;1.0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e40\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e46.81\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e7.0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e117.04%\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e40.06\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e100.15%\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u0026lt;\\u0026thinsp;1.0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e60\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e60.56\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e14.7\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e100.94%\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e62.682\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e104.47%\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"2\\\" rowspan=\\\"3\\\"\\u003e \\u003cp\\u003eBaby Chinese cabbage\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u0026lt;\\u0026thinsp;1.0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e20\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e21.80\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e16.1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e109.00%\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e20.264\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e101.32%\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u0026lt;\\u0026thinsp;1.0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e40\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e36.50\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e5.8\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e91.25%\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e36.384\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e90.96%\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u0026lt;\\u0026thinsp;1.0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e60\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e50.46\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e5.1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e84.11%\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e57.318\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e95.53%\\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 \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.6 Towards the universal immunoassay platform for CLO detection\\u003c/h2\\u003e \\u003cp\\u003eTo showcase its versatility, the fiber-embedded optofluidic biochip was deployed to detect CLO in various food items, specifically milk and Chinese cabbage. The performance of the biochip in food detection was evaluated under these real-world conditions. Given the complexity of milk's composition, which could potentially interfere with antigen-antibody interactions, the milk samples were extracted using a solution containing acetonitrile and trifluoroacetic acid (0.2%). After filtration, the supernatant was analyzed using the fiber-embedded optofluidic biochip, and no CLO was detected in these samples (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). For the detection of CLO in Chinese cabbage, the cabbage was squeezed to extract its juice, which was then filtered through a 0.22 \\u0026micro;m PTFE filter film. The filtered solution was subsequently tested with the biochip, and again, no CLO was also detected (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eTo further assess the biochip's accuracy, two types of food samples were spiked with various concentrations of CLO (20, 40, and 60 \\u0026micro;g/L). As indicated in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e, the recovery rates of CLO in these spiked food samples ranged from 80 to 120%, which were also verified via HPLC (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e) with a strong relationship observed between the measured results obtained using the biochip and HPLC (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). These results indicate that the fiber-embedded optofluidic biochip offers satisfactory accuracy and reliability for detecting CLO in actual food samples. Thus, the fiber-embedded optofluidic biochip proves to be a viable tool for the rapid decentralized screening of CLO in food products, extending its utility beyond environmental samples to include food safety applications.