Sensitive and facile detection of vitamin D based on fluorescent labeled aptamer probe and exonuclease I-assisted signal amplification

Sensitive and facile detection of vitamin D based on fluorescent labeled aptamer probe and exonuclease I-assisted signal amplification | 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 Sensitive and facile detection of vitamin D based on fluorescent labeled aptamer probe and exonuclease I-assisted signal amplification Xinqiu Xu, Chaofan Jia, Fengjiao Zhang, Hao Li, Weilei Gong, Changqin Wang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6029304/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Mar, 2025 Read the published version in Journal of Fluorescence → Version 1 posted 7 You are reading this latest preprint version Abstract The combination of nanomaterials and biomolecular recognition units, and the signal transduction based on fluorescence, have broad prospects in the development of small molecule optical adaptive sensors. In this study, we systematically studied a rapid and simple aptamer-based fluorescence sensor that uses fullerene as a quencher for the detection of vitamin D3. The surface of fullerene consists of a π-electron cloud, which makes it a powerful electron acceptor material capable of accepting or transferring excited electrons from fluorophores such as carboxyfluorescein (FAM). The aptamer labeled with fluorescein (5’6-FAM) is adsorbed onto the fullerene surface through hydrogen bonding and π-π stacking interactions, leading to fluorescence quenching due to Förster resonance energy transfer (FRET). However, in the presence of vitamin D3, it can specifically bind to FAM-ssDNA, forming a vitamin D3-aptamer-hairpin structure that cannot be adsorbed onto the fullerene surface. Under the optimal experimental conditions, the linear detection range for vitamin D3 was 0–600 nM, with a detection limit of 200 nM. When exonuclease I was used, the detection limit was improved to 50 nM. Furthermore, the recovery rate of vitamin D3 in water samples was 88.4–96.3%. The feasibility of the sensor was validated by successfully detecting vitamin D3 in water samples. Vitamin D3 Aptamer Fullerene Fluorescence Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Vitamin D represents a crucial substance necessary for the body to sustain its overall well-being and prevent chronic illnesses[ 1 , 2 ]. The compound cholecalciferol, also known as vitamin D3, is a fat-soluble nutrient that can be obtained through exposure to sunlight or by consuming certain foods[ 3 , 4 ]. Inside the human body, cholecalciferol gets converted into the active form of vitamin D. This active form is vital in controlling the absorption and usage of calcium and phosphorus to ensure the best possible bone health[ 5 , 6 ]. Therefore, the measurement of Vitamin D3 levels can be regarded as a significant indicator for evaluating the quality of life in a population. The standard quantitative measuring tools for Vit-D3 levels involve high performance liquid chromatography (HPLC)[ 7 – 9 ], enzyme linked immunosorbent assay (ELISA)[ 10 – 12 ], radioimmunoassay (RIA)[ 13 , 14 ], chemiluminescent immunoassay (CLIA)[ 15 – 17 ] and high-performance liquid chromatography-mass spectrometry (HPLC-MS)[ 18 , 19 ]. These methods are limited to specialized laboratories, and ELISA faces challenges such as instability under harsh conditions, high production costs, and the need for sophisticated procedures. Consequently, there is a pressing demand for rapid, effective and sensitive techniques to detect Vit-D3 suitable for just-in-time diagnostic application. Lee et al. created a gold nanoparticle-based colorimetric aptamer sensor for detecting vitamin D, using VDBA 14 aptamers, with a detection limit of 1 µM[ 20 ]. Colorimetric methods are straightforward and do not need advanced instruments, but they often experience significant data fluctuations. Carlucci et al. showcased vitamin D detection through Surface-Enhanced Raman Spectroscopy (SRP) and electrochemical biosensors. SRP was used to directly measure vitamin D levels with a vitamin D antibody, achieving a detection limit (LOD) of 2 µg/ml. However, detecting Vitamin D3 was often hindered by interference from other vitamins, due to their similar oxidation potentials[ 21 ]. Seongjae Jo et al. developed a local surface plasmon resonance (LSPR) aptamer sensor using polyethylene glycol (PEG)-free gold nanorods (AuNRs) for large-scale, direct detection of 25-hydroxyvitamin D3[ 22 ]. However, the scarcity of precious metals also limits their large-scale application. Khalid E. Alzahrani et al. modified a microcantilever beam with Bruno adsorbent, and the deflection and frequency of the microcantilever beam were changed by the presence of vitamin D. By measuring these changes, the concentration of vitamin D can be quantitatively analyzed, with a detection limit of about 0.3 nM[ 23 ]. While the sensor demonstrates fast and sensitive detection in laboratory conditions with good performance, its effectiveness in complex biological samples, such as serum or plasma, has not been validated, making it challenging to apply in clinical trials. The fluorescent aptamer sensor detection has many advantages including high sensitivity, high selectivity, real-time monitoring ability, and applicability to complex samples[ 24 – 26 ], which already arouse attention. Herein, this article developed a novel aptamer-based fluorescence strategy based on fullerene and FAM labeled aptamer for the detection of vitamin D3. Aptamers are selected single-stranded oligonucleotides of DNA or RNA sequences[ 27 ] with molecular weights ranging from 10 to 30 kDa[ 28 ]. Aptamers possess advantageous characteristics for their utilization in biomedical applications: Firstly, aptamers demonstrate exceptional stability across diverse environments. Secondly, in contrast to antibodies, aptamers can be manufactured at a cost-effective rate. Finally, these molecules have a long shelf life and can be regenerated after denaturation. Aptamers have a stable three-dimensional structure and are generated by an evolutionary analogy in vitro, called ligand exponential enrichment phylogenetic evolution (SELEX)[ 29 ]. It has high affinity and specificity to the target and becomes the best substitute to antibodies[ 30 ]. It can bind to various target molecules and undergo adaptive conformational changes, driven by hydrophobic interactions, van der Waals forces, and shape complementarity in the intermolecular interactions with the target. Nucleic acid aptamer is a kind of nucleic acid aptamer developed in recent years for the detection of biological matter level, which is used in fluorescence, electrochemistry, colorimetry, electrochemical luminescence and other analytical methods[ 31 ]. The field of biosensors has witnessed a significant research focus on the incorporation of fluorescent substances into nucleic acid aptamers, resulting in the development of highly notable fluorescent aptamer sensors. Recently, a variety of nanomaterials, such as Graphene and its derivatives, carbon nanotubes, polymer nanoparticles, metal nanoparticles and silica nanoparticles have been used to exploit new types of analytical sensor tools[ 32 ]. Composed of 60 sp2 hybrid carbon atoms, fullerenes (or C60) are truncated icosahedral spherical molecules arranged into systematic hexagons and pentagons to form a closed hollow cage, similar to a football[ 33 ]. The three-dimensional highly delocalized electron conjugated molecular structure gives C60 excellent optical properties, which is expected to be applied in optical signal processing and control. The nanoscale and abundant oxygen-containing functional groups of fullerene enable it to form strong non-covalent interactions with DNA probes, thus significantly improving the immobilization efficiency of DNA probes. Consequently, the aptamer can be bound by fullerene and subsequently lead to fluorescence quenching[ 34 ]. At present, several studies have demonstrated fullerene have been successfully applied to aptamer fluorescence biosensors, highlighting its excellent performance in biomolecule recognition and detection. However, no research has focused on constructing an aptasensor for the detection of Vitamin D3 using fluorescent probes. As far as we know, there is still a lack of research on the detection of vitamin D3 using fullerenes at present. In this study, fullerene is used as quenching agent and an aptamer as recognition element for vitamin D detection, which is the first application of fullerene in vitamin D detection. Fullerene is a π-rich nanocarbon that effectively quenches the fluorescence of FAM-DNA through energy transfer and Förster resonance energy transfer, making it an ideal quencher[ 35 ]. A DNA sequence that specifically binds vitamin D was selected as the aptamer. When the target vitamin D3 is present, FAM-DNA binds to it to form an atypical B-type DNA structure that cannot be attached to the fullerenes[ 36 ], and the fluorescence of aptamer would not be quenched[ 37 ]. Therefore, the quantification of vitamin D can be evaluated according to the fluorescence response of the detection method. In addition, this study not only provides an effective sensing approach of fullerene fluorescence analysis, but also broadens a new application for clinical vitamin D level detection. 2. Experimental section 2.1 Materials and chemicals Vitamin D3 aptamer was synthesized from Shanghai Sangon Biotechnology Co., LTD. (Shanghai, China). The oligonucleotide sequence is as follows: 5′-AGCAG CACAG AGGTC ATGGG GGGTG TGACT TTGGT GTGCC TATGCG TGCTA CGGAA-3′, which was selected by Lee, Nguyen, and Gu[ 20 ]. The fullerene aqueous dispersion was purchased from Suzhou Tanfeng Materials Tech Co. Ltd. (Jiangsu, China). Exonuclease I and its 10 × reaction buffer (670 mM glycine-KOH (pH 9.5, 25°C) with 67 mM MgCl2 and 10 mM DTT) were purchased from Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). Vitamin D3 was obtained from Durai Biology Co. Ltd (Nanjing China). Tris-HCL was obtained from Phygene Biotechnology Co. Ltd (Fuzhou, China). Other vitamins were purchased from Qiansheng Biotechnology Co. Ltd (Anhui, China). All other chemicals were of analytically grade and used without any further purification. 