Femtosecond-Laser-Inscribed Dual-Resonance LPFG Biosensor for Ultrasensitive (1nM) Aflatoxin B1 Screening in Food

Femtosecond-Laser-Inscribed Dual-Resonance LPFG Biosensor for Ultrasensitive (1nM) Aflatoxin B1 Screening in Food | 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 Femtosecond-Laser-Inscribed Dual-Resonance LPFG Biosensor for Ultrasensitive (1nM) Aflatoxin B1 Screening in Food Jiuli Shi, Yufan Tian, Duanduan Wu, Qiushun Zou, Shixun Dai, Peiqing Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7492626/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Food safety monitoring demands rapid and sensitive aflatoxin detection technologies that surpass current national standards (10⁻⁸ mol/L). Here we report a femtosecond-laser-Inscribed dual-resonance LPFG biosensor achieving 1nM(10⁻ 9 mol/L) detection limit. Through point-by-point grating inscription and optimized silanization-mediated antibody immobilization, our platform demonstrates that the sensor exhibits excellent selectivity for AFB1 detection,good repeatability, and exceptional sensitivity with a response of 3.768 nm/nM in the critical 5–15 nM detection range, significantly surpassing conventional approaches,in addition, the sensor demonstrated excellent recovery rates of 80.5%-102.9% for AFB1 in complex food matrices, confirming its robust performance even in challenging sample environments. This study provides a highly efficient and scalable solution for food safety monitoring and holds great promise for applications in the field of food quality detection. Femtosecond laser direct writing Optical fiber sensor Long-period fiber grating AFB1 detection and antigen–antibody binding Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Aflatoxin is an exceptionally carcinogenic mycotoxin produced by molds such as Aspergillus flavus and Aspergillus parasiticus in warm and humid environments[ 1 ]. Among the aflatoxins, AFB1 is the most toxic and potent[ 2 ], significantly more harmful than other variants like AFB2, AFG1, and AFG2[ 3 ]. It primarily contaminates grains (including corn, wheat, and rice), peanuts, nuts, legumes, dairy products, and animal feed[ 4 , 5 ]. Ingesting high doses may cause acute aflatoxicosis, resulting in liver failure, hemorrhage[ 6 ], ascites, and even death[ 7 ]. Chronic exposure to low doses is linked to liver cirrhosis, liver cancer, weakened immunity, and malnutrition. In developing countries, AFB1 is recognized as one of the major risk factors for hepatocellular carcinoma (HCC)[ 8 , 9 ]. When poultry and livestock ingest contaminated feed, AFB1 can be biotransformed into aflatoxin M1 (AFM1)[ 10 ], which may subsequently accumulate in milk, dairy products, meat, and eggs[ 11 , 12 ]. Consequently, developing a sensor to detect aflatoxin B1 is of paramount importance. Rapid and accurate detection of AFB1 is essential for ensuring food safety. Currently, common detection methods for AFB1 include chromatographic techniques, immunoassays, optical sensing, optical fiber sensing, and electrochemical sensing[ 13 – 16 ]. Jihea Moon et al. developed a rapid and simple immunochromatographic assay for the detection of AFB1, based on a modified competitive binding format using colloidal gold and polyclonal antibody (Pab) conjugates, which enabled the detection of 10 µg/mL AFB1 in less than 10 minutes[ 17 ]. Peng Chen et al. employed a distance-based readout paper-based microfluidic chip (DPMC), incorporating DNA hydrogel-responsive valves and paper-based capillary channels, to quantitatively detect AFB1 concentrations in the range of 100 to 1000 pM, with a detection limit of 17.64 pM[ 15 ]. Tathagata Pal et al. developed an ultrasensitive AFB1 sensor based on plastic optical fiber (POF), exploring the optical properties of polyaniline (PAni) on the polymethyl methacrylate (PMMA) surface of the POF[ 18 ]. This sensor demonstrated effective performance across picomolar to micromolar concentrations with reasonable accuracy. Although these methods achieve high sensitivity, they often require specialized expertise and expensive instrumentation. As a novel biosensor platform, long-period fiber gratings (LPFGs) offer significant advantages such as high sensitivity, specificity, stability, and ease of fabrication[ 19 ], making them increasingly attractive for widespread applications[ 20 ]. LPFGs are specialized optical devices based on mode coupling, characterized by their high sensitivity, broadband resonance[ 21 ], low insertion loss, and absence of back-reflection[ 22 ]. These features make LPFGs widely applicable in various fields such as biosensing, chemical sensing, environmental monitoring, and structural health monitoring[ 23 – 26 ]. Integrating LPFGs into a porous detection chamber enables the simultaneous analysis of multiple samples, significantly enhancing detection efficiency. This configuration is particularly well-suited for large-scale food quality screening in scenarios such as factory quality control, agricultural procurement and storage, food supplier evaluation, import and export inspections, market sampling, and laboratory automation. It offers a practical and scalable solution for industrial food safety monitoring. To achieve specific detection of AFB1, this study employs silanization to functionalize the surface of the optical fiber[ 27 ]. Silanization is an efficient surface modification technique that introduces organic functional groups onto material surfaces, enabling the stable immobilization of antibodies on the fiber surface. When the antigen specifically binds to the immobilized antibody on the surface, it induces a shift in the transmission spectrum of the long-period fiber grating (LPFG)[ 28 ]. By analyzing the spectral shift, the concentration of AFB1 can be accurately demodulated, enabling high-sensitivity detection[ 29 ]. In this work, we have developed an antibody-functionalized sensor based on dual-peak LPFG, integrated with a porous detection chamber, enabling rapid, specific, accurate, and high-throughput detection of AFB1. By immobilizing antibodies on the fiber surface, the antigen–antibody interaction induces changes in the transmission spectrum of the optical fiber sensor. Encapsulating the LPFG within a porous detection chamber allows for the simultaneous analysis of multiple food samples, significantly saving time and improving operational efficiency. This approach demonstrates high sensitivity, with a detection limit of 1nM, along with excellent repeatability and specificity, showing great promise as a powerful tool for future food safety screening applications. 