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"4. Conclusion\",\"content\":\"\\u003cp\\u003eIn this study, an innovative and decentralized testing method for CLO employing a reusable fiber-embedded optofluidic biochip is described through integrating evanescent wave fluorescence, indirect competition immunoassay, and optofluidic biochip technology. The combination of all-fiber optical structure and tapered fiber nano-biosensor significantly improves the fluorescence excitation and collection efficiency, as well as its on-site detection ability and scalability. The nanopore structure of the fiber nano-biosensor enhanced the number of biorecognition molecules immobilized on its surface and the interaction between the enhanced evanescent field and dye, allowing a further increase in the detection sensitivity. The all-fiber optical structure and the plug-and-play biochip allows it to achieve rapid on-site screening of target pollutants, and unaffected by stringent operating conditions, thus providing timely feedback in a decentralized and rapid fashion. The fiber-embedded optofluidic biochip was successfully used for rapid on-site screening of the CLO in the surface waters and food in Beijing-Tianjin area. Although no positive results that was validated by HPLC were obtained, the spiked surface waters and food samples were detected with satisfactory recovery rates. Several outstanding features of the fiber-embedded optofluidic biochip, such as rapidity, compact size, portability, reusability, user-friendliness, and cost-efficiency, allow it an ideal solution for rapid on-site screening of CLO, towards improving symptom management and enhancing health-related quality of life, particularly in resource-limited environments. This new biosensing platform is highly versatile and easily extended to detect other trace pollutants using the corresponding functionalized fiber nano-biosensors and antibodies.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eCRediT authorship contribution statement\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eLaiya Lu: \\u003c/strong\\u003eConceptualization, Methodology, Validation, Writing-original draft, Funding acquisition. \\u003cstrong\\u003eTianxiang Ji \\u003c/strong\\u003eand\\u003cstrong\\u003e Min Wang:\\u003c/strong\\u003e Methodology, Investigation. \\u003cstrong\\u003eSiyan Liu and Yuxin Zhuo:\\u003c/strong\\u003e Investigation, Data curation. \\u003cstrong\\u003eFeng Long: \\u003c/strong\\u003eSupervision, Funding acquisition, Methodology, Writing-review\\u0026amp;editing.\\u003c/p\\u003e\\n\\n\\u003cp\\u003e\\u003cstrong\\u003eDeclaration of competing interest\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare no competing interests.\\u003c/p\\u003e\\n\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgments\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was financially supported by Beijing Science and Technology Planning Project (Z221100007122001), and the Natural Science Foundation of Beijing (8242031).\\u003c/p\\u003e\\n\\n\\n\\n\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eNanda, S., Ganguly, A., Mandi, M., et al., 2024. Chronic sub-lethal exposure to clothianidin triggers organismal and sub-organismal-level health hazards in a non-target organism, Drosophila melanogaster. Sci. Total Environ. 932, 172783.\\u003c/li\\u003e\\n\\u003cli\\u003eSales-Alba, A., Cruz-Alcalde, A., L\\u0026oacute;pez-Vinent, N., et al., 2023. Removal of neonicotinoid insecticide clothianidin from water by ozone-based oxidation: Kinetics and transformation products. Sep. Purif. Technol. 316, 123735.\\u003c/li\\u003e\\n\\u003cli\\u003eHirano, T., Ohno, S., Ikenaka, Y., et al., 2024. Quantification of the tissue distribution and accumulation of the neonicotinoid pesticide clothianidin and its metabolites in maternal and fetal mice. Toxicology and Applied Pharmacology 484, 116847.\\u003c/li\\u003e\\n\\u003cli\\u003eNaumann, T., Bento, C.P.M., Wittmann, A., et al., 2022. Occurrence and ecological risk assessment of neonicotinoids and related insecticides in the Bohai Sea and its surrounding rivers, China. Water Res. 209, 117912.