2.2 Instrumentation Drops of fullerene dispersion are placed on a specific copper net and naturally dried in a clean environment to prepare samples for transmission electron microscopy (TEM) measurements. Transmission electron microscopy (TEM) observation was measured using a FEI Tecnai G2 12 instrument (FEI, USA). The functional groups of fullerenes were measured by Nicolet IS10 (Nicolet USA) infrared spectroscopic instrument. The nucleic acid sequences were centrifuged by High Speed Refrigerated Centrifuge (Thermo Fisher, Shanghai, China) before dissolved by EDTA to prepare reserve solution. Puxi TU-1901 spectrophotometer was employed to test the UV–Vis absorbance spectrum. Fluorescence intensities were recorded on fluorescence spectrophotometer FL-2700 (Hitachi Hi-Tech Science Nako Co. LTD. Japan). The fluorescence was monitored at 520 nm with an excitation at 485 nm and slit widths of excitation and emission were set at 5 nm. 2.3 Optimization of the detection conditions In order to obtain the best performance sensor, the following parameters are optimized: (a) fullerenes in a concentration range of 0–3.0 µg/ml; (b) The quenching time of fullerenes on aptamer fluorescence is 0–40 minutes; (c) Incubation time range of aptamer and target was 0 min-50 min; (d) The influence of different pH values of Tris-HCL buffer 6.0–8.0 on the sensor; (e) Influence of exonuclease I concentration range 5U-60U on the sensor. 2.4 Construction of a fluorescence signal sensor In a typical vitamin D3 assay, a 59 µL aptamer reserve fluid (100µM) is first swirled for 1 minute, thoroughly mixed, and then used. A working solution containing FAM labeled aptamers was prepared by diluting the reserve solution to a 2nM concentration using a 10mM Tris-HCl buffer (pH 7.0). The fullerene suspension should then be sufficiently shaken to ensure uniform dispersion by adding 40µL fullerene suspension (about 2.4µg/mL) to the working solution. After 25 minutes, the target Vitamin D3 at an appropriate concentration and Exonuclease I (45 U) were added simultaneously. In order to promote the dissolution of vitamin D3, we added an appropriate amount of ethanol to prepare a reaction mixture with a total volume of 5 mL. The mixture was then incubated for 50 minutes at room temperature. Fluorescence measurements were recorded using the FL-2700 fluorescence spectrophotometer (Hitachi Hi-Tech Science Nako Co. Ltd., Japan) at 520 nm. 2.5 Specificity of the assay To further confirm the selectivity and specificity of this sensor, seven different substances were investigated with FAM-DNA/fullerene at a final concentration of 500 nM each under the optimal experimental conditions, including vitamin M, vitamin B1, vitamin B3, vitamin B5, vitamin B6, vitamin B12, and vitamin C. Three control experiments were also conducted to assess the specificity of the assay. Vitamin D3 at a concentration of 500 nM was used as a positive control. The emission spectra of the resultant mixtures were scanned from 510 nm to 660 nm with an excitation at 485 nm, the fluorescence recovery intensity was used to evaluate the Vit-D3 sensing performance of this method. Meanwhile, the sensors were prepared with 6µM of vitamin D3 and stored at 4°C away from light for 6 days, with the fluorescence intensity measured continuously to evaluate the stability of the sensor. And multiple measurements were taken to ensure the reliability of the results, and vitamin D3 at a concentration of 1µM was subjected to six independent experiments (n = 6) to assess reproducibility. 2.6 Detection of the actual samples To further verify the practical applicability and reliability of our biosensors, the sensing platform was applied to real water samples. Tap water was collected directly from the water system at the School of Public Health, Jining Medical College. After allowing a consistent flow for at least 1 minute, the tap water was collected in a clean 10-mL colorimetric tube. Before detection, the collected water samples were filtered through a 0.22µm filter to remove precipitation and impurities, and then mixed with Tris-HCl buffer at a 1:1 ratio and adjust the pH to 7.0. A fixed concentration of FAM-labeled ssDNA, fullerene and exonuclease I were added, followed by the addition of 400 nM, 500 nM, and 600 nM of vitamin D3 for detection. 3. Results and discussion 3.1 The feasibility of the aptasensor The Transmission Electron Microscopy (TEM) image of fullerene sample displayed a well-defined spherical shape of the fullerene nanoparticles (Fig. 1 (a)). The individual fullerene particles are observed to be uniform in size, with diameters typically in the range of 14 nm to 26 nm[ 38 ]. Fourier Transform Infrared (FT-IR) spectra of fullerenes are shown Fig. 1 (b), the characteristic peak at 573 cm − 1 can be attributed to a absorption band of the C-C stretching vibration pattern[ 39 ]. The peaks at 1181 and 1427 cm − 1 can be attributed to the double bond stretching vibration of C = C[ 40 ]. The peak at approximately 3432 cm − 1 could be confirmed the presence of OH stretching vibrations characteristic absorption bands[ 41 ]. Prior to the detection of Vit-D3, we validated the feasibility of using fullerene-aptamer hybrids as sensing. Figure 2 (A) shows how the degree of fluorescence bursting changes under different conditions in a Tris-HCl buffer solution. The aptamer exhibited strong fluorescence signal at 520 nm (curve c), in the presence of fullerene, the significant fluorescence quenching (curve a). This observed quenching phenomenon is likely to be aptamer stacked on fullerene due to the fluorescence resonance energy transfer effect. Figure 2 (B) shows when presented with excited light at 485 nm, the aptamer fluoresces at an emission maximum of 520 nm. Meanwhile fullerene have an absorption at 520 nm, the absorption spectrum overlaps with aptamer emission, it is in resonance with the excited state of aptamer. Hence, the fluorescence signal of aptamer can be quenched by FRET. In the pioneering work of Anton W. Jensen and Liu D et al., the negatively charged luciferine and positively charged fullerene molecules are brought closer together due to electrostatic interaction, which increases the electronic quenching of the excited state of luciferine and transfers energy to the fullerene[ 42 , 43 ]. Fullerene molecules is a kind of π-rich nanocarbon and have low-energy non-bonding orbitals (LUMO), which allow them to readily accept electrons[ 44 ]. The singlet excited state of the dye transfers energy to the fullerene, leaving the fullerene in an excited state while the dye returns to the ground state. Meanwhile, the singlet excited state of the dye transfers electrons to the fullerenes, causing them to form radical anions and the dye to form radical cations[ 45 ]. A strong π-π interaction may exist between FAM-DNA and the football-shaped fullerene, enabling the ssDNA to adsorb to the surface of fullerene, thereby quenching the fluorescence of FAM-DNA. Following the addition of Vit-D3 (curve b), fluorescence signal enhancement is observed. The aptamer's strong affinity for vitamin D3 promotes a dominant ring structure and a smaller hairpin conformation in the probe, leading to the dissociation of the complex from the fullerene surface and hindering energy or electron transfer between FAM and fullerenes. The fundamental distinction arises due to the aptamer's ability to sufficiently unwind, exposing its bases, whereas the vitamin D3-aptamer complex maintains a stable atypical B structure that hinders the necessary unwinding for base exposure[ 20 , 46 ]. In the presence of Vit-D3 and 45 U exonuclease I (Curve d), the amplification signal using exonuclease I was greater than that of the 1:1 binding strategy using the same concentration of Vit-D3 (Curve b). 3.2 Design strategy for vitamin D3 sensor Scheme 1 describes the design strategy for the aptamer/fullerene fluorescent biosensor. The π-π interactions between DNA bases and fullerene ensure that FAM is in close proximity to the fullerene surface, facilitating efficient fluorescence quenching. This strong interaction between the aptamer and fullerene brings the fluorophores closer to the fullerene, enabling efficient energy transfer between the dyes and fullerene [47, [ 47 ]. In the presence of Vit-D3, the aptamer's affinity for Vit-D3 causes it to mainly bind to the target molecule, and their specific recognition can resist the adsorption of fullerene, resulting in increased fluorescence intensity[ 48 ]. The assay allowed for increasing the fluorescence value of the Vit-D3 concentration. By detecting the fluorescence signal varies, we can quantitatively determine the concentration of the Vit-D3. In addition, the exonuclease I[ 49 ] assisted amplification catalysis of the Vit-D3-ssDNA complex and releases the target Vit-D3 that could bind to another FAM-labeled ssDNA[ 50 ]. In the classic experimental protocol, each target molecule binds to only a single aptamer[ 51 ], inhibiting signal enhancement and limiting the sensitivity of detection. The sensitivity can be further enhanced by introducing an exonuclease I-catalyzed amplification strategy. When exposed to Vit-D3, the aptamers exhibit a higher affinity towards vitamin D3 and dissociate from the surface of fullerene. Subsequently, the exonuclease I cleaves the Vitamin D3-aptamer complex, resulting in the release of the fluorophore and ultimately liberating Vitamin D3. The released vitamin D3, which then binds to another aptamer on the fullerene, causes the fullerene to continuously release the dye-labeled aptamer, which results in a significant amplification of the signal. This process enables the recovery of the vitamin D3 (Vit-D3) analyte, which can then freely form complexes with other aptamer. Consequently, a small amount of vitamin D3 (Vit-D3) can effectively induce the release of a large quantity of single-stranded DNA (ssDNA) from the surface of the fullerene[ 52 ], this consequently leads to fluorescence amplification. These changes in the fluorescence intensity of the ssDNA/fullerene constitute the basis for the detection of Vit-D3 proposed herein[ 53 ]. 