Enhancing the UV Radiation Protection of Bacillus thuringiensis Formulation using Sulfur Quantum Dots: A Biotechnological Approach

Enhancing the UV Radiation Protection of Bacillus thuringiensis Formulation using Sulfur Quantum Dots: A Biotechnological Approach | 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 Enhancing the UV Radiation Protection of Bacillus thuringiensis Formulation using Sulfur Quantum Dots: A Biotechnological Approach Elham Jalali, Shahab Maghsoudi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4009872/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Low stability against ultraviolet (UV) radiation is one of the drawbacks of biological pesticides such as Bacillus thuringiensis (Bt). The persistence of Bt crystals against insect pests is thus deactivated. Bt plays a key role in the control of microbial pests. In this study, Bt spores and crystals were protected from UV radiation by sulfur quantum dots (SQDs). These were synthesized by treating sublimated sulfur powders with an alkali using polyethylene glycol 400 (PEG-400). Their effect on the formulation of Bt was investigated to improve its resistance to UV radiation. Excellent aqueous dispersibility and superior photostability were observed for the synthesized SQDs. Properly dispersed SQDs with mean size distributions of 3.27 nm and 6.07 nm were obtained for 120 and 72 h, respectively. The findings indicate that SQDs perform very well in encapsulated formulations prepared by the Pickering emulsion method compared to non-encapsulated formulations. Spore viability and mortality of second-instar Ephestia kuehniella larvae under UV-A radiation were studied. The unique properties of SQDs are believed to reduce the degradation of Bt against UV radiation. Our results showed that these SQDs can be used to improve the stability and resistance of Bt in SQD-stabilized microcapsule formulations. UV resistance Quantum dots Microcapsule Pickering emulsion Spore viability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction A microbial insecticide based on bacteria, Bacillus thuringiensis (Bt) is one of the most widely used pest control products in agriculture, and forestry because of its safety for humans, and the environment. Insecticidal crystal proteins by Bt are toxic to thousands of insect species including Lepidoptera, Diptera, Coleoptera, Hemiptera, Nematoda, and others. Bt persistence is a critical factor in determining the effectiveness of this product as a pest control agent(Federici, 2022 ; Ortiz & Sansinenea, 2023 ). When exposed to ultraviolet radiation in sunlight, the spores and crystals of Bt are susceptible to degradation. Consequently, its persistence is shortened, resulting in a reduction in its activity, and therefore its use as a pesticide is limited(Lahlali et al., 2022 ). A major objective of most studies based on formulations containing Bt is to increase their effectiveness and persistence in the field. Additionally, the new formulations are more persistent, which enables changes in crop management, for example, reducing the frequency of applications(Alkassab, Beims, Janke, & Pistorius, 2022 ). In the last few years, encapsulation has increasingly attracted attention due to the potential for controlling the release rate of products and extending their residual effect(de Oliveira et al., 2022 ). Pickering emulsions are stabilized by solid particles, as opposed to conventional surfactant-stabilized emulsions(Yaakov et al., 2022 ). If particles have proper partial dual wettability, they will accumulate in the oil-water interface spontaneously, and particles will likely be embedded in the continuous phase on the side of the interface that has an oil-water interface. So, the adsorbed particle layer functions as an extremely effective steric barrier to prevent emulsion droplets from coalescing, which differs fundamentally from surfactant molecules that stabilize them(C. Wang et al., 2022 ). These Pickering emulsions result in improved properties of the fabricated materials by providing excellent templates for different morphologies, for example, solid/porous spheres, and hollow capsules(T. Zhang, Liu, Wu, & Ngai, 2022 ). Usually, polymerization is used in the process to preserve the structures after the emulsion templates are removed. When the emulsion template is removed after polymerization, the particles remain at the oil-water interface, functionalizing the surface of the resulting materials and providing the composites with novel properties(Mao et al., 2022 ; Zhu, Lei, Li, & Zhu, 2018 ). Nanoscale systems have several advantages because their small size gives them better dispersion, plus their large surface area makes them more attractive to targets(Rajput, Sevalkar, Pardeshi, & Pingale, 2023 ). Utilizing quantum dots (QDs) in Bt formulations can help overcome these limitations and allow for commercial production. QDs are promising materials due to their unique quantum confinement properties, which are coupled with optical properties(Maholiya et al., 2023 ). A new class of zero-dimensional luminescent material free of metal ions is sulfur quantum dots (SQDs), which exhibit bright photoluminescence (PL), as well as UV-visible absorption. As a result, SQDs have been considered green nanomaterials because of their exceptional electrical and electronic properties, stable photoluminescence low toxicity, high biocompatibility, and excellent solubilities(S. Wang, Bao, Gao, & Li, 2019 ; H. Zhang et al., 2017 ). The functional groups on the surface of the SQDs provide electrons that can enhance the interaction of other compounds. However, in comparison to the exploration of pure elemental QDs, such as carbon, silicon, and phosphorus, SQDs exploration is at an early stage(Gao, Huang, Tan, Lv, & Zhou, 2022 ; C. Lu et al., 2022 ). This study tried to resolve this highly controversial issue by developing a new formulation with higher activity and longer persistence using nanotechnology. We fabricated a Pickering emulsion encapsulated by Bt and stabilized by SQD without any surfactant stabilizers. The principal objective of this study was to improve the persistence of spores and crystals to environmental stresses such as sunlight. 2. Materials and methods 2.1. Materials Ethanol (99.5%), and polyethylene glycol-400 (PEG-400) were purchased from Sigma Aldrich (Steinheim, Germany). Thiourea, sublimated sulfur powder, sodium hydroxide, copper nitrate, methyl methacrylate, methacrylic acid, sodium dodecyl sulfate, ammonium persulfate, and sodium chloride were purchased from Merck (Darmstadt, Germany). 2.2. Synthesis of the SQDs SQDs were synthesized according to the research carried out by Arshad and Palashuddin, with some modifications(Arshad & Sk, 2020 ). Briefly, 3 mL of PEG-400, 4.0 g of sodium hydroxide, and 50 mL of ultrapure DI water were mixed and then 1.4 g of sublimated sulfur powder was added and stirred at a constant temperature at 70°C for a predetermined time (100-125h). As the reaction progresses, sulfur powder gradually dissolves and its color changes over time to dark red. The prepared product was centrifuged at 3000rpm to separate reactants from sulfur QDs, which are then referred to as SQDs. 2.3. Preparation of poly (methyl methacrylate-co-methacrylic acid) [P (MMA- co -MA)] Synthesis of the P (MMA-co-MA) was performed according to our previous work(Jalali, Maghsoudi, & Noroozian, 2020b ). In brief, a 250 mL round bottom flask was filled with 50 mL DI water, 10.65 g of methyl methacrylate, 4.24 g of methacrylic acid, 0.12 g of ammonium persulfate, and 0.20 g of sodium dodecyl sulfate. After stirring at 400 rpm, the mixture was degassed using nitrogen for 30 minutes. The liquid phase was then heated to 80°C, and the process was covered with nitrogen to prevent oxygen from entering. Following 10 h of reaction time, the P (MMA-co-MA) was collected. 