\\u003c/li\\u003e\\n\\u003cli\\u003eSchaafsma, A., Limay-Rios, V., Baute, T., et al., 2015. Neonicotinoid Insecticide Residues in Surface Water and Soil Associated with Commercial Maize (Corn) Fields in Southwestern Ontario. PLoS One 10, e0118139.\\u003c/li\\u003e\\n\\u003cli\\u003eAddy-Orduna, L.M., Brodeur, J.C., Mateo, R., 2019. Oral acute toxicity of imidacloprid, thiamethoxam and clothianidin in eared doves: A contribution for the risk assessment of neonicotinoids in birds. Sci. Total Environ. 650, 1216-1223.\\u003c/li\\u003e\\n\\u003cli\\u003eGibbons, D., Morrissey, C., Mineau, P., 2015. A review of the direct and indirect effects of neonicotinoids and fipronil on vertebrate wildlife. Environmental Science and Pollution Research 22, 103-118.\\u003c/li\\u003e\\n\\u003cli\\u003eWang, Y., Shen, J., Lang, H., et al., 2024. Elevated temperature magnifies the acute and chronic toxicity of clothianidin to Eisenia fetida. Environ. Pollut. 355, 124210.\\u003c/li\\u003e\\n\\u003cli\\u003eWang, X., Anad\\u0026oacute;n, A., Wu, Q., et al., 2018. Mechanism of Neonicotinoid Toxicity: Impact on Oxidative Stress and Metabolism. Annual Review of Pharmacology and Toxicology 58, 471-507.\\u003c/li\\u003e\\n\\u003cli\\u003eZhou, Y., Zhang, Z., Jing, J., et al., 2023. Integrating environmental carry capacity based on pesticide risk assessment in soil management: A case study for China. J. Hazard. Mater. 460, 132341.\\u003c/li\\u003e\\n\\u003cli\\u003eMahai, G., Wan, Y., Wang, A., et al., 2023. Exposure to multiple neonicotinoid insecticides, oxidative stress, and gestational diabetes mellitus: Association and potential mediation analyses. Environ. Int. 179, 108173.\\u003c/li\\u003e\\n\\u003cli\\u003eZhang, D., Lu, S., 2022. Human exposure to neonicotinoids and the associated health risks: A review. Environ. Int. 163, 107201.\\u003c/li\\u003e\\n\\u003cli\\u003eWood, T.J., Goulson, D., 2017. The environmental risks of neonicotinoid pesticides: a review of the evidence post 2013. Environmental Science and Pollution Research 24, 17285-17325.\\u003c/li\\u003e\\n\\u003cli\\u003eAlford, A.M., Krupke, C.H., 2019. Movement of the Neonicotinoid Seed Treatment Clothianidin into Groundwater, Aquatic Plants, and Insect Herbivores. Environ. Sci. Technol. 53, 14368-14376.\\u003c/li\\u003e\\n\\u003cli\\u003eZhao, R., Zhang, Z., Yu, M., et al., 2024. New strategy for improving the rainfastness and control effect on Monochamus alternatus of clothianidin via a castor oil-based polyurethane nanoemulsion. Environmental Technology \\u0026amp; Innovation 34, 103564.\\u003c/li\\u003e\\n\\u003cli\\u003eWoodward, E.E., Hladik, M.L., Main, A.R., et al., 2022. Comparing imidacloprid, clothianidin, and azoxystrobin runoff from lettuce fields using a soil drench or treated seeds in the Salinas Valley, California. Environ. Pollut. 315, 120325.\\u003c/li\\u003e\\n\\u003cli\\u003ePook, C., Gritcan, I., 2019. Validation and application of a modified QuEChERS method for extracting neonicotinoid residues from New Zealand maize field soil reveals their persistence at nominally hazardous concentrations. Environ. Pollut. 255, 113075.\\u003c/li\\u003e\\n\\u003cli\\u003eJeschke, P., Nauen, R., Schindler, M., et al., 2011. Overview of the Status and Global Strategy for Neonicotinoids. Journal of Agricultural and Food Chemistry 59, 2897-2908.\\u003c/li\\u003e\\n\\u003cli\\u003eKim, B.M., Park, J.-S., Choi, J.-H., et al., 2012. Residual determination of clothianidin and its metabolites in three minor crops via tandem mass spectrometry. Food Chem. 131, 1546-1551.\\u003c/li\\u003e\\n\\u003cli\\u003eLiu, L., Peng, M., Xu, K., et al., 2024. Fluorescence Quenching Detection of Clothianidin in Fruit and Vegetable Samples Using MAPbBr3 Perovskite Quantum Dots. ACS Applied Nano Materials 7, 9176-9183.\\u003c/li\\u003e\\n\\u003cli\\u003eLiang, Z., Mahmoud Abdelshafy, A., Luo, Z., et al., 2022. Occurrence, detection, and dissipation of pesticide residue in plant-derived foodstuff: A state-of-the-art review. Food Chem. 384, 132494.