3.3 Optimization for vitamin D3 detection conditions The experimental parameters influencing the sensing process were relevant assessed. First, the pH of the Tris–HCl buffer solution was optimized. To examine the effect of pH, the maximum fluorescence peak intensity was measured in the pH range of 6.0–8.0 (Fig. 3 (a)). At pH 6.0, the FAM molecules gradually become protonated, reducing the conjugation effect within the molecules and decreasing both the light absorption and emission efficiency of the fluorophore, which led to a decrease in fluorescence intensity. Significant changes in fluorescence intensity were observed in the pH range of 6.5 to 8.0. A pH of 7.0 was selected for further experiments, as it produced the maximum vitamin D3-induced fluorescence reaction. Second, the incubation time for vitamin D3–aptamer binding was tested over a range of 10 to 50 minutes (Fig. 3 (b)). The fluorescence intensity increased during the early stages of the reaction, reaching a maximum at 50 minutes, after which the fluorescence intensity plateaued. This phenomenon is probably a result of the aptamer and vitamin D3 hybridizing to the point of equilibrium and saturation. To achieve optimal results, an incubation time of 50 minutes was selected and used in all subsequent experiments. The quenching amount was observed by mixing the fam aptamer with fullerene and then vortexizing. The quenching of fluorophore is proportional to the increase of fullerene concentration. As shown in Figure S1 , we inferred that the quenching rate exceeded 76% when the dosage of fullerenes was 2.4µg/mL. Therefore, 2.4µg/mL was chosen as the optimal concentration for the quenching study. The quenching time of fullerene was investigated (Fig. S2). When fullerene were added for 5 minutes, a rapid attenuation of fluorescence was observed, and a stable fluorescence was achieved after 25 minutes. Consequently, 25 minutes was chosen as the incubation time for the quenching of fullerene. To improve the sensitivity of the detection system, an exonuclease I-based signal amplification approach was explored. Fluorescence emission was recorded at 520 nm with excitation at 485 nm, using different concentrations of exonuclease I ranging from 0 to 50 U. As depicted in Fig. 3 (c), the fluorescence intensity increased notably with the concentration of exonuclease I, leveling off at concentrations of 50 U. At the same time, in order to save costs, 45 U of exonuclease I was selected for use in all subsequent experiments. 3.4 Detection of Vitamin D3 The assay involved combining a FAM-tagged aptamer with fullerene to create an aptamer/fullerene complex. Subsequently, Vitamin D3 (Vit-D3) and exonuclease I (45U) were added together, and the sample was incubated for 50 minutes prior to fluorescence measurement. This setup was used to assess the aptamer/fullerene complex's ability to detect Vit-D3 quantitatively. Figure 4 illustrates that the fluorescence intensity of the complex increased with Vit-D3 concentrations from 0 to 1000.0 µM. The intensity began to rise with the mere addition of 0.05 µM Vit-D3, and it continued to climb with higher Vit-D3 concentrations. The inset of Fig. 4 depicts a strong linear correlation between fluorescence intensity at the maximum emission wavelength (520 nm) and Vit-D3 concentration within the range of 0-0.6µM (R² = 0.994). The fluorescence intensity reached a plateau at 1000 µM Vit-D3, suggesting saturation. The limit of detection (LOD) was calculated as 3σ / k, with σ being the standard deviation of the blank measurements (n = 3) and k the slope of the calibration curve. The calculated detection limit is 49 nM, and the actual measured value is 50nM, which is four times lower than the traditional unamplified strategy. 3.5 The selectivity of the aptasensor To test the selectivity of the sensor, various common vitamins and their mixtures with vitamin D3 (vitamin B1, vitamin B3, vitamin B5, vitamin C, vitamin B6, vitamin M, and vitamin B12, as well as a mixture of all the above vitamins) were detected according to the experimental procedure described in Section 2.5 . As shown in Fig. 5 (a), except for vitamin D3, none of the other vitamins caused significant changes in the fluorescence signal. Moreover, as shown in Fig. 5 (b), when these common vitamins were mixed with vitamin D3, the fluorescence recovery intensity remained essentially the same as that of vitamin D3 alone. The fluorescence difference was calculated as: FL intensity (aptamer/fullerene + vitamins) - FL intensity (aptamer/fullerene). The results demonstrate that the proposed fluorescent method can detect vitamin D3 with high specificity and strong anti-interference ability. 3.6 Repeatability and stability To evaluate the reproducibility, six independent batches of aptamer sensors were adopted to detect 1µM of Vit-D3 (Fig. 5 (c)). The relative standard deviations (RSD) was 3.27%. The above results implied that the reproducibility of the aptasensor was desirable. In addition, we also studied the stability of the constructed aptasensord (Fig. 5 (d)). Store for 6 days in a dark, temperature-controlled environment at 4°C, the fluorescence intensity at 520 nm was still 90%, further demonstrating its excellent stability. 3.7 Analytical application The aptamer sensor was used to detect vitamin D3 in water samples, and its potential application value was investigated. The sample is pretreated according to the procedure outlined in Section 2.6 . As shown in Table 1 , the recovery rates of vitamin D3 in municipal water ranged from 88.4–96.3%, and the relative standard deviations (RSDs) for vitamin D3 were 1.3–8.1% (n = 3). The recoveries and RSDs assays indicate the feasibility of the proposed method for real sample analysis. These results confirm that the sensing system can reliably determine vitamin D3 in real water samples, it is expected to be an alternative method for vitamin D3 testing. In comparison to other vitamin D3 aptasensors, our approach provides a similar limit of detection (LOD) and an excellent detection range (see Table 2 ), positioning it as a promising method for rapid vitamin D3 detection. The ligand sensor we developed is highly sensitive, stable, and easier to operate than other detection methods. It utilizes cost-effective materials, offering greater potential for developing simpler and more affordable sensors. The low production cost and ease of manufacturing make it ideal for simple and efficient vitamin D3 detection in water. This method is easy to use and does not require complex processing steps. It allows for the quantitative analysis of vitamin D3 in real samples, achieving high specificity due to the involvement of vitamin D3 aptamers. Detection methods for fluorescent aptamers have rarely been reported in the field of vitamin D3 detection, so this work is innovative in the field. 4. Conclusions Detecting vitamin D through biosensors remains a challenge. In summary, we present the first ssDNA assay system for the detection of vitamin D3 with fullerene, and the first to achieve the detection of vitamin D3 in water samples. This method relies on Vit-D3-triggered release of single-labeled fluorescent aptamer from fullerene, coupled with exonuclease I-catalyzed Vit-D3 recycling. This single-labeled fluorescent oligonucleotide probe enables vitamin D detection in buffer with a simple mixing step, without requiring complex procedures. The detection platform showed excellent specificity and was able to eliminate interference from multiple other vitamins. The probe is easy to design, offers high detection efficiency, and has great potential for detecting other targets. We expect that this strategy may provide greater application potential for the construction of subsequent sensing platforms. Declarations Author Contribution Xinqiu Xu: software, writing – original draft, validation, visualization. Chaofan Jia , Fengjiao Zhang: Methodology; software. Weilei Gong , Changqin Wang: Data curation; formal analysis. Hao Li: Project administration. Yin Wei: conceptualization, writing – review and editing, funding acquisition, resources. Data Availability Data can be obtained from the first author References Nandakumar M, Das P, Sathyapalan T, Butler AE, Atkin SL (2024) A Cross-Sectional Exploratory Study of Cardiovascular Risk Biomarkers in Non-Obese Women with and without Polycystic Ovary Syndrome: Association with Vitamin D. Int J Mol Sci Prante M, Schüling T, Roth B, Bremer K, Walter J (2019) Characterization of an Aptamer Directed against 25-Hydroxyvitamin D for the Development of a Competitive Aptamer-Based Assay. Biosensors 9:134. https://doi.org/10.3390/bios9040134 Chau Y-Y, Kumar J (2012) Vitamin D in Chronic Kidney Disease. 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Bioelectron Pang X, Liu W, Zheng Z, Zheng X, Wang J, Wang Q, Niu L, Gao F (2024) Hybridization-driven synchronous regeneration of biosensing interfaces for Listeria monocytogenes based on recognition of fullerol to single- and double-stranded DNA. Food Chem 461:140906. https://doi.org/10.1016/j.foodchem.2024.140906 Prante M, Schüling T, Roth B, Bremer K, Walter J (2019) Characterization of an Aptamer Directed against 25-Hydroxyvitamin D for the Development of a Competitive Aptamer-Based Assay. Biosensors 9:134. https://doi.org/10.3390/bios9040134 Gupta R, Kaul S, Singh V, Kumar S, Singhal NK (2021) Graphene oxide and fluorescent aptamer based novel biosensor for detection of 25-hydroxyvitamin D3. Sci Rep 11:23456. https://doi.org/10.1038/s41598-021-02837-4 Hong SK, Lee JH, Ko WB (2011) Synthesis of [60]Fullerene-ZnO Nanocomposite Under Electric Furnace and Photocatalytic Degradation of Organic Dyes. 