2. Theoretical analysis Long-period fiber grating (LPFG) is a specialized optical fiber device that operates based on periodic modulation of the refractive index within the fiber[ 30 ]. This modulation enables coupling between the core mode and cladding modes when specific phase-matching conditions are met. At certain wavelengths that satisfy these conditions, light energy is transferred from the core mode to the cladding modes, resulting in a decrease in transmitted intensity at those wavelengths and the appearance of distinct resonance dips in the transmission spectrum[ 31 ]. LPFGs feature a periodic refractive index modulation structure, typically with a period ranging from 100 to 1000 µm. The phase-matching condition between the effective propagation constants of the core and cladding modes can be described by the following equation[ 32 , 33 ]: $$\:{\beta\:}_{core}-{\beta\:}_{clad}^{m}=\frac{2\pi\:}{\varLambda\:}$$ 1 Where \(\:{\beta\:}_{core}\) and \(\:{\beta\:}_{clad}^{m}\) represent the propagation constants of the fundamental core mode and the m-th cladding mode, respectively. The resonance wavelength of the LPFG depends on the effective refractive indices of the fundamental core mode and the cladding mode, and can be obtained by the following expression: $$\:{\lambda\:}_{res}=\left({n}_{eff}^{co}-{n}_{eff}^{cl,m}\right){\Lambda\:}$$ 2 Where \(\:{\lambda\:}_{res}\) is the resonance wavelength, \(\:{n}_{eff}^{co}\) and \(\:{n}_{eff}^{cl,m}\) represent the effective refractive indices of the fundamental core mode and the m-th order cladding mode of the fiber, respectively, \(\:{\Lambda\:}\) is the grating period. Based on the phase matching condition, changes in the surrounding refractive index (SRI) lead to variations in the effective refractive index of the cladding mode, thereby causing a corresponding shift in the resonance wavelength of the LPFG. When the LPFG detects aflatoxin B1, it binds with the antibody, resulting in a change of the external refractive index. For an LPFG with a fixed period, the refractive index sensitivity can be expressed as: $$\:\frac{d{\lambda\:}_{res}}{d{n}_{amb}}={\lambda\:}_{res}\:\frac{\raisebox{1ex}{$d{\lambda\:}_{res}$}\!\left/\:\!\raisebox{-1ex}{$d{\Lambda\:}$}\right.}{{n}_{eff}^{co}-{n}_{eff}^{cl,m}}\:{{\Gamma\:}}_{amb}$$ 3 $$\:{{\Gamma\:}}_{amb}=-\frac{{u}_{m}^{2}{\lambda\:}_{res}^{3}{n}_{anb}}{8\pi\:{r}_{cl}^{3}{n}_{cl}\left({n}_{eff}^{co}-{n}_{eff}^{cl,m}\right){\left({n}_{cl}^{2}-{n}_{amb}^{2}\right)}^{\raisebox{1ex}{$3$}\!\left/\:\!\raisebox{-1ex}{$2$}\right.}}$$ 4 3. Experiment 3.1 Fabrication of LPFG LPFG is an important optical fiber device that has been widely applied in optical communications, fiber sensing, and biosensing. Femtosecond laser processing, with its ultrashort pulse duration, high peak power, and precise material processing capability, offers an efficient, flexible, and maskless approach for the fabrication of LPFGs. Periodic structures were inscribed inside a standard single-mode fiber (SMF-28) using femtosecond laser direct writing. Prior to inscription, the protective coating of the fiber was removed using a stripping tool (Vintool CF-3), and the bare fiber was thoroughly cleaned with alcohol several times to eliminate surface contaminants. The single-mode fiber (SMF) was then mounted on a high-precision XYZ translation stage to ensure accurate alignment and processing. A femtosecond laser with a central wavelength of 1030 nm was employed to induce periodic refractive index modulations within the fiber core. The desired grating period was achieved by precisely controlling the laser focus, pulse energy, exposure time, and translation step size. A 20× objective lens with a numerical aperture NA of 0.45 (Nikon, Japan) was used to focus femtosecond laser pulses with a repetition rate of 166 kHz and pulse energy of 0.36 µJ into the core of the SMF-28 fiber for grating fabrication. The evolution of the grating transmission spectrum in the range of 1100 nm to 1700 nm was monitored in real time using a supercontinuum SC source (YSL, SC-5, China) and a spectrometer (Flame-NIR, China). The LPFG was fabricated with a grating period of 163µm, corresponding to the LP 0,12 cladding mode, and operated at the phase-matching turning point, thereby enabling maximum sensitivity for this cladding mode. The duty cycle was 50%, and the total grating length was 26080µm, consisting of 160 grating elements. 3.2 Functionalization of LPFG The stable immobilization of antibodies on the optical fiber surface is one of the key techniques to ensure the sensitivity and specificity of a biosensor. Silanization is employed to introduce an organic layer with reactive functional groups onto the fiber surface, enabling subsequent attachment of biomolecules such as antibodies for specific recognition. This method offers the advantages of covalent bonding, providing high stability while remaining simple and effective. First, the cleaned LPFG is incubated in 0.1 M NaOH solution for 60 minutes. NaOH removes surface impurities and increases the number of hydroxyl groups (–OH), which facilitates the subsequent silanization process. After the generation of hydroxyl groups, the grating region of the optical fiber is immersed in a 2% v/v APTES ethanol solution and allowed to react at room temperature for 60 minutes. The fiber is then rinsed three times with ethanol to remove unreacted silane molecules. This process results in the formation of a silane monolayer containing functional groups on the fiber surface through Si–O–Si bonding, thereby anchoring APTES firmly to the surface. Subsequently, the silanized fiber is immersed in a 2% v/v glutaraldehyde solution and incubated at room temperature for 1 hour. The glutaraldehyde crosslinks with the amino groups introduced by APTES, generating aldehyde groups on the surface to provide binding sites for antibodies. The fiber grating region is then immersed in an anti-aflatoxin B1 antibody solution (0.2 mg/mL) and incubated at room temperature for 12 hours. Finally, 1% bovine serum albumin (BSA) is used to block unreacted aldehyde groups and reduce non-specific adsorption. The fiber is rinsed three times with phosphate-buffered saline (PBS) to remove unbound antibodies. A schematic diagram of the antibody immobilization process is shown in Fig. 1. 3.3 Detection setup for Aflatoxin B1 The experimental setup for aflatoxin B1 detection is shown in Fig. 2. Since LPFGs are highly sensitive to external environmental disturbances such as temperature and vibration, the entire experiment was conducted at room temperature to minimize thermal cross-sensitivity. One end of the fiber was fixed at one side of a custom-designed groove, while the other end was tensioned to straighten the fiber, thereby preventing experimental interference from vibration and mechanical strain. The antibody-functionalized optical fiber was fixed inside a porous detection chamber. A specified volume of AFB1 solution was carefully introduced into the chamber through a small opening at the top using a micropipette. A stock solution of AFB1 at a concentration of 1 × 10⁻³ M was prepared. In accordance with national food safety standards for AFB1 limits in food, a series of test solutions with concentrations of 0nM, 1nM, 5nM, 10nM, 15nM, and 20nM were prepared by serial dilution using PBS buffer to obtain low-concentration analytes. The two ends of the optical fiber were connected to single-mode FC/PC patch cords using a fiber fusion splicer (Sumitomo Electric Industries Ltd., Osaka, Japan). The input end of the patch cord was connected to a supercontinuum light source (YSL, SC-5, China) covering a wavelength range of 470–2400 nm, while the output end was connected to a spectrometer (Flame-NIR, China). The detection experiments were carried out sequentially from the lowest to the highest concentration. 