\\u003c/li\\u003e\\n\\u003cli\\u003eYang, H., Xia, L., Zheng, J., et al., 2023. Screening and identification of a DNA aptamer to construct the label-free fluorescent aptasensor for ultrasensitive and selective detection of clothianidin residue in agricultural products. Talanta 262, 124712.\\u003c/li\\u003e\\n\\u003cli\\u003eLi, M., Hua, X., Ma, M., et al., 2014. Detecting clothianidin residues in environmental and agricultural samples using rapid, sensitive enzyme-linked immunosorbent assay and gold immunochromatographic assay. Sci. Total Environ. 499, 1-6.\\u003c/li\\u003e\\n\\u003cli\\u003eXu, Z.-L., Sun, W.-J., Yang, J.-Y., et al., 2012. Development of a Solid-Phase Extraction Coupling Chemiluminescent Enzyme Immunoassay for Determination of Organophosphorus Pesticides in Environmental Water Samples. Journal of Agricultural and Food Chemistry 60, 2069-2075.\\u003c/li\\u003e\\n\\u003cli\\u003eSheng, E., Shi, H., Zhou, L., et al., 2016. Dual-labeled time-resolved fluoroimmunoassay for simultaneous detection of clothianidin and diniconazole in agricultural samples. Food Chem. 192, 525-530.\\u003c/li\\u003e\\n\\u003cli\\u003eLai, X., Cao, W., Zhang, G., et al., 2024. Traffic signal-inspired fluorescence lateral flow immunoassay utilizing self-assembled AIENP@Ni/EC for simultaneous multi-pesticide residue detection. Chem. Eng. J. 501, 157565.\\u003c/li\\u003e\\n\\u003cli\\u003eBorah, P., Biswas, R., 2023. Impactful analytical schemes for assessing pesticides in tea: A comprehensive review. Measurement 221, 113505.\\u003c/li\\u003e\\n\\u003cli\\u003eTaitt, Chris R., Anderson, G.P., Ligler, F.S., 2016. Evanescent wave fluorescence biosensors: Advances of the last decade. Biosensors and Bioelectronics 76, 103-112.\\u003c/li\\u003e\\n\\u003cli\\u003eFernandez-Cuesta, I., Llobera, A., Ramos-Pay\\u0026aacute;n, M., 2022. Optofluidic systems enabling detection in real samples: A review. Anal. Chim. Acta 1192, 339307.\\u003c/li\\u003e\\n\\u003cli\\u003eLi, W., Wang, H., Yang, R., et al., 2018. Integrated multichannel all-fiber optofluidic biosensing platform for sensitive and simultaneous detection of trace analytes. Anal. Chim. Acta 1040, 112-119.\\u003c/li\\u003e\\n\\u003cli\\u003eChen, L., Yu, L., Liu, Y., et al., 2023. Valve-Adjustable Optofluidic Bio-Imaging Platform for Progressive Stenosis Investigation. ACS Sensors 8, 3104-3115.\\u003c/li\\u003e\\n\\u003cli\\u003eWang, Y., Gao, R., Zhan, C., et al., 2025. SERS-based microfluidic sensor for sensitive detection of circulating tumor markers: A critical review. Coord. Chem. Rev. 523, 216289.\\u003c/li\\u003e\\n\\u003cli\\u003ePidenko, S.A., Burmistrova, N.A., Shuvalov, A.A., et al., 2018. Microstructured optical fiber-based luminescent biosensing: Is there any light at the end of the tunnel? - A review. Anal. Chim. Acta 1019, 14-24.\\u003c/li\\u003e\\n\\u003cli\\u003eZhao, Y., Hu, X.-g., Hu, S., et al., 2020. Applications of fiber-optic biochemical sensor in microfluidic chips: A review. Biosensors and Bioelectronics 166, 112447.\\u003c/li\\u003e\\n\\u003cli\\u003eAralekallu, S., Boddula, R., Singh, V., 2023. Development of glass-based microfluidic devices: A review on its fabrication and biologic applications. Materials \\u0026amp; Design 225, 111517.\\u003c/li\\u003e\\n\\u003cli\\u003eYin, M.-j., Gu, B., An, Q.-F., et al., 2018. Recent development of fiber-optic chemical sensors and biosensors: Mechanisms, materials, micro/nano-fabrications and applications. Coord. Chem. Rev. 376, 348-392.\\u003c/li\\u003e\\n\\u003cli\\u003eChen, D., Xu, W., Lu, Y., et al., 2024. Rapid and sensitive parallel on-site detection of antibiotics and resistance genes in aquatic environments using evanescent wave dual-color fluorescence fiber-embedded optofluidic nanochip. Biosensors and Bioelectronics 257, 116281.\\u003c/li\\u003e\\n\\u003cli\\u003eLong, F., He, M., Zhu, A.N., et al., 2009. Portable optical immunosensor for highly sensitive detection of microcystin-LR in water samples. Biosensors and Bioelectronics 24, 2346-2351.