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Bioorg Med Chem 4:767–779. https://doi.org/10.1016/0968-0896(96)00081-8 Liu D, Kowashi S, Nakamuro T, Lungerich D, Yamanouchi K, Harano K, Nakamura E (2022) Ionization and electron excitation of C 60 in a carbon nanotube: A variable temperature/voltage transmission electron microscopic study, Proc. Natl. Acad. Sci. 119 e2200290119. https://doi.org/10.1073/pnas.2200290119 Shirai Y, Guerrero JM, Sasaki T, He T, Ding H, Vives G, Yu B-C, Cheng L, Flatt AK, Taylor PG, Gao Y, Tour JM (2009) Fullerene/Thiol-Terminated Molecules J Org Chem 74:7885–7897. https://doi.org/10.1021/jo901701j Diacon A, Krupka O, Hudhomme P (2022) Fullerene-Perylenediimide (C60-PDI) Based Systems: An Overview and Synthesis of a Versatile Platform for Their Anchor Engineering. Molecules 27:6522. https://doi.org/10.3390/molecules27196522 Bruno JG, Carrillo MP, Phillips T, Edge A (2012) Serum inverts and improves the fluorescence response of an aptamer beacon to various vitamin D analytes. Luminescence 27:51–58. https://doi.org/10.1002/bio.1324 Wei Y, Li B, Wang X, Duan Y (2014) A nano-graphite–DNA hybrid sensor for magnified fluorescent detection of mercury(ii) ions in aqueous solution. Analyst 139:1618. https://doi.org/10.1039/c3an01482g Shetti NP, Mishra A, Basu S, Aminabhavi TM (2021) Versatile fullerenes as sensor materials. Mater Today Chem 20:100454. https://doi.org/10.1016/j.mtchem.2021.100454 Lan Y (2020) Exonuclease I-assisted fluorescence aptasensor for tetrodotoxin. Ecotoxicol Environ Saf Fan Y, Amin K, Jing W, Lyu B, Wang S, Fu H, Yu H, Yang H, Li J (2024) A novel Recjf Exo signal amplification strategy based on bioinformatics-assisted truncated aptamer for efficient fluorescence detection of AFB1. Int J Biol Macromol 254:128061. https://doi.org/10.1016/j.ijbiomac.2023.128061 Liao Y, Zhang N, Chai D, Liu B, Li J, Fang Y, Zhang D, Liu R, Li Z (2023) Rational design of a ratiometric fluorescent aptasensor for patulin in traditional Chinese Cite this: DOI: 10.1039/d3an00923h medicine through the studies of the interaction mechanism between its DNA aptamer and the target molecule Wang J, Li Z, Li S, Qi W, Liu P, Liu F, Ye Y, Wu L, Wang L, Wu W (2013) Adsorption of Cu(II) on Oxidized Multi-Walled Carbon Nanotubes in the Presence of Hydroxylated and Carboxylated Fullerenes. PLoS ONE 8. e72475. https://doi.org/10.1371/journal.pone.0072475 Wei Y (2015) Amplified fluorescent aptasensor through catalytic recycling for highly sensitive detection of ochratoxin A, Biosens. Bioelectron Men K, Chen Y, Liu J, Wei D (2017) Electrochemical Detection of Vitamin D2 and D3 Based on a AuPd Modified Glassy Carbon Electrode. Int J Electrochem Sci 12:9555–9564. https://doi.org/10.20964/2017.10.15 Prakasam S, Anthonysamy E, Krishnan G, Chinnathambi S (2023) Impact of boron doping on microporous carbon for enhancing the electrochemical sensitivity of vitamin D3. Mater Chem Phys 296:127353. https://doi.org/10.1016/j.matchemphys.2023.127353 Alsager OA, Alotaibi KM, Alswieleh AM, Alyamani BJ (2018) Colorimetric Aptasensor of Vitamin D3: A Novel Approach to Eliminate Residual Adhesion between Aptamers and Gold Nanoparticles. Sci Rep 8:12947. https://doi.org/10.1038/s41598-018-31221-y Tables Table 1 Measurements of Vit-D3 in municipal water samples (n = 3) Sample Added (nM) Detected(nM) Recovery (%) RSD (%) 1 400 353.45 88.36 8.08 2 500 476.08 95.22 1.365 3 600 577.60 96.27 1.297 Table 2 Comparison of different methods for the detection of vitamin D3 Detection method Linear range Limit of detection Ref. Electrochemistry 5–50 µM 0.18 µM [ 54 ] 0.5 µM-42 µM 1.45 µM [ 55 ] Colorimetric 0-1000 nM 1 nM [ 56 ] Fluorescence ------------- 4 µM [ 46 ] 0-1.25 µg/mL 0.075 µg/mL [ 37 ] 0-600 nM 50 nM This work Scheme Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Scheme.png Scheme. 1. Aptasensor and the detection mechanism for Vit-D3 Supplementarymaterial.docx Cite Share Download PDF Status: Published Journal Publication published 28 Mar, 2025 Read the published version in Journal of Fluorescence → Version 1 posted Editorial decision: Revision requested 03 Mar, 2025 Reviews received at journal 01 Mar, 2025 Reviewers agreed at journal 01 Mar, 2025 Reviewers invited by journal 24 Feb, 2025 Editor assigned by journal 14 Feb, 2025 Submission checks completed at journal 14 Feb, 2025 First submitted to journal 14 Feb, 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. 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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-6029304","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":415800526,"identity":"77b67981-a5ca-47e4-9aa2-3719ee1ec805","order_by":0,"name":"Xinqiu Xu","email":"","orcid":"","institution":"a School of Public Health, Shandong First Medical University, Shandong Academy of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xinqiu","middleName":"","lastName":"Xu","suffix":""},{"id":415800528,"identity":"0a6bfa6a-2697-4adf-a35d-67e15d28259b","order_by":1,"name":"Chaofan Jia","email":"","orcid":"","institution":"Binzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chaofan","middleName":"","lastName":"Jia","suffix":""},{"id":415800530,"identity":"536e8bf4-9ecf-41da-8738-48884e1701a6","order_by":2,"name":"Fengjiao Zhang","email":"","orcid":"","institution":"a School of Public Health, Shandong First Medical University, Shandong Academy of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Fengjiao","middleName":"","lastName":"Zhang","suffix":""},{"id":415800532,"identity":"60410b35-e248-49ec-9375-1e7741c6e3d8","order_by":3,"name":"Hao Li","email":"","orcid":"","institution":"Jining Medical College","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Li","suffix":""},{"id":415800534,"identity":"c9b01fb5-4998-465a-8eb6-f8980f59e11e","order_by":4,"name":"Weilei Gong","email":"","orcid":"","institution":"Jining Medical University","correspondingAuthor":false,"prefix":"","firstName":"Weilei","middleName":"","lastName":"Gong","suffix":""},{"id":415800535,"identity":"6ef657b9-7799-4631-b16f-707ddb73fcef","order_by":5,"name":"Changqin Wang","email":"","orcid":"","institution":"Jining Medical University","correspondingAuthor":false,"prefix":"","firstName":"Changqin","middleName":"","lastName":"Wang","suffix":""},{"id":415800537,"identity":"c7f1e91e-4f29-4e1a-a769-b2cde584c5eb","order_by":6,"name":"Yin Wei","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYFACxgaGBAYJOX5m5gMHPvwgXouFsWR7W+LBmT3EW1WRuOHMGePDHGxEqDU43twm8XCHBGPDjZwPhxl4GOT5xQ4Q0HLmYJtE4hkJZsYZuRsOF1gwGM6cnYBfi9mNRKCWNgk2Zgmglhk8DAkGtwlpuf8QrIWHTSLnwWEeNmK03GAEa5Hg4TnDQJwW+zOJzRZALQYS7G0GwECWIOwXyfbjD2/+bKur33+Y+fGHDz9s5PmlCWgBAhYJJI4ETmXIgPkDUcpGwSgYBaNg5AIAw1hIkfqzD5IAAAAASUVORK5CYII=","orcid":"","institution":"Jining Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yin","middleName":"","lastName":"Wei","suffix":""}],"badges":[],"createdAt":"2025-02-14 09:38:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6029304/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6029304/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10895-025-04282-2","type":"published","date":"2025-03-28T15:57:48+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":76464032,"identity":"1c989bef-1bac-4222-9a6f-0834442c0583","added_by":"auto","created_at":"2025-02-17 12:07:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":626504,"visible":true,"origin":"","legend":"\u003cp\u003e(a) TEM image of fullerene\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(b) FT-IR spectra of fullerene\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6029304/v1/1c0eca50416869851f543c18.png"},{"id":76465212,"identity":"4471c268-28bc-41b2-b841-fe2bdb4bb22f","added_by":"auto","created_at":"2025-02-17 12:15:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":164974,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The fluorescence signal of 2nM FAM-aptamer under different solutions (pH: 7.0): (a) FAM-labeled aptamer+fullerene (b) FAM-labeled aptamer+fullerene+500nM Vit-D3 (c) FAM-labeled aptamer+fullerene+500 nM Vit-D3+45U Exonuclease I (d) FAM-labeled aptamer. Fig.2. (B) (a) The absorption spectrum of fullerenes. (b) The emission spectrum of FAM-aptamer. Fullerene absorbance (UV-Vis data) and fluorescence spectral data of FAM normalized processing for comparison. Y\u003csub\u003enormalized\u003c/sub\u003e=Y\u003csub\u003emax\u003c/sub\u003e−Y\u003csub\u003emin\u003c/sub\u003e/Y−Y\u003csub\u003emin.\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6029304/v1/4efbb71ef46e9b0ad1d4115b.png"},{"id":76464029,"identity":"0f7667e8-3069-47e6-a5bc-00c5f2f49c35","added_by":"auto","created_at":"2025-02-17 12:07:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":74703,"visible":true,"origin":"","legend":"\u003cp\u003eOptimization of experimental conditions. (a) The influence of different pH values of Tris-HCL buffer on the sensor. (b) Incubation time of aptamer and vitamin D3. (c) Effect of different concentrations of exonuclease I on sensor. Error bars represent means ± SD from three experiments.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6029304/v1/6460619a497f0a7f15b473c0.png"},{"id":76464030,"identity":"03e6b6ab-c33d-42d0-8168-505b30f9cc15","added_by":"auto","created_at":"2025-02-17 12:07:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":109421,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence spectra of 2 nM aptamer/fullerene in the presence of exonuclease I and\u0026nbsp; different concentrations of Vit-D3 (range from bottom to top: 0, 0.05, 0.4, 0.5, 0.6, 2, 3, 4, 6, 8, 10 μM) in the Tris–HCl buffer solution (pH: 7.0). The calibration line (R\u003csup\u003e2\u003c/sup\u003e=0.994) between fluorescence intensity at 520 nm and vitamin D3 concentration range of 0 to 0.6 μM. Error rod represent means ± SD from three experiments.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6029304/v1/0a7cd440e604eb7be5d79e5b.png"},{"id":76464034,"identity":"332bf84f-3854-4dd0-b223-690b3e96a41d","added_by":"auto","created_at":"2025-02-17 12:07:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":207664,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Specificity and interference of the sensor towards other vitamins. Difference=FL Intensity (aptamer/fullerene+vitamins)-FL Intensity (aptamer/fullerene). ([vitamin D3]: 500nM; [other vitamins]: 500 nM). Error bars represent means ± SD from three experiments. (b) Specificity of the FAM-DNA/fullerene system toward Vit-D3 and against other interfering substance. From left to right were vitamin B1, vitamin B3, vitamin B5, vitamin C, vitamin B6, vitamin M, vitamin B12, vitamin D3. The concentration of each substance was 500 nM. (c) The repeatability of the sensor for measuring 1μM of vitamin D3 continuously for 6 times. (d) Continuously measure for 6 days to obtain the stability of the sensor.