4. Results and discussions 4.1 Surface topography of LPFG sensor After completing the surface functionalization of the optical fiber, the microscopic morphology of the fiber surface was characterized using scanning electron microscopy (SEM). The unmodified fiber surface exhibited relatively smooth and uniform features, whereas the fiber surface after silanization and antibody immobilization showed a significant increase in surface roughness, with irregular granular structures attached, as shown in Fig. 3(b). These particles can be attributed to the successful immobilization of antibody molecules. When aflatoxin B1 binds to the thin and uniform chemical layer, the external refractive index of the fiber changes. At the same time, the transmission spectrum of the LPFG sensor also undergoes variation before and after surface functionalization, as illustrated in Fig. 3(a). After functionalization, both resonance peaks of the LPFG exhibited noticeable wavelength shifts. These results indicate that the functionalization process not only altered the surface morphology of the fiber and achieved successful antibody modification, but also provided an effective platform for subsequent biological recognition. 4.2 Performance characterization of the LPFG The working principle of the LPFG sensor is based on the shift in the resonance wavelengths of its transmission spectrum in response to changes in the external refractive index, enabling sensitive detection of refractive index variations. Therefore, in the initial stage of the experiment, the performance of the LPFG sensor was characterized by immersing it in glycerol solutions of varying concentrations and observing the corresponding changes in the transmission spectrum. Glycerol solutions with refractive indices ranging from 1.34 to 1.42 were prepared. As shown in Fig. 4, both resonance peaks exhibited noticeable shifts with increasing refractive index: the left peak experienced a blue shift of 27.41 nm, while the right peak exhibited a red shift of 62.62 nm. A fitting of the experimental data revealed that within the refractive index range of 1.333 to 1.42, the LPFG demonstrated a refractive index sensitivity of up to 1238.72 nm/RIU, confirming the high sensitivity of the fabricated LPFG and its readiness for subsequent sensing applications. 4.3 Detection of AFB1 By detecting AFB1 samples at different concentrations, the sensor enables qualitative analysis of the analyte solutions. Measurements were carried out sequentially from low to high concentrations, ranging from 0nM to 20nM. To minimize experimental errors during the sensing process, the sensing region was rinsed with PBS when switching between different concentrations to reduce cross-contamination. The LPFG transmission spectra were collected using a spectrometer and recorded on a computer. As shown in Fig. 5, the sensitivity S of the fiber is defined as S = Δλ /Δc, where Δλ represents the wavelength shift and c is the concentration of aflatoxin B1. Both resonance peaks of the LPFG sensor exhibited shifts with changes in concentration, as illustrated in Figs. 5(b) and 5(c). The sensitivities at AFB1 concentrations of 5nM and 15nM were 3.768 nm/nM and 1.835 nm/nM, respectively. The sensor demonstrated a detection limit as low as 1nM, which is one order of magnitude more sensitive than the national safety standard. These experimental results confirm that the fabricated LPFG sensor exhibits high sensitivity for the detection of AFB1. 4.4 Repeatability The repeatability of a sensor is one of the key indicators for transitioning optical fiber sensors from laboratory research to practical applications, as it determines the reliability of detection. To evaluate the repeatability of the functionalized long-period fiber grating sensor for detecting AFB1, four functionalized LPFGs were selected and tested in AFB1 solutions at three different concentrations (1nM, 10nM, and 20nM). For each concentration, the resonance wavelength shift was independently measured for each fiber. By comparing the detection responses of different fibers under the same concentration, the consistency and repeatability of the sensor were assessed. The results showed that the four fibers exhibited good reproducibility of resonance wavelength shifts at all concentration levels, with small standard deviations, confirming the stability and detection reliability of the fabricated functionalized optical fibers. 4.5 Stability and specificity Standard deviation (SD) is an important indicator for evaluating the repeatability and background fluctuation of a sensor. To assess the stability of the functionalized LPFG sensor, nine measurements were performed under stable conditions at the lowest AFB1 concentration (0nM). As shown in Fig. 7(a), the calculated SD was 0.083, indicating that the sensor exhibits excellent signal stability and repeatability, thereby providing a reliable basis for subsequent specificity detection. In the detection process using functionalized LPFG sensors, specificity is one of the key factors determining sensor performance. To verify the specificity of the functionalized LPFG for AFB1, control solutions containing deoxynivalenol (DON), zearalenone (ZEN), glucose, and bovine serum albumin (BSA) were prepared, with concentrations equivalent to that of AFB1. The sensor was used to detect both AFB1 and the control solutions individually. As shown in Fig. 7(b), the other interfering substances induced only slight shifts in the LPFG resonance wavelength, whereas AFB1 caused a significant wavelength shift. This is attributed to the specific binding between AFB1 and the antibodies immobilized on the fiber surface. The sensor’s response to AFB1 was significantly higher than that to other interfering substances, demonstrating that the antibody possesses high selectivity for AFB1 and that the sensor exhibits excellent specificity in target recognition. 4.6 Real sample detection To evaluate the applicability of the functionalized LPFG sensor in real-world samples, this study selected fruit juice as the detection matrix for AFB1 analysis. In the experiment, the fruit juice samples were first centrifuged, and the supernatant was collected to remove pulp and other impurities. Then, known concentrations of AFB1 standard solutions (1nM, 10nM, and 20nM) were spiked into the juice as the experimental groups, while juice samples without AFB1 served as the blank control group. The functionalized LPFG sensor was used to detect each of the four sample groups, and the resonance wavelength shifts were recorded. As shown in Fig. 8(b), compared with the blank juice samples, the fruit juice solutions spiked with AFB1 exhibited significant dual-peak wavelength shifts. The recovery rates for AFB1 in the spiked samples ranged from 80.5–102.9%. These results demonstrate that the sensor possesses strong recognition ability and high sensitivity toward the target toxin AFB1. Furthermore, the functionalized LPFG maintained specific responsiveness to AFB1 even in complex food matrices, thereby confirming the sensor’s feasibility for food detection and laying a technical foundation for future intelligent food production and regulatory monitoring. 