2.4. Preparation of Pickering emulsions stabilized by SQDs The encapsulated formulation was prepared by the Pickering emulsion method. To fabricate emulsions, SQDs particles with a 1:1 oil/ aqueous phase ratio were prepared. The oil phase was prepared from SQDs in olive oil and ethanol at a concentration of 10 v/v%. The aqueous phase was prepared by dispersing the 0.5g Bt, 2.5 mL of P (MMA-co-MA), 2.5 mL DI water, and 0.073 g NaCl using a homogenizer (Hielscher, UP400St, 20 kHz, 400 W, Germany) for 2 min. Then, the aqueous phase was added dropwise to the oil phase. The mixture was suspended with a homogenizer for 5 min. The ratio of the oil to aqueous phase was 10:4, 10:6, 10:8, and 1:1. Finally, a 1:1 oil-to-aqueous phase ratio was selected as the best ratio. 2.5. Exposure of emulsions to UV radiation Diluted encapsulated formulation with Bt and free spore (non-encapsulated formulation) were used. A 385 nm UVA tube (Philips, 15 W) was mounted 140 mm above the open Petri dishes to provide radiation for 7 days. The spore count was calculated by counting colony-forming units (CFUs). Before the determination of spore count, the volume of each sample was adjusted with sterile distilled water to compensate for evaporation losses. Three replications were performed on three separate days for all the experiments. As follows is the Equation for calculating spore viability: V spore (%) = [CFU rad ∕CFU 0 ] The CFU 0 represents the non-irradiated spore count (free or encapsulated formulation), and the CFU rad represents the irradiated spore count (free or encapsulated formulation). 2.6. Characterization The fluorescence properties were measured using a F96 Pro Fluorescent Spectrophotometer with a 10mm quartz cell. The ultraviolet-visible absorption spectra were recorded on a Specord 250 PLUS spectrophotometer (Analytik Jena, Germany). The crystalline structure of the SQDs was characterized by X-ray diffraction (XRD) using an X'pert PRO model. Cu-Ka radiation was used in the 2θ angular range of 10°-80°. Fourier transform infrared (FTIR) spectra was recorded using a Tensor 27 spectrometer (Bruker, Saarbrücken, Germany). The spectra were recorded from 4000 to 400 cm − 1 . Transmission electron microscope (TEM) images were achieved by a Philips EM208S transmission electron microscope with an accelerating voltage of 100 kV. High-resolution transmission electron microscopy (HRTEM) was performed with a FEI Tecnai G2 F20 SuperTwin with an accelerating voltage of 200 kV. Scanning electron microscopy (FE-SEM, Sigma, Zeiss) was used to study the morphology. 3. Results and Discussion 3.1. Characterization of SQDs The FT-IR spectrum of the SQDs and the polyethylene glycol is shown in Fig.1. The main peaks of PEG-400 include absorption bands at 3361 cm -1 , 2864 cm -1, and 1297 cm -1 . The absorption band at 3442 cm -1 is related to the stretching vibration of the O-H bond. C-H stretching and C-H bending vibrations are responsible for the absorption bands observed at 2934 cm -1 and 1467 cm -1 , respectively. The absorption band observed at 1342 cm -1 is related to the C=O group. The broad absorption band at 1622 cm -1 in PEG was transformed into two absorption bands at 1648 cm -1 and 1115 cm -1 in SQDs. These bands are related to the C-O-H and C-O-C stretching vibrations(H. Lu, Zhang, Li, & Gan, 2021). The absorption band at 615 cm -1 is also related to the S-S stretching vibration(Tan, An, Pan, Liu, & Hu, 2021). Weak absorption bands between 500 cm -1 and 800 cm -1 are related to the PEG structure(Kolhe & Kannan, 2003; Shameli et al., 2012). In the FT-IR spectrum of SQDs, all the main characteristic peaks of PEG were observed and no new peaks were added except for a decrease in intensity or overlapping of some peaks. Therefore, there was no chemical reaction between PEG and SQDs. According to these results, PEG is effective in discussing SQD formation mechanisms. As PEG is physically absorbed by SQDs, this absorption layer prevents self-aggregation of the particles and is therefore essential for the maintenance of SQD stability. The XRD pattern of the SQDs is shown in Fig. 2. The diffraction peaks at 18.77º, 22.22º, 23.12º, 25.37º, 31.22º and 33.72º correspond to planes (202), (220), (222), (133), (044) and (242) respectively. This indicates the possibility of the formation of a polycrystalline phase of sulfur(Xie et al., 2012). Fig. 3 shows the changes in the size and shape of the SQDs at different times and scales using TEM images. For the 72h sample, as shown in Fig.3, the synthesized SQDs are monodispersed with almost spherical morphology. This can be attributed to the electrostatic repulsion between the anionic groups on the surface of the SQDs. Therefore, it can be concluded that the S-dots in the 72h sample are monodispersed and not aggregated. Even though the particles have self-aggregated within the 96h sample, there is a large change or non-uniformity in size. As the duration increased from 100 to 120 h, the fission of the accumulated particles likely resulted in smaller quantum dots with more defined and distinct boundaries. As shown in Fig. 4., a histogram of the particle size distribution is shown for 72 h and 120 h SQDs, respectively. The particles in 72 h are mainly distributed in the range of 2.23 nm to 9.77 nm with an average size of 6.07 nm, while in 120 h they are distributed in the range of 2.09 nm to 4.41 nm with an average size of 3.27 nm. Fig. 5 shows the HRTEM image of SQDs synthesized in 120 h, confirming the uniform distribution of particles. The UV absorption spectra of the synthesized SQDs is shown in Fig. 6. The UV spectrum has been recorded in two separate areas to better show the spectral characteristics. A peak at 216 nm is observed, which is probably related to the n → σ* transfer of non-bonded sulfur electrons(Fu et al., 2020). The peak at 333 nm is also related to the direct transition of the forbidden band of S[0], indicating the presence of the oxidation reaction of S x -2 ions to S[0](Li et al., 2014; Xiao et al., 2019). The fluorescence spectrum of the SQDs is shown in Fig. (7. a). As is well known, the maximum emission wavelength of sulfur quantum dots is around 440 nm, which occurs at an excitation wavelength of 360 nm, and the blue fluorescence is characteristic of sulfur quantum dots. SQDs are transparent yellow in normal light and emit blue light in UV light at a wavelength of 360 nm. Figure (7. b) shows the emission spectrum of SQDs synthesized at different times. The quantum size effect may be responsible for this dependence of the emission spectrum on particle size as fluorescence intensity has increased with synthesis time. There has also been a slight blue shift in the emission wavelength with an increase in synthesis time. Since quantum dots have size-dependent emission, from the blue shift in the emission wavelength between samples synthesized at different times, which is attributed to the quantum size effect of the quantum dots, it can be concluded that the size of the particles gradually changes as the synthesis time increases. With increasing synthesis time, the particle size of SQDs decreases and as a result, the size of SQDs can be controlled. TEM images and emission fluorescence spectra indicate that particle size changes with increasing duration. Over time, particle distribution changes from monodispersed to non-uniform. Gradually, the quantum dots' border becomes clearer and the size of SQDs decreases. It appears that during SQD formation, aggregation and fission compete with each other until a dynamic equilibrium is achieved. When PEG is added to a reaction, it physically absorbs on the surface of the sulfur, preventing particles from aggregating. The high surface energy of the particles tends to aggregate the particles as the reaction progresses(Xie et al., 2012). As a result, even the absorption layer of PEG on the surface cannot prevent quantum dots from agglomeration. In the synthesis with a duration of 72 h, a dynamic balance is established between aggregation and fission. Finally, after 96 h, the aggregation effect is weakened and the fission effect plays the main role, leading to an increasingly clear view of SQDs in TEM images. Fig. (8. a) shows an image of Bt microcapsule particles composed of polymer latex particles and SQDs examined under a 40X microscope. FESEM images of P (MMA-co-MA) particles are shown in Figure 8. b. The particles are hollow and the aggregates are stabilizing the microcapsule. 