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6029304/v1/a220058641f31a096e9ecd99.png"},{"id":79605422,"identity":"1b706732-9c60-46d6-a4ec-f1987df46c53","added_by":"auto","created_at":"2025-03-31 16:11:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1898776,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6029304/v1/e14ba841-5ee0-4b25-9478-44955310f525.pdf"},{"id":76464026,"identity":"9595319d-ad92-463f-ae05-f66dd6ce86b9","added_by":"auto","created_at":"2025-02-17 12:07:27","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":119353,"visible":true,"origin":"","legend":"\u003cp\u003eScheme. 1. Aptasensor and the detection mechanism for Vit-D3\u003c/p\u003e","description":"","filename":"Scheme.png","url":"https://assets-eu.researchsquare.com/files/rs-6029304/v1/c20e7da2c245df14ad8569b8.png"},{"id":76465213,"identity":"726d42b1-2b9b-40e5-a39f-af817ff44cd4","added_by":"auto","created_at":"2025-02-17 12:15:28","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":385229,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6029304/v1/2efa2045b05a4de3bea520d0.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sensitive and facile detection of vitamin D based on fluorescent labeled aptamer probe and exonuclease I-assisted signal amplification","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eVitamin D represents a crucial substance necessary for the body to sustain its overall well-being and prevent chronic illnesses[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The compound cholecalciferol, also known as vitamin D3, is a fat-soluble nutrient that can be obtained through exposure to sunlight or by consuming certain foods[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Inside the human body, cholecalciferol gets converted into the active form of vitamin D. This active form is vital in controlling the absorption and usage of calcium and phosphorus to ensure the best possible bone health[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, the measurement of Vitamin D3 levels can be regarded as a significant indicator for evaluating the quality of life in a population. The standard quantitative measuring tools for Vit-D3 levels involve high performance liquid chromatography (HPLC)[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], enzyme linked immunosorbent assay (ELISA)[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], radioimmunoassay (RIA)[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], chemiluminescent immunoassay (CLIA)[\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and high-performance liquid chromatography-mass spectrometry (HPLC-MS)[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These methods are limited to specialized laboratories, and ELISA faces challenges such as instability under harsh conditions, high production costs, and the need for sophisticated procedures. Consequently, there is a pressing demand for rapid, effective and sensitive techniques to detect Vit-D3 suitable for just-in-time diagnostic application.\u003c/p\u003e \u003cp\u003eLee et al. created a gold nanoparticle-based colorimetric aptamer sensor for detecting vitamin D, using VDBA 14 aptamers, with a detection limit of 1 \u0026micro;M[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Colorimetric methods are straightforward and do not need advanced instruments, but they often experience significant data fluctuations. Carlucci et al. showcased vitamin D detection through Surface-Enhanced Raman Spectroscopy (SRP) and electrochemical biosensors. SRP was used to directly measure vitamin D levels with a vitamin D antibody, achieving a detection limit (LOD) of 2 \u0026micro;g/ml. However, detecting Vitamin D3 was often hindered by interference from other vitamins, due to their similar oxidation potentials[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Seongjae Jo et al. developed a local surface plasmon resonance (LSPR) aptamer sensor using polyethylene glycol (PEG)-free gold nanorods (AuNRs) for large-scale, direct detection of 25-hydroxyvitamin D3[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, the scarcity of precious metals also limits their large-scale application. Khalid E. Alzahrani et al. modified a microcantilever beam with Bruno adsorbent, and the deflection and frequency of the microcantilever beam were changed by the presence of vitamin D. By measuring these changes, the concentration of vitamin D can be quantitatively analyzed, with a detection limit of about 0.3 nM[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. While the sensor demonstrates fast and sensitive detection in laboratory conditions with good performance, its effectiveness in complex biological samples, such as serum or plasma, has not been validated, making it challenging to apply in clinical trials. The fluorescent aptamer sensor detection has many advantages including high sensitivity, high selectivity, real-time monitoring ability, and applicability to complex samples[\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], which already arouse attention. Herein, this article developed a novel aptamer-based fluorescence strategy based on fullerene and FAM labeled aptamer for the detection of vitamin D3.\u003c/p\u003e \u003cp\u003eAptamers are selected single-stranded oligonucleotides of DNA or RNA sequences[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] with molecular weights ranging from 10 to 30 kDa[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Aptamers possess advantageous characteristics for their utilization in biomedical applications: Firstly, aptamers demonstrate exceptional stability across diverse environments. Secondly, in contrast to antibodies, aptamers can be manufactured at a cost-effective rate. Finally, these molecules have a long shelf life and can be regenerated after denaturation. Aptamers have a stable three-dimensional structure and are generated by an evolutionary analogy in vitro, called ligand exponential enrichment phylogenetic evolution (SELEX)[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. It has high affinity and specificity to the target and becomes the best substitute to antibodies[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. It can bind to various target molecules and undergo adaptive conformational changes, driven by hydrophobic interactions, van der Waals forces, and shape complementarity in the intermolecular interactions with the target. Nucleic acid aptamer is a kind of nucleic acid aptamer developed in recent years for the detection of biological matter level, which is used in fluorescence, electrochemistry, colorimetry, electrochemical luminescence and other analytical methods[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The field of biosensors has witnessed a significant research focus on the incorporation of fluorescent substances into nucleic acid aptamers, resulting in the development of highly notable fluorescent aptamer sensors.\u003c/p\u003e \u003cp\u003eRecently, a variety of nanomaterials, such as Graphene and its derivatives, carbon nanotubes, polymer nanoparticles, metal nanoparticles and silica nanoparticles have been used to exploit new types of analytical sensor tools[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Composed of 60 sp2 hybrid carbon atoms, fullerenes (or C60) are truncated icosahedral spherical molecules arranged into systematic hexagons and pentagons to form a closed hollow cage, similar to a football[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The three-dimensional highly delocalized electron conjugated molecular structure gives C60 excellent optical properties, which is expected to be applied in optical signal processing and control. The nanoscale and abundant oxygen-containing functional groups of fullerene enable it to form strong non-covalent interactions with DNA probes, thus significantly improving the immobilization efficiency of DNA probes. Consequently, the aptamer can be bound by fullerene and subsequently lead to fluorescence quenching[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. At present, several studies have demonstrated fullerene have been successfully applied to aptamer fluorescence biosensors, highlighting its excellent performance in biomolecule recognition and detection. However, no research has focused on constructing an aptasensor for the detection of Vitamin D3 using fluorescent probes. As far as we know, there is still a lack of research on the detection of vitamin D3 using fullerenes at present.\u003c/p\u003e \u003cp\u003eIn this study, fullerene is used as quenching agent and an aptamer as recognition element for vitamin D detection, which is the first application of fullerene in vitamin D detection. Fullerene is a π-rich nanocarbon that effectively quenches the fluorescence of FAM-DNA through energy transfer and F\u0026ouml;rster resonance energy transfer, making it an ideal quencher[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. A DNA sequence that specifically binds vitamin D was selected as the aptamer. When the target vitamin D3 is present, FAM-DNA binds to it to form an atypical B-type DNA structure that cannot be attached to the fullerenes[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], and the fluorescence of aptamer would not be quenched[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Therefore, the quantification of vitamin D can be evaluated according to the fluorescence response of the detection method. In addition, this study not only provides an effective sensing approach of fullerene fluorescence analysis, but also broadens a new application for clinical vitamin D level detection.