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"microchimica-acta\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"miac\",\"sideBox\":\"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)\",\"snPcode\":\"604\",\"submissionUrl\":\"https://submission.springernature.com/new-submission/604/3\",\"title\":\"Microchimica Acta\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"Fiber-embedded optofluidic biochip, Clothianidin, Evanescent wave fluorescence, Immunoassay, On-site detection\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-6323851/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-6323851/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"Clothianidin (CLO), a highly effective neonicotinoid insecticide, is globally utilized to combat both sucking and chewing pests. There is an increasing demand for rapid and high-frequency on-site detection of CLO in water and food sources due to its high toxicity to non-target organisms. To address this, we introduce an innovative and decentralized testing method for CLO employing a reusable fiber-embedded optofluidic biochip. This biochip leverages evanescent wave fluorescence, indirect competitive immunoassay, and optofluidic technology to provide reliable, rapid, straightforward, and cost-effective CLO measurements. The integration of an all-fiber optical structure with a tapered fiber nano-biosensor significantly enhances fluorescence excitation and collection efficiency, bolstering the biochip's on-site detection capabilities and scalability. This biochip demonstrated high sensitivity and specificity in detecting CLO, achieving a satisfactory limit of detection of 1.0 µg/L within 12 min. It was successfully applied for rapid on-site screening of CLO in surface waters and food in the Beijing-Tianjin region, offering timely and decentralized feedback. The biochip detected spiked surface waters and food with satisfactory recovery rates. These confirm the biochip's potential as a robust tool for rapid and high-frequency on-site CLO screening in water and food, particularly in settings with limited resources. This biochip is highly adaptable and can be easily expanded to detect other trace pollutants by utilizing the appropriate functionalized fiber biosensors and antibodies.\",\"manuscriptTitle\":\"Rapid and sensitive on-site detection of Clothianidin in surface waters with a reusable fiber-embedded optofluidic biochip\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-04-21 09:41:04\",\"doi\":\"10.21203/rs.3.rs-6323851/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2025-04-18T16:54:43+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-04-17T20:03:00+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"150230794129529271486961831538449587022\",\"date\":\"2025-04-14T15:03:13+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-04-10T12:00:06+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"22155427191847633244909723813017475825\",\"date\":\"2025-04-01T09:39:27+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2025-04-01T08:47:18+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2025-03-31T01:27:18+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2025-03-31T01:25:54+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Microchimica Acta\",\"date\":\"2025-03-28T01:17:28+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"microchimica-acta\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"miac\",\"sideBox\":\"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)\",\"snPcode\":\"604\",\"submissionUrl\":\"https://submission.springernature.com/new-submission/604/3\",\"title\":\"Microchimica Acta\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"90645de9-d358-48e0-be04-920be22817e2\",\"owner\":[],\"postedDate\":\"April 21st, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-05-26T16:08:06+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-6323851\",\"link\":\"https://doi.org/10.1007/s00604-025-07220-y\",\"journal\":{\"identity\":\"microchimica-acta\",\"isVorOnly\":false,\"title\":\"Microchimica Acta\"},\"publishedOn\":\"2025-05-23 15:58:34\",\"publishedOnDateReadable\":\"May 23rd, 2025\"},\"versionCreatedAt\":\"2025-04-21 09:41:04\",\"video\":\"\",\"vorDoi\":\"10.1007/s00604-025-07220-y\",\"vorDoiUrl\":\"https://doi.org/10.1007/s00604-025-07220-y\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-6323851\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-6323851\",\"identity\":\"rs-6323851\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}