3.2. Assessing the UV stability of SQD-based microcapsule formulations 3.2.1. Evaluation of spore viability Figure (9) shows the effect of UV exposure time on the percentage of spore viability of different Bt formulations with SQDs up to 120 h. It can be seen that the percentage of spore viability for the microcapsule formulation stabilized with SQDs is declining at a gentler rate. It has a slightly decreasing slope up to 72 h and then a steeper slope. While the percentage of spore viability in the nanoformulation of SQDs (non-microcapsules) decreases with a greater slope than in the unirradiated condition up to 48 h, and after 72 h the difference is less compared to the formulation of unprotected Bt (as a control). This remarkable difference between the spore viability percentages of the formulations shows that UV radiation has the least effect on the spores in the microcapsule formulation with SQDs. This indicates that the spores are adequately covered by the SQDs and P (MMA-co-MA) particles. Consequently, the microcapsule formulation has better UV performance than the nanoformulation of SQDs (non-microcapsule). Table 1 shows spore viability and mortality due to insecticidal activity of different formulations of SQDs on second instar Ephestia kuehniella larvae after 96h UV exposure. The classification showed that after 96 h of UV exposure, there was a significant difference between the two different formulations of Bt with SQDs and the unprotected Bt formulation. It was found that all three formulations not exposed to UV were in the same category. This means that the difference between them is not significant (p-value=1.000). The spore viability percentages for unprotected Bt, the nano formulation of Bt with SQDs, and the microcapsule formulation of Bt with SQDs were changed to 31.25%, 33.74%, and 57.77%, respectively. As a result, Bt microcapsules formulated with SQDs have the lowest percentage decrease in spore viability. The good coverage of spores in the microcapsule formulation with SQDs is demonstrated by the sharp drop in spore viability percentage in the unprotected Bt formulation (as a control). As the most stable formulation against UV radiation, the microcapsule formulation was chosen. Table 1. Mean ± (standard error) percentage of spore viability and percentage of mortality due to the insecticidal activity of different formulations of SQDs on second instar Ephestia kuehniella larvae after 96 h UV exposure. Formulations Spore Viability% Mortality% Non-irradiated free spore 100 ±0.00 d 100 ±0.00 d Irradiated free spore 31.25 ±0.24 a 38.42 ±0.25 a Non-irradiated SQDs formulation (non-microcapsules) 100 ±0.00 d 100 ±0.00 d Irradiated SQDs formulation (non-microcapsules) 33.74 ±0.65 b 42.34±0.87 b Non-irradiated microencapsulated formulation 100 ±0.00 d 100 ±0.00 d Irradiated microencapsulated formulation 57.77 ±0.51 c 71.22 ±0.29 c Note: Data are the mean of three replicates. For the statistical analysis, ANOVA was performed on the data in each column and the mean values were grouped according to Duncan's method. Significant differences at the 5% level based on Duncan's test (p-value < 0.05) are indicated by different letters in each column. 3.2.2. Assessing formulation insecticidal activity Fig. 10 illustrates the percentage of mortality due to insecticidal activity of different formulations with SQDs on second instar larvae of Ephestia Kuehniella for different exposure times. The significant difference in larval mortality between the Bt microcapsule formulation with SQDs and the other two formulations is an indication of the better performance and greater insecticidal activity of the UV-exposed microcapsule formulation. The decreasing slope of the graph for the microcapsule formulation up to 48 h is very slow. This shows the very high stability of the formulation for up to 48 h. Although the percentage of larval mortality in the nano-formulation of SQDs (non-microcapsules) decreases at a very gradual rate up to 48 h, this is confirmation that the nano-formulation is effective up to 48 h. After a while, however, it decreases with a steep slope and its performance drops. While the percentage of larval mortality caused by insecticidal activity after 48 h is lower in the microcapsule formulation than in the nano-formulation (non-microcapsules). The results confirmed the higher insecticidal activity of the microcapsule formulation with SQDs under UV irradiation. A comparison of the average percentage of mortality of the different formulations in the Duncan's test is shown in Table 1, the separate classification of all three formulations after 96 h of irradiation shows a significant difference between them. (Duncan's test, p-value < 0.05). The percentage of mortality due to the insecticidal activity of different formulations of SQDs after 96 h of UV exposure on second instar larvae of Ephestia Kuehniella was 38.42%, 42.34%, and 71.22% for unprotected Bt formulation (as a control), Bt nanoformulation with SQDs and Bt microcapsule formulation with SQDs, respectively. A sharp decrease in mortality percentage in the unprotected Bt formulation (as a control) indicates a lack of stability and destruction of the crystal against UV radiation, resulting in a decrease in insecticidal activity. The results indicate that in the microcapsule formulation, the crystals were well covered by the SQDs and P (MMA-co-MA) particles, confirming the higher percentage of mortality from insecticidal activity. The SQD-stabilized microcapsule formulation was therefore introduced as a UV-stable formulation with insecticidal activity. Thus, as a UV-stable formulation with insecticidal activity, the SQDs-stabilized microcapsule formulation was introduced. The results showed that the preparation of formulations with SQDs had a more satisfactory performance in the preservation of Bt spores and crystals against UV radiation compared to Bt microcapsule formulations stabilized with graphene oxide nanosheets(Jalali et al., 2020b) and also formulations prepared with other nanoparticles(Jalali, Maghsoudi, & Noroozian, 2020a; Maghsoudi & Jalali, 2017). 