\u003c/p\u003e"},{"header":"2. Experimental section","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials and chemicals\u003c/h2\u003e \u003cp\u003eVitamin D3 aptamer was synthesized from Shanghai Sangon Biotechnology Co., LTD. (Shanghai, China). The oligonucleotide sequence is as follows: 5\u0026prime;-AGCAG CACAG AGGTC ATGGG GGGTG TGACT TTGGT GTGCC TATGCG TGCTA CGGAA-3\u0026prime;, which was selected by Lee, Nguyen, and Gu[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The fullerene aqueous dispersion was purchased from Suzhou Tanfeng Materials Tech Co. Ltd. (Jiangsu, China). Exonuclease I and its 10 \u0026times; reaction buffer (670 mM glycine-KOH (pH 9.5, 25\u0026deg;C) with 67 mM MgCl2 and 10 mM DTT) were purchased from Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). Vitamin D3 was obtained from Durai Biology Co. Ltd (Nanjing China). Tris-HCL was obtained from Phygene Biotechnology Co. Ltd (Fuzhou, China). Other vitamins were purchased from Qiansheng Biotechnology Co. Ltd (Anhui, China). All other chemicals were of analytically grade and used without any further purification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Instrumentation\u003c/h2\u003e \u003cp\u003eDrops of fullerene dispersion are placed on a specific copper net and naturally dried in a clean environment to prepare samples for transmission electron microscopy (TEM) measurements. Transmission electron microscopy (TEM) observation was measured using a FEI Tecnai G2 12 instrument (FEI, USA). The functional groups of fullerenes were measured by Nicolet IS10 (Nicolet USA) infrared spectroscopic instrument. The nucleic acid sequences were centrifuged by High Speed Refrigerated Centrifuge (Thermo Fisher, Shanghai, China) before dissolved by EDTA to prepare reserve solution. Puxi TU-1901 spectrophotometer was employed to test the UV\u0026ndash;Vis absorbance spectrum. Fluorescence intensities were recorded on fluorescence spectrophotometer FL-2700 (Hitachi Hi-Tech Science Nako Co. LTD. Japan). The fluorescence was monitored at 520 nm with an excitation at 485 nm and slit widths of excitation and emission were set at 5 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Optimization of the detection conditions\u003c/h2\u003e \u003cp\u003eIn order to obtain the best performance sensor, the following parameters are optimized: (a) fullerenes in a concentration range of 0\u0026ndash;3.0 \u0026micro;g/ml; (b) The quenching time of fullerenes on aptamer fluorescence is 0\u0026ndash;40 minutes; (c) Incubation time range of aptamer and target was 0 min-50 min; (d) The influence of different pH values of Tris-HCL buffer 6.0\u0026ndash;8.0 on the sensor; (e) Influence of exonuclease I concentration range 5U-60U on the sensor.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Construction of a fluorescence signal sensor\u003c/h2\u003e \u003cp\u003eIn a typical vitamin D3 assay, a 59 \u0026micro;L aptamer reserve fluid (100\u0026micro;M) is first swirled for 1 minute, thoroughly mixed, and then used. A working solution containing FAM labeled aptamers was prepared by diluting the reserve solution to a 2nM concentration using a 10mM Tris-HCl buffer (pH 7.0). The fullerene suspension should then be sufficiently shaken to ensure uniform dispersion by adding 40\u0026micro;L fullerene suspension (about 2.4\u0026micro;g/mL) to the working solution. After 25 minutes, the target Vitamin D3 at an appropriate concentration and Exonuclease I (45 U) were added simultaneously. In order to promote the dissolution of vitamin D3, we added an appropriate amount of ethanol to prepare a reaction mixture with a total volume of 5 mL. The mixture was then incubated for 50 minutes at room temperature. Fluorescence measurements were recorded using the FL-2700 fluorescence spectrophotometer (Hitachi Hi-Tech Science Nako Co. Ltd., Japan) at 520 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Specificity of the assay\u003c/h2\u003e \u003cp\u003eTo further confirm the selectivity and specificity of this sensor, seven different substances were investigated with FAM-DNA/fullerene at a final concentration of 500 nM each under the optimal experimental conditions, including vitamin M, vitamin B1, vitamin B3, vitamin B5, vitamin B6, vitamin B12, and vitamin C. Three control experiments were also conducted to assess the specificity of the assay. Vitamin D3 at a concentration of 500 nM was used as a positive control. The emission spectra of the resultant mixtures were scanned from 510 nm to 660 nm with an excitation at 485 nm, the fluorescence recovery intensity was used to evaluate the Vit-D3 sensing performance of this method. Meanwhile, the sensors were prepared with 6\u0026micro;M of vitamin D3 and stored at 4\u0026deg;C away from light for 6 days, with the fluorescence intensity measured continuously to evaluate the stability of the sensor. And multiple measurements were taken to ensure the reliability of the results, and vitamin D3 at a concentration of 1\u0026micro;M was subjected to six independent experiments (n\u0026thinsp;=\u0026thinsp;6) to assess reproducibility.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Detection of the actual samples\u003c/h2\u003e \u003cp\u003eTo further verify the practical applicability and reliability of our biosensors, the sensing platform was applied to real water samples. Tap water was collected directly from the water system at the School of Public Health, Jining Medical College. After allowing a consistent flow for at least 1 minute, the tap water was collected in a clean 10-mL colorimetric tube. Before detection, the collected water samples were filtered through a 0.22\u0026micro;m filter to remove precipitation and impurities, and then mixed with Tris-HCl buffer at a 1:1 ratio and adjust the pH to 7.0. A fixed concentration of FAM-labeled ssDNA, fullerene and exonuclease I were added, followed by the addition of 400 nM, 500 nM, and 600 nM of vitamin D3 for detection.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 The feasibility of the aptasensor\u003c/h2\u003e \u003cp\u003eThe Transmission Electron Microscopy (TEM) image of fullerene sample displayed a well-defined spherical shape of the fullerene nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(a)). The individual fullerene particles are observed to be uniform in size, with diameters typically in the range of 14 nm to 26 nm[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Fourier Transform Infrared (FT-IR) spectra of fullerenes are shown Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(b), the characteristic peak at 573 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e can be attributed to a absorption band of the C-C stretching vibration pattern[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The peaks at 1181 and 1427 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e can be attributed to the double bond stretching vibration of C\u0026thinsp;=\u0026thinsp;C[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The peak at approximately 3432 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e could be confirmed the presence of OH stretching vibrations characteristic absorption bands[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrior to the detection of Vit-D3, we validated the feasibility of using fullerene-aptamer hybrids as sensing. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (A) shows how the degree of fluorescence bursting changes under different conditions in a Tris-HCl buffer solution. The aptamer exhibited strong fluorescence signal at 520 nm (curve c), in the presence of fullerene, the significant fluorescence quenching (curve a). This observed quenching phenomenon is likely to be aptamer stacked on fullerene due to the fluorescence resonance energy transfer effect. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (B) shows when presented with excited light at 485 nm, the aptamer fluoresces at an emission maximum of 520 nm. Meanwhile fullerene have an absorption at 520 nm, the absorption spectrum overlaps with aptamer emission, it is in resonance with the excited state of aptamer. Hence, the fluorescence signal of aptamer can be quenched by FRET. In the pioneering work of Anton W. Jensen and Liu D et al., the negatively charged luciferine and positively charged fullerene molecules are brought closer together due to electrostatic interaction, which increases the electronic quenching of the excited state of luciferine and transfers energy to the fullerene[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Fullerene molecules is a kind of π-rich nanocarbon and have low-energy non-bonding orbitals (LUMO), which allow them to readily accept electrons[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The singlet excited state of the dye transfers energy to the fullerene, leaving the fullerene in an excited state while the dye returns to the ground state. Meanwhile, the singlet excited state of the dye transfers electrons to the fullerenes, causing them to form radical anions and the dye to form radical cations[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. A strong π-π interaction may exist between FAM-DNA and the football-shaped fullerene, enabling the ssDNA to adsorb to the surface of fullerene, thereby quenching the fluorescence of FAM-DNA. Following the addition of Vit-D3 (curve b), fluorescence signal enhancement is observed. The aptamer's strong affinity for vitamin D3 promotes a dominant ring structure and a smaller hairpin conformation in the probe, leading to the dissociation of the complex from the fullerene surface and hindering energy or electron transfer between FAM and fullerenes. The fundamental distinction arises due to the aptamer's ability to sufficiently unwind, exposing its bases, whereas the vitamin D3-aptamer complex maintains a stable atypical B structure that hinders the necessary unwinding for base exposure[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In the presence of Vit-D3 and 45 U exonuclease I (Curve d), the amplification signal using exonuclease I was greater than that of the 1:1 binding strategy using the same concentration of Vit-D3 (Curve b).