5. Conclusion In summary, this study designed and validated a highly sensitive sensor based on a dual-peak functionalized long-period fiber grating for the detection of aflatoxin B1. The LPFG was fabricated on standard single-mode silica fiber using femtosecond laser direct writing technology. Antibodies were firmly immobilized on the fiber surface through silanization, enabling specific binding between AFB1 and the antibodies, which induced a resonance wavelength shift in the LPFG for quantitative detection of AFB1. Experimental results demonstrated that the fabricated fiber sensor exhibits high sensitivity, with a detection limit of 1nM, along with excellent repeatability, selectivity, and stability. Moreover, the sensor effectively detected AFB1 in juice samples, showing high detection sensitivity suitable for screening within complex food matrices. Compared to other sensors, this sensor offers advantages such as simple fabrication, strong specificity and stability, real-time detection capability, and miniaturization potential, indicating broad application prospects. Declarations Author Contribution Jiulli Shi,Yufan Tian, Duanduan Wu, Qiushun Zou, shixun Dai, Peiging Zhang have performed the research.Peiqing Zhang Zhang was responsible for providing resources and offering supervisory guidance. Jiuli Shi wrote the main manuscript text and prepared the figures.All authors reviewed the manuscript and approved the final manuscript. Acknowledgments This work was supported by the Natural Science Foundation of China (Nos.62475124,62075107), Zhejiang Provincial Natural Science Foundation of China (No.LR24F050001) and Key R&D Project of Ningbo City (No.2023Z105) Data Availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Conflict of interest The authors have no conflicts to disclose. References Lu H, Liu FF, Zhu QQ, Zhang MM, Li T, Chen JM, et al. Journal of The Science of Food and Agriculture; 97 :1910-5. (2017) Wu ZH, Sun DW, Pu HB, Wei QY. Talanta; 252 . 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7492626","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":516729380,"identity":"a572cb95-2550-44dc-b5ad-de84fe9b4423","order_by":0,"name":"Jiuli Shi","email":"","orcid":"","institution":"Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Jiuli","middleName":"","lastName":"Shi","suffix":""},{"id":516729381,"identity":"ae112a80-2610-4cc6-a8ab-faa60b69afcf","order_by":1,"name":"Yufan Tian","email":"","orcid":"","institution":"Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Yufan","middleName":"","lastName":"Tian","suffix":""},{"id":516729382,"identity":"dfd070bf-b04b-41e6-bfc3-dbc8dd5f9600","order_by":2,"name":"Duanduan Wu","email":"","orcid":"","institution":"Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Duanduan","middleName":"","lastName":"Wu","suffix":""},{"id":516729383,"identity":"c33508fb-38bc-4efa-911a-09f613b50442","order_by":3,"name":"Qiushun Zou","email":"","orcid":"","institution":"Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Qiushun","middleName":"","lastName":"Zou","suffix":""},{"id":516729384,"identity":"c818dff8-3e6c-4b11-b5bb-08b1a4684434","order_by":4,"name":"Shixun Dai","email":"","orcid":"","institution":"Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Shixun","middleName":"","lastName":"Dai","suffix":""},{"id":516729385,"identity":"c1f01a83-b889-4697-939d-47cd2ce675a5","order_by":5,"name":"Peiqing Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYDACCQjFw8AMJD8wHABxDIjXwjiDFC1gwMxDjBb52c3PHn7dYSPDd5z38GvbHXcSG9ibt0kw1NzBqYVxzjFzY9kzaTySh/nSrHPPPEts4DlWJsFw7BlOLcwSCWbSkm2HeQwO85gZ57YdTmyQyDGTYGw4jFMLm0T6N6CW/xAtliAt8m/wa+EBmin5se0ASIvxY0awLTz4tUhI5JRJM55JBvqFx4yxt+2ZcRtPWrFFwjHcWuRnpG+T/LnDzp7v/BnjDz/b7sj2sx/eeONDDW4t4CDgbQCSB4D+AvsORCTg1QAM6J8QLcwfCCgcBaNgFIyCEQoAl3tUSf8wO0EAAAAASUVORK5CYII=","orcid":"","institution":"Ningbo University","correspondingAuthor":true,"prefix":"","firstName":"Peiqing","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-08-30 04:23:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7492626/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7492626/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91591021,"identity":"cfe0eb27-096c-4328-821d-9ec6eaa9b827","added_by":"auto","created_at":"2025-09-18 06:35:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":118568,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of the optical fiber functionalization process\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7492626/v1/1d5c3ea50cfa595ccbbfee3f.png"},{"id":91591022,"identity":"67820458-9e22-4d13-b5cc-efae39e69dd0","added_by":"auto","created_at":"2025-09-18 06:35:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":162185,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of the detection setup for aflatoxin B1\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7492626/v1/23d0ec8021605ea5b0002359.png"},{"id":91592085,"identity":"e4be91b8-0edc-4bfd-aa53-308314c0487c","added_by":"auto","created_at":"2025-09-18 06:51:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":338703,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Transmission spectrum before and after functionalization; (b) SEM image of the optical fiber surface after functionalization.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7492626/v1/ce44fa4eb239f3584c9897fd.png"},{"id":91591031,"identity":"b18bc877-d66b-4a14-9184-1336802645a9","added_by":"auto","created_at":"2025-09-18 06:35:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":245172,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Resonance wavelength shifts of the optical fiber in the refractive index range of 1.333~1.42; (b) Linear fitting of the wavelength shift.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7492626/v1/f7130a44b56ba1a0f4ed447f.png"},{"id":91591026,"identity":"65883a61-5d27-47f2-b9f0-1500ca7bdfc9","added_by":"auto","created_at":"2025-09-18 06:35:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":408058,"visible":true,"origin":"","legend":"\u003cp\u003e(a), (b), and (c) show the real-time spectral responses of the DP-LPFG sensor at different concentrations of aflatoxin B1; (d) shows the fitted curve of the wavelength shift of the LPFG sensor for detecting various concentrations of aflatoxin B1.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7492626/v1/10b8904acc6f779a74e280af.png"},{"id":91591829,"identity":"1fbe157a-9646-46dd-8404-6973fbcbd158","added_by":"auto","created_at":"2025-09-18 06:43:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":400224,"visible":true,"origin":"","legend":"\u003cp\u003eWavelength shifts of LPFG sensors (LPFG1, 2, 3, 4) in response to different concentrations of aflatoxin B1.