4. Conclusion This study aims to demonstrate a simple method for synthesizing SQDs to be used in Bt formulations to increase UV stability. The resulting SQDs exhibited excellent dispersion, high stability, and photoluminescent properties as a function of reaction time. The formation of SQDs after the dissolution of sulfur powder was found to involve two forces, aggregation, and fission. The balance and competition between these two forces determine the morphology of these quantum dots. This concept was used to synthesize quantum dots with optimal dispersion after 120 and 72 h, respectively, with average size distributions of 3.27 nm and 6.07 nm. For the first time, SQDs were used to make different formulations to improve the stability of Bt crystals and spores against UV radiation in the second part. Using SQDs was intended to achieve properties such as good solubility in water, high stability, fluorescence properties, environmental compatibility, and low toxicity. The results showed very high performance of SQDs in the formulation of microcapsules prepared by the Pickering emulsion method. The purpose of this study was to demonstrate that SQDs and P (MMA-co-MA) particles with appropriate spore and crystal coatings have reduced their destruction by UV radiation. Microcapsule formulations stabilized with SQDs showed higher levels of spore viability and mortality due to insecticidal activity, indicating that these quantum dots can improve Bt stability and resistance. Declarations Authorship contribution statement Elham Jalali: Conceptualization, Methodology, Software, Data curation, Writing- Original draft preparation, Visualization, Investigation. Shahab Maghsoudi : Supervision, Conceptualization, Methodology, Visualization, Investigation, Validation, Reviewing and Editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability Data will be made available on request. Acknowledgements The authors would like to express their sincere gratitude to Mr. Alireza Afzalipour and Mrs. Fakhereh Saba, the visionaries who founded Shahid Bahonar University of Kerman. Their wise leadership and generous contributions have played a vital role in shaping the education of future leaders. References Alkassab, A. T., Beims, H., Janke, M., & Pistorius, J. (2022). Determination, distribution, and environmental fate of Bacillus thuringiensis spores in various honeybee matrices after field application as plant protection product. Environmental Science and Pollution Research , 29 (17), 25995–26001. Arshad, F., & Sk, M. P. (2020). Luminescent sulfur quantum dots for colorimetric discrimination of multiple metal ions. ACS Applied Nano Materials , 3 (3), 3044–3049. de Oliveira, J. L., Gómez, I., Sánchez, J., Soberón, M., Polanczyk, R. A., & Bravo, A. (2022). Performance of microencapsulated Bacillus thuringiensis Cry pesticidal proteins. Federici, B. (2022). 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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-4009872","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":276487749,"identity":"19f77926-59e1-4e1f-9f64-463656057b8c","order_by":0,"name":"Elham Jalali","email":"","orcid":"","institution":"Shahid Bahonar University of Kerman","correspondingAuthor":false,"prefix":"","firstName":"Elham","middleName":"","lastName":"Jalali","suffix":""},{"id":276487750,"identity":"bd01564a-3a69-43e9-882a-d7c92f89ce7c","order_by":1,"name":"Shahab Maghsoudi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYBAC+QYeBmYQwwBE8BjYMEgwwLnYgcEBVC1pRGhhQNHCcBiqBQ8wYD978HNhjp3ddv7DxyTeFJxPnNnA/PADQ8E93H7pyUuWnrktOXnnjLQ0yTkGtxNnM7AZSzAYFOO25kCOgTTvNuZkgxs8ZtI8QC3zGBjMgLYn4NZy/o3xb95t9ckG58+AtJwDamH/hl/LjRwzoC2H7QwO5IC0HAA6jAe/LQY33phZz9x2PMHgRlqy5RyDZOOZzTzFEgl4tMj35xjfLtxWbW9w/vDBG2/+2MnOON6+8cOHP3gcBgWJDXAmKJoIa2BgsCdCzSgYBaNgFIxUAABnOFCewq7a6AAAAABJRU5ErkJggg==","orcid":"","institution":"Shahid Bahonar University of Kerman","correspondingAuthor":true,"prefix":"","firstName":"Shahab","middleName":"","lastName":"Maghsoudi","suffix":""}],"badges":[],"createdAt":"2024-03-04 00:05:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4009872/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4009872/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52127374,"identity":"b2b19cbd-a9eb-4db9-8ece-b6e3b3cacea4","added_by":"auto","created_at":"2024-03-07 07:05:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":22539,"visible":true,"origin":"","legend":"\u003cp\u003eFT-IR spectrum of a) PEG-400 and b) SQDs\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4009872/v1/4916024efc35ba68972022fc.png"},{"id":52127373,"identity":"1b0c6f13-9faa-45db-8175-e14b344b62e3","added_by":"auto","created_at":"2024-03-07 07:05:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":22060,"visible":true,"origin":"","legend":"\u003cp\u003eXRD pattern of SQDs\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4009872/v1/a3e2ea56c88d299c62a6de0c.png"},{"id":52127375,"identity":"447d6a61-0ceb-457f-8b9b-4cf685587aca","added_by":"auto","created_at":"2024-03-07 07:05:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":411489,"visible":true,"origin":"","legend":"\u003cp\u003eTEM images of SQDs at different synthesis times, a) 72 h, b) 96 h, C) 120 h\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4009872/v1/3554bab2e2e505154a58322c.png"},{"id":52127376,"identity":"a410cac2-49e5-4913-bcfc-53a3597e96a0","added_by":"auto","created_at":"2024-03-07 07:05:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":81115,"visible":true,"origin":"","legend":"\u003cp\u003eHistogram of the particle size distribution of the SQDs synthesized a) after 72 h and b) after 120 h.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4009872/v1/c2e7f065e46cefaafdf8e810.png"},{"id":52127590,"identity":"0877d6d2-88a0-4e72-a3ca-469c874acfe2","added_by":"auto","created_at":"2024-03-07 07:13:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":608441,"visible":true,"origin":"","legend":"\u003cp\u003eHRTEM image of SQDs synthesized in 120 h\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4009872/v1/25ea1b2d85909b6c0a490794.png"},{"id":52127379,"identity":"67aa092e-c7c0-4d20-9583-cd131d9030e4","added_by":"auto","created_at":"2024-03-07 07:05:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":105959,"visible":true,"origin":"","legend":"\u003cp\u003eUV absorption spectra of SQDs in two different concentration ranges, a) diluted 1000 times, b) diluted 60 times with DI water.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4009872/v1/7b5f5a9f8c2caf02e0d1002c.png"},{"id":52127381,"identity":"ac454960-5a05-48f5-9ad6-97eda8d0443e","added_by":"auto","created_at":"2024-03-07 07:05:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":214515,"visible":true,"origin":"","legend":"\u003cp\u003ea) Fluorescence spectrum of SQDs, b) Comparison of the fluorescence spectrum of synthesized SQDs at different times\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4009872/v1/99c40654cfcdd3a02909a649.png"},{"id":52127383,"identity":"52610150-4fa6-419e-8333-f63b91684d15","added_by":"auto","created_at":"2024-03-07 07:05:10","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1186662,"visible":true,"origin":"","legend":"\u003cp\u003ea) Optical microscope image of a microcapsule containing Bt (40X), b) FESEM images of P (MMA-co-MA) particles (scale bar: 200nm)\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4009872/v1/f5aacfc5f5b3be8ba0765a1e.png"},{"id":52127591,"identity":"a1fa52b1-2025-45a0-8ef0-139807352462","added_by":"auto","created_at":"2024-03-07 07:13:09","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":24173,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of UV exposure time on the percentage of spore viability of different formulations of SQDs (spore viability values shown are the average of three replicates).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4009872/v1/9c954512ea17c92b90696f9f.png"},{"id":52127378,"identity":"1fb96585-29ba-4d0e-843d-d0037194628e","added_by":"auto","created_at":"2024-03-07 07:05:09","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":23436,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of UV exposure time on \u003cem\u003eEphestia kuehniella\u003c/em\u003e larvae mortality percentage caused by insecticidal properties of different SQDs formulations (mortality shown as the average of three replicates).