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Design strategy for vitamin D3 sensor\u003c/h2\u003e \u003cp\u003eScheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e describes the design strategy for the aptamer/fullerene fluorescent biosensor. The π-π interactions between DNA bases and fullerene ensure that FAM is in close proximity to the fullerene surface, facilitating efficient fluorescence quenching. This strong interaction between the aptamer and fullerene brings the fluorophores closer to the fullerene, enabling efficient energy transfer between the dyes and fullerene\u003csup\u003e[47,\u003c/sup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In the presence of Vit-D3, the aptamer's affinity for Vit-D3 causes it to mainly bind to the target molecule, and their specific recognition can resist the adsorption of fullerene, resulting in increased fluorescence intensity[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The assay allowed for increasing the fluorescence value of the Vit-D3 concentration. By detecting the fluorescence signal varies, we can quantitatively determine the concentration of the Vit-D3.\u003c/p\u003e \u003cp\u003eIn addition, the exonuclease I[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] assisted amplification catalysis of the Vit-D3-ssDNA complex and releases the target Vit-D3 that could bind to another FAM-labeled ssDNA[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In the classic experimental protocol, each target molecule binds to only a single aptamer[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], inhibiting signal enhancement and limiting the sensitivity of detection. The sensitivity can be further enhanced by introducing an exonuclease I-catalyzed amplification strategy. When exposed to Vit-D3, the aptamers exhibit a higher affinity towards vitamin D3 and dissociate from the surface of fullerene. Subsequently, the exonuclease I cleaves the Vitamin D3-aptamer complex, resulting in the release of the fluorophore and ultimately liberating Vitamin D3. The released vitamin D3, which then binds to another aptamer on the fullerene, causes the fullerene to continuously release the dye-labeled aptamer, which results in a significant amplification of the signal. This process enables the recovery of the vitamin D3 (Vit-D3) analyte, which can then freely form complexes with other aptamer. Consequently, a small amount of vitamin D3 (Vit-D3) can effectively induce the release of a large quantity of single-stranded DNA (ssDNA) from the surface of the fullerene[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], this consequently leads to fluorescence amplification. These changes in the fluorescence intensity of the ssDNA/fullerene constitute the basis for the detection of Vit-D3 proposed herein[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Optimization for vitamin D3 detection conditions\u003c/h2\u003e \u003cp\u003eThe experimental parameters influencing the sensing process were relevant assessed. First, the pH of the Tris\u0026ndash;HCl buffer solution was optimized. To examine the effect of pH, the maximum fluorescence peak intensity was measured in the pH range of 6.0\u0026ndash;8.0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(a)). At pH 6.0, the FAM molecules gradually become protonated, reducing the conjugation effect within the molecules and decreasing both the light absorption and emission efficiency of the fluorophore, which led to a decrease in fluorescence intensity. Significant changes in fluorescence intensity were observed in the pH range of 6.5 to 8.0. A pH of 7.0 was selected for further experiments, as it produced the maximum vitamin D3-induced fluorescence reaction. Second, the incubation time for vitamin D3\u0026ndash;aptamer binding was tested over a range of 10 to 50 minutes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(b)). The fluorescence intensity increased during the early stages of the reaction, reaching a maximum at 50 minutes, after which the fluorescence intensity plateaued. This phenomenon is probably a result of the aptamer and vitamin D3 hybridizing to the point of equilibrium and saturation. To achieve optimal results, an incubation time of 50 minutes was selected and used in all subsequent experiments.\u003c/p\u003e \u003cp\u003eThe quenching amount was observed by mixing the fam aptamer with fullerene and then vortexizing. The quenching of fluorophore is proportional to the increase of fullerene concentration. As shown in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, we inferred that the quenching rate exceeded 76% when the dosage of fullerenes was 2.4\u0026micro;g/mL. Therefore, 2.4\u0026micro;g/mL was chosen as the optimal concentration for the quenching study. The quenching time of fullerene was investigated (Fig. S2). When fullerene were added for 5 minutes, a rapid attenuation of fluorescence was observed, and a stable fluorescence was achieved after 25 minutes. Consequently, 25 minutes was chosen as the incubation time for the quenching of fullerene.\u003c/p\u003e \u003cp\u003eTo improve the sensitivity of the detection system, an exonuclease I-based signal amplification approach was explored. Fluorescence emission was recorded at 520 nm with excitation at 485 nm, using different concentrations of exonuclease I ranging from 0 to 50 U. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(c), the fluorescence intensity increased notably with the concentration of exonuclease I, leveling off at concentrations of 50 U. At the same time, in order to save costs, 45 U of exonuclease I was selected for use in all subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Detection of Vitamin D3\u003c/h2\u003e \u003cp\u003eThe assay involved combining a FAM-tagged aptamer with fullerene to create an aptamer/fullerene complex. Subsequently, Vitamin D3 (Vit-D3) and exonuclease I (45U) were added together, and the sample was incubated for 50 minutes prior to fluorescence measurement. This setup was used to assess the aptamer/fullerene complex's ability to detect Vit-D3 quantitatively. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e illustrates that the fluorescence intensity of the complex increased with Vit-D3 concentrations from 0 to 1000.0 \u0026micro;M. The intensity began to rise with the mere addition of 0.05 \u0026micro;M Vit-D3, and it continued to climb with higher Vit-D3 concentrations. The inset of Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e depicts a strong linear correlation between fluorescence intensity at the maximum emission wavelength (520 nm) and Vit-D3 concentration within the range of 0-0.6\u0026micro;M (R\u0026sup2; = 0.994). The fluorescence intensity reached a plateau at 1000 \u0026micro;M Vit-D3, suggesting saturation. The limit of detection (LOD) was calculated as 3σ / k, with σ being the standard deviation of the blank measurements (n\u0026thinsp;=\u0026thinsp;3) and k the slope of the calibration curve. The calculated detection limit is 49 nM, and the actual measured value is 50nM, which is four times lower than the traditional unamplified strategy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.5 The selectivity of the aptasensor\u003c/h2\u003e \u003cp\u003eTo test the selectivity of the sensor, various common vitamins and their mixtures with vitamin D3 (vitamin B1, vitamin B3, vitamin B5, vitamin C, vitamin B6, vitamin M, and vitamin B12, as well as a mixture of all the above vitamins) were detected according to the experimental procedure described in Section \u003cspan refid=\"Sec7\" class=\"InternalRef\"\u003e2.5\u003c/span\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(a), except for vitamin D3, none of the other vitamins caused significant changes in the fluorescence signal. Moreover, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(b), when these common vitamins were mixed with vitamin D3, the fluorescence recovery intensity remained essentially the same as that of vitamin D3 alone. The fluorescence difference was calculated as: FL intensity (aptamer/fullerene\u0026thinsp;+\u0026thinsp;vitamins) - FL intensity (aptamer/fullerene). The results demonstrate that the proposed fluorescent method can detect vitamin D3 with high specificity and strong anti-interference ability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Repeatability and stability\u003c/h2\u003e \u003cp\u003eTo evaluate the reproducibility, six independent batches of aptamer sensors were adopted to detect 1\u0026micro;M of Vit-D3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(c)). The relative standard deviations (RSD) was 3.27%. The above results implied that the reproducibility of the aptasensor was desirable. In addition, we also studied the stability of the constructed aptasensord (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(d)). Store for 6 days in a dark, temperature-controlled environment at 4\u0026deg;C, the fluorescence intensity at 520 nm was still 90%, further demonstrating its excellent stability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Analytical application\u003c/h2\u003e \u003cp\u003eThe aptamer sensor was used to detect vitamin D3 in water samples, and its potential application value was investigated. The sample is pretreated according to the procedure outlined in Section \u003cspan refid=\"Sec8\" class=\"InternalRef\"\u003e2.6\u003c/span\u003e. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the recovery rates of vitamin D3 in municipal water ranged from 88.4\u0026ndash;96.3%, and the relative standard deviations (RSDs) for vitamin D3 were 1.3\u0026ndash;8.1% (n\u0026thinsp;=\u0026thinsp;3). The recoveries and RSDs assays indicate the feasibility of the proposed method for real sample analysis. These results confirm that the sensing system can reliably determine vitamin D3 in real water samples, it is expected to be an alternative method for vitamin D3 testing.\u003c/p\u003e \u003cp\u003eIn comparison to other vitamin D3 aptasensors, our approach provides a similar limit of detection (LOD) and an excellent detection range (see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), positioning it as a promising method for rapid vitamin D3 detection. The ligand sensor we developed is highly sensitive, stable, and easier to operate than other detection methods. It utilizes cost-effective materials, offering greater potential for developing simpler and more affordable sensors. The low production cost and ease of manufacturing make it ideal for simple and efficient vitamin D3 detection in water. This method is easy to use and does not require complex processing steps. It allows for the quantitative analysis of vitamin D3 in real samples, achieving high specificity due to the involvement of vitamin D3 aptamers. Detection methods for fluorescent aptamers have rarely been reported in the field of vitamin D3 detection, so this work is innovative in the field.