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7492626/v1/52ee0b64d85c0185a5f27caa.png"},{"id":91591027,"identity":"1225fda9-43cb-48d7-a921-8d0f954ced74","added_by":"auto","created_at":"2025-09-18 06:35:11","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":152580,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Verification of the optical fiber's stability; (b) Specificity verification of the LPFG sensor, including responses to DON, ZEN, glucose, and BSA.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7492626/v1/0db831801e50097dc89e465c.png"},{"id":91591037,"identity":"c31e1d2f-824c-4e08-9bc9-04aa878a8859","added_by":"auto","created_at":"2025-09-18 06:35:11","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":273188,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Process diagram for obtaining juice supernatant (b)Detection results of aflatoxin B1 in real samples (fruit juice)\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7492626/v1/bf95dac2da79bb7e68bef27b.png"},{"id":98622300,"identity":"ee4dacf9-952a-4259-919e-3910ba35e0f9","added_by":"auto","created_at":"2025-12-19 16:51:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2560404,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7492626/v1/1459ca9e-fcae-4668-9e41-d9afac298d9c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Femtosecond-Laser-Inscribed Dual-Resonance LPFG Biosensor for Ultrasensitive (1nM) Aflatoxin B1 Screening in Food","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAflatoxin is an exceptionally carcinogenic mycotoxin produced by molds such as Aspergillus flavus and Aspergillus parasiticus in warm and humid environments[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Among the aflatoxins, AFB1 is the most toxic and potent[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], significantly more harmful than other variants like AFB2, AFG1, and AFG2[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. It primarily contaminates grains (including corn, wheat, and rice), peanuts, nuts, legumes, dairy products, and animal feed[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Ingesting high doses may cause acute aflatoxicosis, resulting in liver failure, hemorrhage[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], ascites, and even death[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Chronic exposure to low doses is linked to liver cirrhosis, liver cancer, weakened immunity, and malnutrition. In developing countries, AFB1 is recognized as one of the major risk factors for hepatocellular carcinoma (HCC)[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. When poultry and livestock ingest contaminated feed, AFB1 can be biotransformed into aflatoxin M1 (AFM1)[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], which may subsequently accumulate in milk, dairy products, meat, and eggs[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Consequently, developing a sensor to detect aflatoxin B1 is of paramount importance.\u003c/p\u003e\u003cp\u003eRapid and accurate detection of AFB1 is essential for ensuring food safety. Currently, common detection methods for AFB1 include chromatographic techniques, immunoassays, optical sensing, optical fiber sensing, and electrochemical sensing[\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e–\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Jihea Moon et al. developed a rapid and simple immunochromatographic assay for the detection of AFB1, based on a modified competitive binding format using colloidal gold and polyclonal antibody (Pab) conjugates, which enabled the detection of 10 µg/mL AFB1 in less than 10 minutes[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Peng Chen et al. employed a distance-based readout paper-based microfluidic chip (DPMC), incorporating DNA hydrogel-responsive valves and paper-based capillary channels, to quantitatively detect AFB1 concentrations in the range of 100 to 1000 pM, with a detection limit of 17.64 pM[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Tathagata Pal et al. developed an ultrasensitive AFB1 sensor based on plastic optical fiber (POF), exploring the optical properties of polyaniline (PAni) on the polymethyl methacrylate (PMMA) surface of the POF[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This sensor demonstrated effective performance across picomolar to micromolar concentrations with reasonable accuracy. Although these methods achieve high sensitivity, they often require specialized expertise and expensive instrumentation. As a novel biosensor platform, long-period fiber gratings (LPFGs) offer significant advantages such as high sensitivity, specificity, stability, and ease of fabrication[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], making them increasingly attractive for widespread applications[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eLPFGs are specialized optical devices based on mode coupling, characterized by their high sensitivity, broadband resonance[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], low insertion loss, and absence of back-reflection[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These features make LPFGs widely applicable in various fields such as biosensing, chemical sensing, environmental monitoring, and structural health monitoring[\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e–\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Integrating LPFGs into a porous detection chamber enables the simultaneous analysis of multiple samples, significantly enhancing detection efficiency. This configuration is particularly well-suited for large-scale food quality screening in scenarios such as factory quality control, agricultural procurement and storage, food supplier evaluation, import and export inspections, market sampling, and laboratory automation. It offers a practical and scalable solution for industrial food safety monitoring.\u003c/p\u003e\u003cp\u003eTo achieve specific detection of AFB1, this study employs silanization to functionalize the surface of the optical fiber[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Silanization is an efficient surface modification technique that introduces organic functional groups onto material surfaces, enabling the stable immobilization of antibodies on the fiber surface. When the antigen specifically binds to the immobilized antibody on the surface, it induces a shift in the transmission spectrum of the long-period fiber grating (LPFG)[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. By analyzing the spectral shift, the concentration of AFB1 can be accurately demodulated, enabling high-sensitivity detection[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this work, we have developed an antibody-functionalized sensor based on dual-peak LPFG, integrated with a porous detection chamber, enabling rapid, specific, accurate, and high-throughput detection of AFB1. By immobilizing antibodies on the fiber surface, the antigen–antibody interaction induces changes in the transmission spectrum of the optical fiber sensor. Encapsulating the LPFG within a porous detection chamber allows for the simultaneous analysis of multiple food samples, significantly saving time and improving operational efficiency. This approach demonstrates high sensitivity, with a detection limit of 1nM, along with excellent repeatability and specificity, showing great promise as a powerful tool for future food safety screening applications.