\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-4009872/v1/6e5e64e5434c5167e4864fb4.png"},{"id":52334421,"identity":"4551e0e8-fbc9-4910-aed4-9acb73d19e8a","added_by":"auto","created_at":"2024-03-09 10:21:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2753544,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4009872/v1/2e8b517a-30e2-4862-ad91-591181ce59b7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhancing the UV Radiation Protection of Bacillus thuringiensis Formulation using Sulfur Quantum Dots: A Biotechnological Approach","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eA microbial insecticide based on bacteria, \u003cem\u003eBacillus thuringiensis\u003c/em\u003e (Bt) is one of the most widely used pest control products in agriculture, and forestry because of its safety for humans, and the environment. Insecticidal crystal proteins by Bt are toxic to thousands of insect species including Lepidoptera, Diptera, Coleoptera, Hemiptera, Nematoda, and others. Bt persistence is a critical factor in determining the effectiveness of this product as a pest control agent(Federici, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ortiz \u0026amp; Sansinenea, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). When exposed to ultraviolet radiation in sunlight, the spores and crystals of Bt are susceptible to degradation. Consequently, its persistence is shortened, resulting in a reduction in its activity, and therefore its use as a pesticide is limited(Lahlali et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). A major objective of most studies based on formulations containing Bt is to increase their effectiveness and persistence in the field. Additionally, the new formulations are more persistent, which enables changes in crop management, for example, reducing the frequency of applications(Alkassab, Beims, Janke, \u0026amp; Pistorius, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the last few years, encapsulation has increasingly attracted attention due to the potential for controlling the release rate of products and extending their residual effect(de Oliveira et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Pickering emulsions are stabilized by solid particles, as opposed to conventional surfactant-stabilized emulsions(Yaakov et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). If particles have proper partial dual wettability, they will accumulate in the oil-water interface spontaneously, and particles will likely be embedded in the continuous phase on the side of the interface that has an oil-water interface. So, the adsorbed particle layer functions as an extremely effective steric barrier to prevent emulsion droplets from coalescing, which differs fundamentally from surfactant molecules that stabilize them(C. Wang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These Pickering emulsions result in improved properties of the fabricated materials by providing excellent templates for different morphologies, for example, solid/porous spheres, and hollow capsules(T. Zhang, Liu, Wu, \u0026amp; Ngai, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Usually, polymerization is used in the process to preserve the structures after the emulsion templates are removed. When the emulsion template is removed after polymerization, the particles remain at the oil-water interface, functionalizing the surface of the resulting materials and providing the composites with novel properties(Mao et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhu, Lei, Li, \u0026amp; Zhu, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNanoscale systems have several advantages because their small size gives them better dispersion, plus their large surface area makes them more attractive to targets(Rajput, Sevalkar, Pardeshi, \u0026amp; Pingale, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Utilizing quantum dots (QDs) in Bt formulations can help overcome these limitations and allow for commercial production. QDs are promising materials due to their unique quantum confinement properties, which are coupled with optical properties(Maholiya et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). A new class of zero-dimensional luminescent material free of metal ions is sulfur quantum dots (SQDs), which exhibit bright photoluminescence (PL), as well as UV-visible absorption. As a result, SQDs have been considered green nanomaterials because of their exceptional electrical and electronic properties, stable photoluminescence low toxicity, high biocompatibility, and excellent solubilities(S. Wang, Bao, Gao, \u0026amp; Li, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; H. Zhang et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The functional groups on the surface of the SQDs provide electrons that can enhance the interaction of other compounds. However, in comparison to the exploration of pure elemental QDs, such as carbon, silicon, and phosphorus, SQDs exploration is at an early stage(Gao, Huang, Tan, Lv, \u0026amp; Zhou, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; C. Lu et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study tried to resolve this highly controversial issue by developing a new formulation with higher activity and longer persistence using nanotechnology. We fabricated a Pickering emulsion encapsulated by Bt and stabilized by SQD without any surfactant stabilizers. The principal objective of this study was to improve the persistence of spores and crystals to environmental stresses such as sunlight.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.1. Materials\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eEthanol (99.5%), and polyethylene glycol-400 (PEG-400) were purchased from Sigma Aldrich (Steinheim, Germany). Thiourea, sublimated sulfur powder, sodium hydroxide, copper nitrate, methyl methacrylate, methacrylic acid, sodium dodecyl sulfate, ammonium persulfate, and sodium chloride were purchased from Merck (Darmstadt, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.2. Synthesis of the SQDs\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eSQDs were synthesized according to the research carried out by Arshad and Palashuddin, with some modifications(Arshad \u0026amp; Sk, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Briefly, 3 mL of PEG-400, 4.0 g of sodium hydroxide, and 50 mL of ultrapure DI water were mixed and then 1.4 g of sublimated sulfur powder was added and stirred at a constant temperature at 70\u0026deg;C for a predetermined time (100-125h). As the reaction progresses, sulfur powder gradually dissolves and its color changes over time to dark red. The prepared product was centrifuged at 3000rpm to separate reactants from sulfur QDs, which are then referred to as SQDs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.3. Preparation of poly (methyl methacrylate-co-methacrylic acid) [P (MMA-\u003c/em\u003eco\u003cem\u003e-MA)]\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eSynthesis of the P (MMA-co-MA) was performed according to our previous work(Jalali, Maghsoudi, \u0026amp; Noroozian, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e). In brief, a 250 mL round bottom flask was filled with 50 mL DI water, 10.65 g of methyl methacrylate, 4.24 g of methacrylic acid, 0.12 g of ammonium persulfate, and 0.20 g of sodium dodecyl sulfate. After stirring at 400 rpm, the mixture was degassed using nitrogen for 30 minutes. The liquid phase was then heated to 80\u0026deg;C, and the process was covered with nitrogen to prevent oxygen from entering. Following 10 h of reaction time, the P (MMA-co-MA) was collected.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.4. Preparation of Pickering emulsions stabilized by SQDs\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe encapsulated formulation was prepared by the Pickering emulsion method. To fabricate emulsions, SQDs particles with a 1:1 oil/ aqueous phase ratio were prepared.\u003c/p\u003e \u003cp\u003eThe oil phase was prepared from SQDs in olive oil and ethanol at a concentration of 10 v/v%. The aqueous phase was prepared by dispersing the 0.5g Bt, 2.5 mL of P (MMA-co-MA), 2.5 mL DI water, and 0.073 g NaCl using a homogenizer (Hielscher, UP400St, 20 kHz, 400 W, Germany) for 2 min. Then, the aqueous phase was added dropwise to the oil phase. The mixture was suspended with a homogenizer for 5 min. The ratio of the oil to aqueous phase was 10:4, 10:6, 10:8, and 1:1. Finally, a 1:1 oil-to-aqueous phase ratio was selected as the best ratio.