\u003c/p\u003e "},{"header":"4. Conclusions","content":"\u003cp\u003eDetecting vitamin D through biosensors remains a challenge. In summary, we present the first ssDNA assay system for the detection of vitamin D3 with fullerene, and the first to achieve the detection of vitamin D3 in water samples. This method relies on Vit-D3-triggered release of single-labeled fluorescent aptamer from fullerene, coupled with exonuclease I-catalyzed Vit-D3 recycling. This single-labeled fluorescent oligonucleotide probe enables vitamin D detection in buffer with a simple mixing step, without requiring complex procedures. The detection platform showed excellent specificity and was able to eliminate interference from multiple other vitamins. The probe is easy to design, offers high detection efficiency, and has great potential for detecting other targets. We expect that this strategy may provide greater application potential for the construction of subsequent sensing platforms.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eXinqiu Xu: software, writing \u0026ndash; original draft, validation, visualization. Chaofan Jia , Fengjiao Zhang: Methodology; software. Weilei Gong , Changqin Wang: Data curation; formal analysis. Hao Li: Project administration. Yin Wei: conceptualization, writing \u0026ndash; review and editing, funding acquisition, resources.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData can be obtained from the first author\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNandakumar M, Das P, Sathyapalan T, Butler AE, Atkin SL (2024) A Cross-Sectional Exploratory Study of Cardiovascular Risk Biomarkers in Non-Obese Women with and without Polycystic Ovary Syndrome: Association with Vitamin D. Int J Mol Sci\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrante M, Sch\u0026uuml;ling T, Roth B, Bremer K, Walter J (2019) Characterization of an Aptamer Directed against 25-Hydroxyvitamin D for the Development of a Competitive Aptamer-Based Assay. 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PLoS ONE 8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ee72475. https://doi.org/10.1371/journal.pone.0072475\u003c/span\u003e\u003cspan address=\"e72475. 10.1371/journal.pone.0072475\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei Y (2015) Amplified fluorescent aptasensor through catalytic recycling for highly sensitive detection of ochratoxin A, Biosens. Bioelectron\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMen K, Chen Y, Liu J, Wei D (2017) Electrochemical Detection of Vitamin D2 and D3 Based on a AuPd Modified Glassy Carbon Electrode. Int J Electrochem Sci 12:9555\u0026ndash;9564. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.20964/2017.10.15\u003c/span\u003e\u003cspan address=\"10.20964/2017.10.15\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrakasam S, Anthonysamy E, Krishnan G, Chinnathambi S (2023) Impact of boron doping on microporous carbon for enhancing the electrochemical sensitivity of vitamin D3. Mater Chem Phys 296:127353. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.matchemphys.2023.127353\u003c/span\u003e\u003cspan address=\"10.1016/j.matchemphys.2023.127353\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlsager OA, Alotaibi KM, Alswieleh AM, Alyamani BJ (2018) Colorimetric Aptasensor of Vitamin D3: A Novel Approach to Eliminate Residual Adhesion between Aptamers and Gold Nanoparticles. Sci Rep 8:12947. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-018-31221-y\u003c/span\u003e\u003cspan address=\"10.1038/s41598-018-31221-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\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\u003eMeasurements of Vit-D3 in municipal water samples (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAdded (nM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDetected(nM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRecovery (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRSD (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e353.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e88.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e476.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e95.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.365\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e577.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e96.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.297\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of different methods for the detection of vitamin D3\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDetection method\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLinear range\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLimit of detection\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRef.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eElectrochemistry\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026ndash;50 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.18 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5 \u0026micro;M-42 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.45 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColorimetric\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0-1000 nM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 nM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFluorescence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-------------\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0-1.25 \u0026micro;g/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.075 \u0026micro;g/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0-600 nM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50 nM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThis work\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-fluorescence","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jofl","sideBox":"Learn more about [Journal of Fluorescence](https://www.springer.com/journal/10895)","snPcode":"10895","submissionUrl":"https://submission.nature.com/new-submission/10895/3","title":"Journal of Fluorescence","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Vitamin D3, Aptamer, Fullerene, Fluorescence","lastPublishedDoi":"10.21203/rs.3.rs-6029304/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6029304/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe combination of nanomaterials and biomolecular recognition units, and the signal transduction based on fluorescence, have broad prospects in the development of small molecule optical adaptive sensors. In this study, we systematically studied a rapid and simple aptamer-based fluorescence sensor that uses fullerene as a quencher for the detection of vitamin D3. The surface of fullerene consists of a π-electron cloud, which makes it a powerful electron acceptor material capable of accepting or transferring excited electrons from fluorophores such as carboxyfluorescein (FAM). The aptamer labeled with fluorescein (5\u0026rsquo;6-FAM) is adsorbed onto the fullerene surface through hydrogen bonding and π-π stacking interactions, leading to fluorescence quenching due to F\u0026ouml;rster resonance energy transfer (FRET). However, in the presence of vitamin D3, it can specifically bind to FAM-ssDNA, forming a vitamin D3-aptamer-hairpin structure that cannot be adsorbed onto the fullerene surface. Under the optimal experimental conditions, the linear detection range for vitamin D3 was 0\u0026ndash;600 nM, with a detection limit of 200 nM. When exonuclease I was used, the detection limit was improved to 50 nM. Furthermore, the recovery rate of vitamin D3 in water samples was 88.4\u0026ndash;96.3%. The feasibility of the sensor was validated by successfully detecting vitamin D3 in water samples.\u003c/p\u003e","manuscriptTitle":"Sensitive and facile detection of vitamin D based on fluorescent labeled aptamer probe and exonuclease I-assisted signal amplification","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-17 12:07:22","doi":"10.21203/rs.3.rs-6029304/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-03T13:06:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-02T04:31:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"55004728286603985641604151805256301506","date":"2025-03-02T04:13:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-02-24T13:38:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-14T11:17:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-02-14T11:17:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Fluorescence","date":"2025-02-14T09:29:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-fluorescence","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jofl","sideBox":"Learn more about [Journal of Fluorescence](https://www.springer.com/journal/10895)","snPcode":"10895","submissionUrl":"https://submission.nature.com/new-submission/10895/3","title":"Journal of Fluorescence","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c028ef07-94f4-4f08-b549-2c61c3abf6f5","owner":[],"postedDate":"February 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-31T16:08:45+00:00","versionOfRecord":{"articleIdentity":"rs-6029304","link":"https://doi.org/10.1007/s10895-025-04282-2","journal":{"identity":"journal-of-fluorescence","isVorOnly":false,"title":"Journal of Fluorescence"},"publishedOn":"2025-03-28 15:57:48","publishedOnDateReadable":"March 28th, 2025"},"versionCreatedAt":"2025-02-17 12:07:22","video":"","vorDoi":"10.1007/s10895-025-04282-2","vorDoiUrl":"https://doi.org/10.1007/s10895-025-04282-2","workflowStages":[]},"version":"v1","identity":"rs-6029304","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6029304","identity":"rs-6029304","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}