\u003c/p\u003e"},{"header":"2. Theoretical analysis","content":"\u003cp\u003eLong-period fiber grating (LPFG) is a specialized optical fiber device that operates based on periodic modulation of the refractive index within the fiber[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This modulation enables coupling between the core mode and cladding modes when specific phase-matching conditions are met. At certain wavelengths that satisfy these conditions, light energy is transferred from the core mode to the cladding modes, resulting in a decrease in transmitted intensity at those wavelengths and the appearance of distinct resonance dips in the transmission spectrum[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. LPFGs feature a periodic refractive index modulation structure, typically with a period ranging from 100 to 1000 µm. The phase-matching condition between the effective propagation constants of the core and cladding modes can be described by the following equation[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]:\u003c/p\u003e\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:{\\beta\\:}_{core}-{\\beta\\:}_{clad}^{m}=\\frac{2\\pi\\:}{\\varLambda\\:}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003cp\u003eWhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\beta\\:}_{core}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\beta\\:}_{clad}^{m}\\)\u003c/span\u003e\u003c/span\u003e represent the propagation constants of the fundamental core mode and the m-th cladding mode, respectively. The resonance wavelength of the LPFG depends on the effective refractive indices of the fundamental core mode and the cladding mode, and can be obtained by the following expression:\u003c/p\u003e\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:{\\lambda\\:}_{res}=\\left({n}_{eff}^{co}-{n}_{eff}^{cl,m}\\right){\\Lambda\\:}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003cp\u003eWhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\lambda\\:}_{res}\\)\u003c/span\u003e\u003c/span\u003eis the resonance wavelength, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{n}_{eff}^{co}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{n}_{eff}^{cl,m}\\)\u003c/span\u003e\u003c/span\u003e represent the effective refractive indices of the fundamental core mode and the m-th order cladding mode of the fiber, respectively,\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\Lambda\\:}\\)\u003c/span\u003e\u003c/span\u003e is the grating period. Based on the phase matching condition, changes in the surrounding refractive index (SRI) lead to variations in the effective refractive index of the cladding mode, thereby causing a corresponding shift in the resonance wavelength of the LPFG. When the LPFG detects aflatoxin B1, it binds with the antibody, resulting in a change of the external refractive index. For an LPFG with a fixed period, the refractive index sensitivity can be expressed as:\u003c/p\u003e\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\:\\frac{d{\\lambda\\:}_{res}}{d{n}_{amb}}={\\lambda\\:}_{res}\\:\\frac{\\raisebox{1ex}{$d{\\lambda\\:}_{res}$}\\!\\left/\\:\\!\\raisebox{-1ex}{$d{\\Lambda\\:}$}\\right.}{{n}_{eff}^{co}-{n}_{eff}^{cl,m}}\\:{{\\Gamma\\:}}_{amb}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equ4\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ4\" name=\"EquationSource\"\u003e\n$$\\:{{\\Gamma\\:}}_{amb}=-\\frac{{u}_{m}^{2}{\\lambda\\:}_{res}^{3}{n}_{anb}}{8\\pi\\:{r}_{cl}^{3}{n}_{cl}\\left({n}_{eff}^{co}-{n}_{eff}^{cl,m}\\right){\\left({n}_{cl}^{2}-{n}_{amb}^{2}\\right)}^{\\raisebox{1ex}{$3$}\\!\\left/\\:\\!\\raisebox{-1ex}{$2$}\\right.}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e4\u003c/div\u003e\u003c/div\u003e"},{"header":"3. Experiment","content":"\u003cp\u003e3.1 Fabrication of LPFG\u003c/p\u003e\u003cp\u003eLPFG is an important optical fiber device that has been widely applied in optical communications, fiber sensing, and biosensing. Femtosecond laser processing, with its ultrashort pulse duration, high peak power, and precise material processing capability, offers an efficient, flexible, and maskless approach for the fabrication of LPFGs. Periodic structures were inscribed inside a standard single-mode fiber (SMF-28) using femtosecond laser direct writing. Prior to inscription, the protective coating of the fiber was removed using a stripping tool (Vintool CF-3), and the bare fiber was thoroughly cleaned with alcohol several times to eliminate surface contaminants. The single-mode fiber (SMF) was then mounted on a high-precision XYZ translation stage to ensure accurate alignment and processing. A femtosecond laser with a central wavelength of 1030 nm was employed to induce periodic refractive index modulations within the fiber core. The desired grating period was achieved by precisely controlling the laser focus, pulse energy, exposure time, and translation step size. A 20× objective lens with a numerical aperture NA of 0.45 (Nikon, Japan) was used to focus femtosecond laser pulses with a repetition rate of 166 kHz and pulse energy of 0.36 µJ into the core of the SMF-28 fiber for grating fabrication. The evolution of the grating transmission spectrum in the range of 1100 nm to 1700 nm was monitored in real time using a supercontinuum SC source (YSL, SC-5, China) and a spectrometer (Flame-NIR, China). The LPFG was fabricated with a grating period of 163µm, corresponding to the LP\u003csub\u003e0,12\u003c/sub\u003e cladding mode, and operated at the phase-matching turning point, thereby enabling maximum sensitivity for this cladding mode. The duty cycle was 50%, and the total grating length was 26080µm, consisting of 160 grating elements.\u003c/p\u003e\u003cp\u003e3.2 Functionalization of LPFG\u003c/p\u003e\u003cp\u003eThe stable immobilization of antibodies on the optical fiber surface is one of the key techniques to ensure the sensitivity and specificity of a biosensor. Silanization is employed to introduce an organic layer with reactive functional groups onto the fiber surface, enabling subsequent attachment of biomolecules such as antibodies for specific recognition. This method offers the advantages of covalent bonding, providing high stability while remaining simple and effective.