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. \u003cem\u003eExposure of emulsions to UV radiation\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eDiluted encapsulated formulation with Bt and free spore (non-encapsulated formulation) were used. A 385 nm UVA tube (Philips, 15 W) was mounted 140 mm above the open Petri dishes to provide radiation for 7 days. The spore count was calculated by counting colony-forming units (CFUs). Before the determination of spore count, the volume of each sample was adjusted with sterile distilled water to compensate for evaporation losses. Three replications were performed on three separate days for all the experiments. As follows is the Equation for calculating spore viability:\u003c/p\u003e \u003cp\u003eV spore (%) = [CFU\u003csub\u003erad\u003c/sub\u003e∕CFU\u003csub\u003e0\u003c/sub\u003e]\u003c/p\u003e \u003cp\u003eThe CFU\u003csub\u003e0\u003c/sub\u003e represents the non-irradiated spore count (free or encapsulated formulation), and the CFU\u003csub\u003erad\u003c/sub\u003e represents the irradiated spore count (free or encapsulated formulation).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Characterization\u003c/h2\u003e \u003cp\u003eThe fluorescence properties were measured using a F96 Pro Fluorescent Spectrophotometer with a 10mm quartz cell. The ultraviolet-visible absorption spectra were recorded on a Specord 250 PLUS spectrophotometer (Analytik Jena, Germany). The crystalline structure of the SQDs was characterized by X-ray diffraction (XRD) using an X'pert PRO model. Cu-Ka radiation was used in the 2θ angular range of 10\u0026deg;-80\u0026deg;. Fourier transform infrared (FTIR) spectra was recorded using a Tensor 27 spectrometer (Bruker, Saarbr\u0026uuml;cken, Germany). The spectra were recorded from 4000 to 400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Transmission electron microscope (TEM) images were achieved by a Philips EM208S transmission electron microscope with an accelerating voltage of 100 kV. High-resolution transmission electron microscopy (HRTEM) was performed with a FEI Tecnai G2 F20 SuperTwin with an accelerating voltage of 200 kV. Scanning electron microscopy (FE-SEM, Sigma, Zeiss) was used to study the morphology.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.1. Characterization of SQDs\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe FT-IR spectrum of the SQDs and the polyethylene glycol is shown in Fig.1. The main peaks of PEG-400 include absorption bands at 3361 cm\u003csup\u003e-1\u003c/sup\u003e, 2864 cm\u003csup\u003e-1,\u003c/sup\u003e and 1297 cm\u003csup\u003e-1\u003c/sup\u003e. The absorption band at\u0026nbsp;3442\u0026nbsp;cm\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eis related to the stretching vibration of the O-H bond. C-H stretching and C-H bending vibrations are\u0026nbsp;responsible\u0026nbsp;for the absorption bands observed at\u0026nbsp;2934\u0026nbsp;cm\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eand 1467 cm\u003csup\u003e-1\u003c/sup\u003e, respectively. The absorption band observed at 1342 cm\u003csup\u003e-1\u003c/sup\u003e is related to the C=O group. The broad absorption band at 1622 cm\u003csup\u003e-1\u003c/sup\u003e in PEG was transformed into two absorption bands at 1648 cm\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eand 1115 cm\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003ein SQDs. These bands are related to the C-O-H and C-O-C stretching vibrations(H. Lu, Zhang, Li, \u0026amp; Gan, 2021). The absorption band at\u0026nbsp;615\u0026nbsp;cm\u003csup\u003e-1\u003c/sup\u003e is also related to the S-S stretching vibration(Tan, An, Pan, Liu, \u0026amp; Hu, 2021). Weak absorption bands between 500 cm\u003csup\u003e-1\u003c/sup\u003e and 800 cm\u003csup\u003e-1\u003c/sup\u003e are related to the PEG structure(Kolhe \u0026amp; Kannan, 2003; Shameli et al., 2012). In the FT-IR spectrum of SQDs, all the main characteristic peaks of PEG were observed and no new peaks were added except for a decrease in intensity or overlapping of some peaks. Therefore, there was no chemical reaction between PEG and SQDs. According to these results, PEG is effective in discussing SQD formation mechanisms. As PEG is physically absorbed by SQDs, this absorption layer prevents self-aggregation of the particles and is therefore essential for the maintenance of SQD stability.\u003c/p\u003e\n\u003cp\u003eThe XRD pattern of the SQDs is shown in Fig. 2. The diffraction peaks at 18.77\u0026ordm;, 22.22\u0026ordm;, 23.12\u0026ordm;, 25.37\u0026ordm;, 31.22\u0026ordm; and 33.72\u0026ordm; correspond to planes (202), (220), (222), (133), (044) and (242) respectively. This indicates the possibility of the formation of a polycrystalline phase of sulfur(Xie et al., 2012).\u003c/p\u003e\n\u003cp\u003eFig. 3 shows the changes in the size and shape of the SQDs at different times and scales using TEM images. For the 72h sample, as shown in Fig.3, the synthesized SQDs are monodispersed with almost spherical morphology. This can be attributed to the electrostatic repulsion between the anionic groups on the surface of the SQDs. Therefore, it can be concluded that the S-dots in the 72h sample are monodispersed and not aggregated. Even though the particles have self-aggregated within the 96h sample, there is a large change or non-uniformity in size. As the duration increased from 100 to 120 h, the fission of the accumulated particles likely resulted in smaller quantum dots with more defined and distinct boundaries.\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. 4., a histogram of the particle size distribution is shown for 72 h and 120 h SQDs, respectively. The particles in 72 h are mainly distributed in the range of 2.23 nm to 9.77 nm with an average size of 6.07 nm, while in 120 h they are distributed in the range of 2.09 nm to 4.41 nm with an average size of 3.27 nm.\u003c/p\u003e\n\u003cp\u003eFig. 5 shows the HRTEM image of SQDs synthesized in 120 h, confirming the uniform distribution of particles.\u003c/p\u003e\n\u003cp\u003eThe UV absorption spectra of the synthesized SQDs is shown in Fig. 6. The UV spectrum has been recorded in two separate areas to better show the spectral characteristics. A peak at 216 nm is observed, which is probably related to the n \u0026rarr; \u0026sigma;* transfer of non-bonded sulfur electrons(Fu et al., 2020).\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eThe peak at 333 nm is also related to the direct transition of the forbidden band of S[0], indicating the presence of the oxidation reaction of S\u003csub\u003ex\u003c/sub\u003e\u003csup\u003e-2\u003c/sup\u003e ions to S[0](Li et al., 2014; Xiao et al., 2019).\u003c/p\u003e\n\u003cp\u003eThe fluorescence spectrum of the SQDs is shown in Fig. (7. a). As is well known, the maximum emission wavelength of sulfur quantum dots is around 440 nm, which occurs at an excitation wavelength of 360 nm, and the blue fluorescence is characteristic of sulfur quantum dots. SQDs are transparent yellow in normal light and emit blue light in UV light at a wavelength of 360 nm. Figure (7. b) shows the emission spectrum of SQDs synthesized at different times. The quantum size effect may be responsible for this dependence of the emission spectrum on particle size as fluorescence intensity has increased with synthesis time. There has also been a slight blue shift in the emission wavelength with an increase in synthesis time. Since quantum dots have size-dependent emission, from the blue shift in the emission wavelength between samples synthesized at different times, which is attributed to the quantum size effect of the quantum dots, it can be concluded that the size of the particles gradually changes as the synthesis time increases. With increasing synthesis time, the particle size of SQDs decreases and as a result, the size of SQDs can be controlled.