Structure and properties of SQDsThe 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 vibrations25. The absorption band at 615 cm-1 is also related to the S–S stretching vibration26. Weak absorption bands between 500 cm-1 and 800 cm-1 are related to the PEG structure27,28. In addition to the characteristic bands for PEG surface capping, sulfur-induced absorption bands were observed in SQDs at 668 cm−1 and 519 cm−1, corresponding to S–O bending and S–S stretching, respectively. 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.Figure 1FT-IR spectrum of (a) PEG-400 and (b) SQDs.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 sulfur29.Figure 2Figure 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.Figure 3TEM images of SQDs at different synthesis times, (a) 72 h, (b) 96 h, (c) 120 h.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.Figure 4Histogram of the particle size distribution of the SQDs synthesized (a) after 72 h and (b) after 120 h.Figure 5 shows the HRTEM image of SQDs synthesized in 120 h, confirming the uniform distribution of particles.Figure 5HRTEM image of SQDs synthesized in 120 h.The UV absorption spectra of the synthesized SQDs are 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 electrons30. 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 Sx-2 ions to S[0]31,32.Figure 6UV absorption spectra of SQDs in two different concentration ranges, (a) diluted 1000 times, (b) diluted 60 times with DI water.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.Figure 7(a) Fluorescence spectrum of SQDs, (b) Comparison of the fluorescence spectrum of synthesized SQDs at different times.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 progresses29. 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.Figure 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 Fig. 8 b The particles are hollow and the aggregates are stabilizing the microcapsule.Figure 8(a) Optical microscope image of a microcapsule containing Bt (40X), (b) FESEM images of P (MMA-co-MA) particles (scale bar: 200nm).Assessing the UV stability of SQD-based formulationsEvaluation of spore viabilityFigure 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 SQDs formulation (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 (free spore, 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 SQDs formulation (non-microcapsule).Figure 9The 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).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 (free spore) 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 (free spore), the SQDs formulation (non-microcapsule, 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 (free spore, as a control). The enhanced UV stability of the microcapsule formulation can be attributed to the protective role of the SQDs and P(MMA-co-MA) particles, which shield the spores from UV radiation. This protective mechanism is likely due to the ability of SQDs to absorb and dissipate UV energy, thereby preventing damage to the spores. Additionally, the microcapsule structure provides a physical barrier, further enhancing UV resistance. The significant difference in spore viability percentages observed between the formulations demonstrates that UV radiation has the least impact on the spores in the microcapsule formulation with SQDs. This suggests that the spores are effectively covered by the SQDs and P(MMA-co-MA) particles. Consequently, the microcapsule formulation exhibits superior UV performance compared to the SQDs formulation (non-microcapsule). As the most stable formulation against UV radiation, the microcapsule formulation was chosen. The main factor contributing to the enhanced UV stability is the powerful UV absorption properties of SQDs. These SQDs not only effectively absorb UV light, but also emit it at a different wavelength, thereby minimizing the direct UV exposure on the Bt spores. Additionally, the P(MMA-co-MA) particles play a role in creating a robust and long-lasting coating that provides further protection against UV degradation for the spores.
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.Assessing SQD-based formulations’ insecticidal activityFigure 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 SQDs formulation (non-microcapsules) decreases at a very gradual rate up to 48 h, this is confirmation that the SQDs formulation (non-microcapsules) is effective up to 48 h. After a while, however, it decreases with a steep slope and its performance drops. The percentage of larval mortality caused by insecticidal activity after 48 h is lower in the microcapsule formulation than in the SQDs formulation (non-microcapsules). The results confirmed the higher insecticidal activity of the microcapsule formulation with SQDs under UV irradiation.Figure 10Effect of UV exposure time on Ephestia kuehniella larvae mortality percentage caused by insecticidal properties of different SQDs formulations (mortality shown as the average of three replicates).A comparison of the average percentage of mortality of the different formulations in 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 (free spore, as a control), SQDs formulation (non-microcapsules) and microcapsule formulation with SQDs, respectively. A sharp decrease in mortality percentage in the unprotected Bt formulation (free spore, 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 microcapsule formulation with SQDs was therefore introduced as a UV-stable formulation with insecticidal activity. Thus, as a UV-stable formulation with insecticidal activity, the microcapsule formulation with SQDs was introduced. The enhanced insecticidal activity and UV stability of the microcapsule formulation can be attributed to the optimal synthesis conditions and the effective encapsulation of spores and crystals by SQDs. The protective function of SQDs, which absorb and dissipate UV energy, safeguards against spore damage and preserves their insecticidal efficacy.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 nanosheets33 and also formulations prepared with other nanoparticles34,35. Our results confirm that the microcapsule formulation with SQDs exhibits better UV stability and insecticidal activity compared to formulations using other nanomaterials. Previous studies have shown varying degrees of success with different nanoparticles36, but SQDs provide a unique combination of strong UV absorption, low cost, low toxicity, and environmental compatibility.The effectiveness of the microcapsule formulation with SQDs was evaluated by comparing it with other nanomaterials33,34,35, including the Pickering emulsion method stabilized with graphene oxide nanosheets. Our results showed that after 96 h of UV exposure, the microcapsule formulation with SQDs exhibited a higher rate of spore viability and mortality due to insecticidal activity compared to the graphene oxide-stabilized formulation. While the microcapsule formulation with graphene oxide demonstrated some protective effect, microcapsule formulation with SQDs showed superior performance, particularly within the first 48 h of UV exposure. The mortality of the larvae exposed to the microcapsule formulation with SQDs remained very high for up to 48 h, indicating an exceptional resistance of the formulation. After this period, although there was a decline, the microcapsule formulation with SQDs continued to retain its protective and absorptive properties to a greater extent than the graphene oxide-stabilized method. This remarkable performance can be attributed to the unique properties of SQDs, which provide strong UV absorption and stability. The enhanced UV resistance and insecticidal efficacy of the microcapsule formulation with SQDs suggest that they are more effective in protecting Bt spores and crystals compared to formulations stabilized with graphene oxide nanosheets.These findings illustrate the potential of utilizing microcapsule formulation with SQDs to improve UV stability and efficacy in agricultural applications. This presents a promising alternative to other formulations based on nanoparticles.
Enhancing UV radiation protection of Bacillus thuringiensis formulations using sulfur quantum dots: synthesis and efficacy evaluation