\u003c/p\u003e\u003cp\u003eFirst, the cleaned LPFG is incubated in 0.1 M NaOH solution for 60 minutes. NaOH removes surface impurities and increases the number of hydroxyl groups (–OH), which facilitates the subsequent silanization process. After the generation of hydroxyl groups, the grating region of the optical fiber is immersed in a 2% v/v APTES ethanol solution and allowed to react at room temperature for 60 minutes. The fiber is then rinsed three times with ethanol to remove unreacted silane molecules. This process results in the formation of a silane monolayer containing functional groups on the fiber surface through Si–O–Si bonding, thereby anchoring APTES firmly to the surface. Subsequently, the silanized fiber is immersed in a 2% v/v glutaraldehyde solution and incubated at room temperature for 1 hour. The glutaraldehyde crosslinks with the amino groups introduced by APTES, generating aldehyde groups on the surface to provide binding sites for antibodies. The fiber grating region is then immersed in an anti-aflatoxin B1 antibody solution (0.2 mg/mL) and incubated at room temperature for 12 hours. Finally, 1% bovine serum albumin (BSA) is used to block unreacted aldehyde groups and reduce non-specific adsorption. The fiber is rinsed three times with phosphate-buffered saline (PBS) to remove unbound antibodies. A schematic diagram of the antibody immobilization process is shown in Fig.\u0026nbsp;1.\u003c/p\u003e\u003cp\u003e3.3 Detection setup for Aflatoxin B1\u003c/p\u003e\u003cp\u003eThe experimental setup for aflatoxin B1 detection is shown in Fig.\u0026nbsp;2. Since LPFGs are highly sensitive to external environmental disturbances such as temperature and vibration, the entire experiment was conducted at room temperature to minimize thermal cross-sensitivity. One end of the fiber was fixed at one side of a custom-designed groove, while the other end was tensioned to straighten the fiber, thereby preventing experimental interference from vibration and mechanical strain. The antibody-functionalized optical fiber was fixed inside a porous detection chamber. A specified volume of AFB1 solution was carefully introduced into the chamber through a small opening at the top using a micropipette. A stock solution of AFB1 at a concentration of 1 × 10⁻³ M was prepared. In accordance with national food safety standards for AFB1 limits in food, a series of test solutions with concentrations of 0nM, 1nM, 5nM, 10nM, 15nM, and 20nM were prepared by serial dilution using PBS buffer to obtain low-concentration analytes. The two ends of the optical fiber were connected to single-mode FC/PC patch cords using a fiber fusion splicer (Sumitomo Electric Industries Ltd., Osaka, Japan). The input end of the patch cord was connected to a supercontinuum light source (YSL, SC-5, China) covering a wavelength range of 470–2400 nm, while the output end was connected to a spectrometer (Flame-NIR, China). The detection experiments were carried out sequentially from the lowest to the highest concentration.\u003c/p\u003e"},{"header":"4. Results and discussions","content":"\u003cp\u003e4.1 Surface topography of LPFG sensor\u003c/p\u003e\u003cp\u003eAfter completing the surface functionalization of the optical fiber, the microscopic morphology of the fiber surface was characterized using scanning electron microscopy (SEM). The unmodified fiber surface exhibited relatively smooth and uniform features, whereas the fiber surface after silanization and antibody immobilization showed a significant increase in surface roughness, with irregular granular structures attached, as shown in Fig.\u0026nbsp;3(b). These particles can be attributed to the successful immobilization of antibody molecules. When aflatoxin B1 binds to the thin and uniform chemical layer, the external refractive index of the fiber changes. At the same time, the transmission spectrum of the LPFG sensor also undergoes variation before and after surface functionalization, as illustrated in Fig.\u0026nbsp;3(a). After functionalization, both resonance peaks of the LPFG exhibited noticeable wavelength shifts. These results indicate that the functionalization process not only altered the surface morphology of the fiber and achieved successful antibody modification, but also provided an effective platform for subsequent biological recognition.\u003c/p\u003e\u003cp\u003e4.2 Performance characterization of the LPFG\u003c/p\u003e\u003cp\u003eThe working principle of the LPFG sensor is based on the shift in the resonance wavelengths of its transmission spectrum in response to changes in the external refractive index, enabling sensitive detection of refractive index variations. Therefore, in the initial stage of the experiment, the performance of the LPFG sensor was characterized by immersing it in glycerol solutions of varying concentrations and observing the corresponding changes in the transmission spectrum. Glycerol solutions with refractive indices ranging from 1.34 to 1.42 were prepared. As shown in Fig.\u0026nbsp;4, both resonance peaks exhibited noticeable shifts with increasing refractive index: the left peak experienced a blue shift of 27.41 nm, while the right peak exhibited a red shift of 62.62 nm. A fitting of the experimental data revealed that within the refractive index range of 1.333 to 1.42, the LPFG demonstrated a refractive index sensitivity of up to 1238.72 nm/RIU, confirming the high sensitivity of the fabricated LPFG and its readiness for subsequent sensing applications.\u003c/p\u003e\u003cp\u003e4.3 Detection of AFB1\u003c/p\u003e\u003cp\u003eBy detecting AFB1 samples at different concentrations, the sensor enables qualitative analysis of the analyte solutions. Measurements were carried out sequentially from low to high concentrations, ranging from 0nM to 20nM. To minimize experimental errors during the sensing process, the sensing region was rinsed with PBS when switching between different concentrations to reduce cross-contamination. The LPFG transmission spectra were collected using a spectrometer and recorded on a computer. As shown in Fig.\u0026nbsp;5, the sensitivity S of the fiber is defined as S = Δλ /Δc, where Δλ represents the wavelength shift and c is the concentration of aflatoxin B1. Both resonance peaks of the LPFG sensor exhibited shifts with changes in concentration, as illustrated in Figs.\u0026nbsp;5(b) and 5(c). The sensitivities at AFB1 concentrations of 5nM and 15nM were 3.768 nm/nM and 1.835 nm/nM, respectively. The sensor demonstrated a detection limit as low as 1nM, which is one order of magnitude more sensitive than the national safety standard. These experimental results confirm that the fabricated LPFG sensor exhibits high sensitivity for the detection of AFB1.\u003c/p\u003e\u003cp\u003e4.4 Repeatability\u003c/p\u003e\u003cp\u003eThe repeatability of a sensor is one of the key indicators for transitioning optical fiber sensors from laboratory research to practical applications, as it determines the reliability of detection. To evaluate the repeatability of the functionalized long-period fiber grating sensor for detecting AFB1, four functionalized LPFGs were selected and tested in AFB1 solutions at three different concentrations (1nM, 10nM, and 20nM). For each concentration, the resonance wavelength shift was independently measured for each fiber. By comparing the detection responses of different fibers under the same concentration, the consistency and repeatability of the sensor were assessed. The results showed that the four fibers exhibited good reproducibility of resonance wavelength shifts at all concentration levels, with small standard deviations, confirming the stability and detection reliability of the fabricated functionalized optical fibers.