\u003c/p\u003e\n\u003cp\u003eTEM images and emission fluorescence spectra indicate that particle size changes with increasing duration. Over time, particle distribution changes from monodispersed to non-uniform. Gradually, the quantum dots\u0026apos; border becomes clearer and the size of SQDs decreases. It appears that during SQD formation, aggregation and fission compete with each other until a dynamic equilibrium is achieved. When PEG is added to a reaction, it physically absorbs on the surface of the sulfur, preventing particles from aggregating. The high surface energy of the particles tends to aggregate the particles as the reaction progresses(Xie et al., 2012). As a result, even the absorption layer of PEG on the surface cannot prevent quantum dots from agglomeration. In the synthesis with a duration of 72 h, a dynamic balance is established between aggregation and fission. Finally, after 96 h, the aggregation effect is weakened and the fission effect plays the main role, leading to an increasingly clear view of SQDs in TEM images.\u003c/p\u003e\n\u003cp\u003eFig. (8. a) shows an image of Bt microcapsule particles composed of polymer latex particles and SQDs examined under a 40X microscope. FESEM images of P (MMA-co-MA) particles are shown in Figure 8. b. The particles are hollow and the aggregates are stabilizing the microcapsule.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.2. Assessing the UV stability of SQD-based microcapsule formulations\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.2.1. Evaluation of spore viability\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure (9) shows the effect of UV exposure time on the percentage of spore viability of different Bt formulations with SQDs up to 120 h. It can be seen that the percentage of spore viability for the microcapsule formulation stabilized with SQDs is declining at a gentler rate. It has a slightly decreasing slope up to 72 h and then a steeper slope.\u0026nbsp;While the percentage of spore viability in the nanoformulation of SQDs (non-microcapsules) decreases with a greater slope than in the unirradiated condition up to 48 h, and after 72 h the difference is less compared to the formulation of unprotected Bt (as a control). This remarkable difference between the spore viability percentages of the formulations shows that UV radiation has the least effect on the spores in the microcapsule formulation with SQDs. This indicates that the spores are adequately covered by the SQDs and\u0026nbsp;P (MMA-co-MA) particles. Consequently, the microcapsule formulation has better UV performance than the nanoformulation of SQDs (non-microcapsule).\u003c/p\u003e\n\u003cp\u003eTable 1 shows spore viability and mortality due to insecticidal activity of different formulations of SQDs on second instar \u003cem\u003eEphestia kuehniella\u003c/em\u003e larvae after 96h UV exposure. The classification showed that after 96 h of UV exposure, there was a significant difference between the two different formulations of Bt with SQDs and the unprotected Bt formulation. It was found that all three formulations not exposed to UV were in the same category. This means that the difference between them is not significant (p-value=1.000). The spore viability percentages for unprotected Bt, the nano formulation of Bt with SQDs, and the microcapsule formulation of Bt with SQDs were changed to 31.25%, 33.74%, and 57.77%, respectively. As a result, Bt microcapsules formulated with SQDs have the lowest percentage decrease in spore viability. The good coverage of spores in the microcapsule formulation with SQDs is demonstrated by the sharp drop in spore viability percentage in the unprotected Bt formulation (as a control). As the most stable formulation against UV radiation, the microcapsule formulation was chosen.\u003c/p\u003e\n\u003cp\u003eTable 1. Mean \u0026plusmn; (standard error) percentage of spore viability and percentage of mortality due to the insecticidal activity of different formulations of SQDs on second instar \u003cem\u003eEphestia kuehniella\u003c/em\u003e larvae after 96 h UV exposure.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFormulations\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpore Viability%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMortality%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNon-irradiated free spore\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e100 \u0026plusmn;0.00\u003csup\u003e\u0026nbsp;d\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e100 \u0026plusmn;0.00\u003csup\u003e\u0026nbsp;d\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eIrradiated free spore\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e31.25 \u0026plusmn;0.24\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e38.42 \u0026plusmn;0.25\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNon-irradiated\u0026nbsp;SQDs\u0026nbsp;formulation\u0026nbsp;(non-microcapsules)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e100 \u0026plusmn;0.00\u003csup\u003e\u0026nbsp;d\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e100 \u0026plusmn;0.00\u003csup\u003e\u0026nbsp;d\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eIrradiated\u0026nbsp;SQDs\u0026nbsp;formulation\u0026nbsp;(non-microcapsules)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e33.74 \u0026plusmn;0.65\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e42.34\u0026plusmn;0.87\u003csup\u003e\u0026nbsp;b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNon-irradiated microencapsulated formulation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e100 \u0026plusmn;0.00\u003csup\u003e\u0026nbsp;d\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e100 \u0026plusmn;0.00\u003csup\u003e\u0026nbsp;d\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eIrradiated microencapsulated formulation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e57.77 \u0026plusmn;0.51\u003csup\u003e\u0026nbsp;c\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e71.22 \u0026plusmn;0.29\u003csup\u003e\u0026nbsp;c\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: Data are the mean of three replicates. For the statistical analysis, ANOVA was performed on the data in each column and the mean values were grouped according to Duncan\u0026apos;s method. Significant differences at the 5% level based on Duncan\u0026apos;s test (p-value \u0026lt; 0.05) are indicated by different letters in each column.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.2.2. Assessing formulation insecticidal activity\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFig. 10 illustrates the percentage of mortality due to insecticidal activity of different formulations with SQDs on second instar larvae of \u003cem\u003eEphestia Kuehniella\u003c/em\u003e for different exposure times. The significant difference in larval mortality between the Bt microcapsule formulation with SQDs and the other two formulations is an indication of the better performance and greater insecticidal activity of the UV-exposed microcapsule formulation. The decreasing slope of the graph for the microcapsule formulation up to 48 h is very slow. This shows the very high stability of the formulation for up to 48 h. Although the percentage of larval mortality in the nano-formulation of SQDs (non-microcapsules) decreases at a very gradual rate up to 48 h, this is confirmation that the nano-formulation is effective up to 48 h. After a while, however, it decreases with a steep slope and its performance drops. While the percentage of larval mortality caused by insecticidal activity after 48 h is lower in the microcapsule formulation than in the nano-formulation (non-microcapsules). The results confirmed the higher insecticidal activity of the microcapsule formulation with SQDs under UV irradiation.\u003c/p\u003e\n\u003cp\u003eA comparison of the average percentage of mortality of the different formulations in the Duncan\u0026apos;s test is shown in Table 1, the separate classification of all three formulations after 96 h of irradiation shows a significant difference between them. (Duncan\u0026apos;s test, p-value \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003eThe percentage of mortality due to the insecticidal activity of different formulations of SQDs after 96 h of UV exposure on second instar larvae of \u003cem\u003eEphestia Kuehniella\u003c/em\u003e was 38.42%, 42.34%, and 71.22% for unprotected Bt formulation (as a control), \u003cem\u003eBt\u003c/em\u003e nanoformulation with SQDs and \u003cem\u003eBt\u0026nbsp;\u003c/em\u003emicrocapsule formulation with SQDs, respectively. A sharp decrease in mortality percentage in the unprotected Bt formulation (as a control) indicates a lack of stability and destruction of the crystal against UV radiation, resulting in a decrease in insecticidal activity. The results indicate that in the microcapsule formulation, the crystals were well covered by the SQDs and P (MMA-co-MA) particles, confirming the higher percentage of mortality from insecticidal activity. The SQD-stabilized\u0026nbsp;microcapsule formulation was therefore introduced as a UV-stable formulation with insecticidal activity. Thus, as a UV-stable formulation with insecticidal activity, the SQDs-stabilized\u0026nbsp;microcapsule formulation was introduced.\u003c/p\u003e\n\u003cp\u003eThe results showed that the preparation of formulations with SQDs had a more satisfactory performance in the preservation of Bt spores and crystals against UV radiation compared to Bt microcapsule formulations stabilized with graphene oxide nanosheets(Jalali et al., 2020b) and also formulations prepared with other nanoparticles(Jalali, Maghsoudi, \u0026amp; Noroozian, 2020a; Maghsoudi \u0026amp; Jalali, 2017).\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study aims to demonstrate a simple method for synthesizing SQDs to be used in Bt formulations to increase UV stability. The resulting SQDs exhibited excellent dispersion, high stability, and photoluminescent properties as a function of reaction time. The formation of SQDs after the dissolution of sulfur powder was found to involve two forces, aggregation, and fission. The balance and competition between these two forces determine the morphology of these quantum dots. This concept was used to synthesize quantum dots with optimal dispersion after 120 and 72 h, respectively, with average size distributions of 3.27 nm and 6.07 nm. For the first time, SQDs were used to make different formulations to improve the stability of Bt crystals and spores against UV radiation in the second part. Using SQDs was intended to achieve properties such as good solubility in water, high stability, fluorescence properties, environmental compatibility, and low toxicity. The results showed very high performance of SQDs in the formulation of microcapsules prepared by the Pickering emulsion method. The purpose of this study was to demonstrate that SQDs and P (MMA-co-MA) particles with appropriate spore and crystal coatings have reduced their destruction by UV radiation. Microcapsule formulations stabilized with SQDs showed higher levels of spore viability and mortality due to insecticidal activity, indicating that these quantum dots can improve Bt stability and resistance.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eElham Jalali:\u003c/strong\u003e Conceptualization, Methodology, Software, Data curation, Writing- Original draft preparation, Visualization, Investigation. \u003cstrong\u003eShahab Maghsoudi\u003c/strong\u003e: Supervision, Conceptualization, Methodology, Visualization, Investigation, Validation, Reviewing and Editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to express their sincere gratitude to Mr. Alireza Afzalipour and Mrs. Fakhereh Saba, the visionaries who founded Shahid Bahonar University of Kerman. Their wise leadership and generous contributions have played a vital role in shaping the education of future leaders.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAlkassab, A. T., Beims, H., Janke, M., \u0026amp; Pistorius, J. (2022). Determination, distribution, and environmental fate of Bacillus thuringiensis spores in various honeybee matrices after field application as plant protection product. \u003cem\u003eEnvironmental Science and Pollution Research\u003c/em\u003e, \u003cem\u003e29\u003c/em\u003e(17), 25995\u0026ndash;26001.\u003c/li\u003e\n \u003cli\u003eArshad, F., \u0026amp; Sk, M. P. (2020). Luminescent sulfur quantum dots for colorimetric discrimination of multiple metal ions. \u003cem\u003eACS Applied Nano Materials\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e(3), 3044\u0026ndash;3049.\u003c/li\u003e\n \u003cli\u003ede Oliveira, J. L., G\u0026oacute;mez, I., S\u0026aacute;nchez, J., Sober\u0026oacute;n, M., Polanczyk, R. 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Pickering emulsions stabilized by biocompatible particles: A review of preparation, bioapplication, and perspective. \u003cem\u003eParticuology\u003c/em\u003e, \u003cem\u003e64\u003c/em\u003e, 110\u0026ndash;120.\u003c/li\u003e\n \u003cli\u003eZhu, H., Lei, L., Li, B.-G., \u0026amp; Zhu, S. (2018). Development of novel materials from polymerization of Pickering emulsion templates. \u003cem\u003ePolymer Reaction Engineering of Dispersed Systems: Volume I\u003c/em\u003e, 101\u0026ndash;119.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"UV resistance, Quantum dots, Microcapsule, Pickering emulsion, Spore viability","lastPublishedDoi":"10.21203/rs.3.rs-4009872/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4009872/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLow stability against ultraviolet (UV) radiation is one of the drawbacks of biological pesticides such as \u003cem\u003eBacillus thuringiensis\u003c/em\u003e (Bt). The persistence of Bt crystals against insect pests is thus deactivated. Bt plays a key role in the control of microbial pests. In this study, Bt spores and crystals were protected from UV radiation by sulfur quantum dots (SQDs). These were synthesized by treating sublimated sulfur powders with an alkali using polyethylene glycol 400 (PEG-400). Their effect on the formulation of Bt was investigated to improve its resistance to UV radiation. Excellent aqueous dispersibility and superior photostability were observed for the synthesized SQDs. Properly dispersed SQDs with mean size distributions of 3.27 nm and 6.07 nm were obtained for 120 and 72 h, respectively. The findings indicate that SQDs perform very well in encapsulated formulations prepared by the Pickering emulsion method compared to non-encapsulated formulations. Spore viability and mortality of second-instar \u003cem\u003eEphestia kuehniella\u003c/em\u003e larvae under UV-A radiation were studied. The unique properties of SQDs are believed to reduce the degradation of Bt against UV radiation. Our results showed that these SQDs can be used to improve the stability and resistance of Bt in SQD-stabilized microcapsule formulations.\u003c/p\u003e","manuscriptTitle":"Enhancing the UV Radiation Protection of Bacillus thuringiensis Formulation using Sulfur Quantum Dots: A Biotechnological Approach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-07 07:05:04","doi":"10.21203/rs.3.rs-4009872/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"764bc8ef-e392-4c0c-956c-5f28e7f79b4c","owner":[],"postedDate":"March 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-09T10:20:03+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-07 07:05:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4009872","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4009872","identity":"rs-4009872","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}