\u003c/p\u003e\u003cp\u003e4.5 Stability and specificity\u003c/p\u003e\u003cp\u003eStandard deviation (SD) is an important indicator for evaluating the repeatability and background fluctuation of a sensor. To assess the stability of the functionalized LPFG sensor, nine measurements were performed under stable conditions at the lowest AFB1 concentration (0nM). As shown in Fig.\u0026nbsp;7(a), the calculated SD was 0.083, indicating that the sensor exhibits excellent signal stability and repeatability, thereby providing a reliable basis for subsequent specificity detection.\u003c/p\u003e\u003cp\u003eIn the detection process using functionalized LPFG sensors, specificity is one of the key factors determining sensor performance. To verify the specificity of the functionalized LPFG for AFB1, control solutions containing deoxynivalenol (DON), zearalenone (ZEN), glucose, and bovine serum albumin (BSA) were prepared, with concentrations equivalent to that of AFB1. The sensor was used to detect both AFB1 and the control solutions individually. As shown in Fig.\u0026nbsp;7(b), the other interfering substances induced only slight shifts in the LPFG resonance wavelength, whereas AFB1 caused a significant wavelength shift. This is attributed to the specific binding between AFB1 and the antibodies immobilized on the fiber surface. The sensor’s response to AFB1 was significantly higher than that to other interfering substances, demonstrating that the antibody possesses high selectivity for AFB1 and that the sensor exhibits excellent specificity in target recognition.\u003c/p\u003e\u003cp\u003e4.6 Real sample detection\u003c/p\u003e\u003cp\u003eTo evaluate the applicability of the functionalized LPFG sensor in real-world samples, this study selected fruit juice as the detection matrix for AFB1 analysis. In the experiment, the fruit juice samples were first centrifuged, and the supernatant was collected to remove pulp and other impurities. Then, known concentrations of AFB1 standard solutions (1nM, 10nM, and 20nM) were spiked into the juice as the experimental groups, while juice samples without AFB1 served as the blank control group. The functionalized LPFG sensor was used to detect each of the four sample groups, and the resonance wavelength shifts were recorded. As shown in Fig.\u0026nbsp;8(b), compared with the blank juice samples, the fruit juice solutions spiked with AFB1 exhibited significant dual-peak wavelength shifts. The recovery rates for AFB1 in the spiked samples ranged from 80.5–102.9%. These results demonstrate that the sensor possesses strong recognition ability and high sensitivity toward the target toxin AFB1. Furthermore, the functionalized LPFG maintained specific responsiveness to AFB1 even in complex food matrices, thereby confirming the sensor’s feasibility for food detection and laying a technical foundation for future intelligent food production and regulatory monitoring.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn summary, this study designed and validated a highly sensitive sensor based on a dual-peak functionalized long-period fiber grating for the detection of aflatoxin B1. The LPFG was fabricated on standard single-mode silica fiber using femtosecond laser direct writing technology. Antibodies were firmly immobilized on the fiber surface through silanization, enabling specific binding between AFB1 and the antibodies, which induced a resonance wavelength shift in the LPFG for quantitative detection of AFB1. Experimental results demonstrated that the fabricated fiber sensor exhibits high sensitivity, with a detection limit of 1nM, along with excellent repeatability, selectivity, and stability. Moreover, the sensor effectively detected AFB1 in juice samples, showing high detection sensitivity suitable for screening within complex food matrices. Compared to other sensors, this sensor offers advantages such as simple fabrication, strong specificity and stability, real-time detection capability, and miniaturization potential, indicating broad application prospects.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJiulli Shi,Yufan Tian, Duanduan Wu, Qiushun Zou, shixun Dai, Peiging Zhang have performed the research.Peiqing Zhang Zhang was responsible for providing resources and offering supervisory guidance. Jiuli Shi wrote the main manuscript text and prepared the figures.All authors reviewed the manuscript and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eThis work was supported by the Natural Science Foundation of China (Nos.62475124,62075107), Zhejiang Provincial Natural Science Foundation of China (No.LR24F050001) and Key R\u0026amp;D Project of Ningbo City (No.2023Z105)\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\u003cp\u003eConflict of interest\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLu H, Liu FF, Zhu QQ, Zhang MM, Li T, Chen JM, et al. Journal of The Science of Food and Agriculture;\u003cstrong\u003e97\u003c/strong\u003e:1910-5. 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(2022)\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Femtosecond laser direct writing, Optical fiber sensor, Long-period fiber grating, AFB1 detection and antigen–antibody binding","lastPublishedDoi":"10.21203/rs.3.rs-7492626/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7492626/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFood safety monitoring demands rapid and sensitive aflatoxin detection technologies that surpass current national standards (10⁻⁸ mol/L). Here we report a femtosecond-laser-Inscribed dual-resonance LPFG biosensor achieving 1nM(10⁻\u003csup\u003e9\u003c/sup\u003e mol/L) detection limit. Through point-by-point grating inscription and optimized silanization-mediated antibody immobilization, our platform demonstrates that the sensor exhibits excellent selectivity for AFB1 detection,good repeatability, and exceptional sensitivity with a response of 3.768 nm/nM in the critical 5\u0026ndash;15 nM detection range, significantly surpassing conventional approaches,in addition, the sensor demonstrated excellent recovery rates of 80.5%-102.9% for AFB1 in complex food matrices, confirming its robust performance even in challenging sample environments. This study provides a highly efficient and scalable solution for food safety monitoring and holds great promise for applications in the field of food quality detection.\u003c/p\u003e","manuscriptTitle":"Femtosecond-Laser-Inscribed Dual-Resonance LPFG Biosensor for Ultrasensitive (1nM) Aflatoxin B1 Screening in Food","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-18 06:35:06","doi":"10.21203/rs.3.rs-7492626/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f32bdaba-d035-4e84-9278-ae4bc16b49df","owner":[],"postedDate":"September 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-10T01:23:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-18 06:35:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7492626","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7492626","identity":"rs-7492626","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}