Pancakes are fast food snacks that are generally made with wheat flour as the basic ingredients, which is an imported commodity and detrimental for people who are allergic to gluten. To reduce the use of wheat, alternative raw materials derived from local commodities are used, such as modified cassava flour (mocaf), arrowroot flour, and suweg flour. The experiment was carried out by mixing mocaf flour, arrowroot flour, and suweg flour to produce composite flour with a ratio of 70:15:15 (CF1), 70:20:10 (CF2), and 70:20:5 (CF3). The result showed that the ratio of mocaf flour, arrowroot flour, and suweg flour had a significant effect on pasting temperature, peak viscosity, hold viscosity, breakdown viscosity, setback, L*, a*, hue, whiteness, ∆E, as well as swelling volume and solubility on the characteristics of the composite flour. There was also a significant effect on the texture characteristics of hardness, adhesiveness, chewiness, color characteristics L*, a*, whiteness, ∆E, and flavor preference for the gluten-free pancake products. The best formulation to produce pancakes that have characteristics similar to wheat flour-based pancakes was 70% mocaf flour, 15% arrowroot flour, and 15% suweg flour. Flour is a raw material that plays an important role in Indonesian food and is widely used in food processing. The flour that is most often used in Indonesia is wheat flour, which is derived from wheat. Until now, Indonesia has needed to import wheat to fulfill high domestic demand. Importing wheat is also necessary because the wheat plant can only grow in subtropical regions. To reduce dependence on wheat imports, the price of which is continuously rising, it is necessary for the food sector to diversify.Another reason that food diversification is necessary is the gluten content of wheat flour. Gluten is a protein that can be found in wheat . Gluten Tubers are a source of carbohydrates that can be processed into alternative flours. Tuber flour has some advantages, such as being gluten-free and having raw materials that are available in Indonesia. However, the absence of gluten content in flour also has drawbacks, as products made without it have poorer characteristics than those cooked with it. Modifying the characteristics of non-gluten flours used is necessary to improve the products. Several previous studies have reported that starch/flour modification treatment can improve the flour’s characteristics and expand its application in the food industry ,7,8,9. AOne application of composite flour is an ingredient in gluten-free pancake production. Pancakes are fast food snacks with a flat, round shape, and are generally made with wheat flour as a basic ingredients. Good quality pancakes have perfect swelling power, and dough must always be fresh to meet good quality standards. Pancakes are generally consumed as an easy and quick breakfast to make at home; dough is made by mixing flour with milk, eggs, sugar, and baking powder, and is then fried on a pan . DespiteThe main ingredients used in this study were commercial wheat flour , commercial mocaf flour , 7–8-month-old arrowroot tuber, and 10–12-month-old suweg tuber. Arrowroot tuber was obtained from Ciamis, Indonesia, and suweg tuber was obtained from a local farmer in Madiun, Indonesia.Arrowroot flour was prepared according to Marta et al.  with a sSuweg flour was prepared according to Marta et al.  with a sMocaf flour (MF), arrowroot flour (AF), and suweg flour (SF) were mixed and sieved to make a homogeneous composite flour (CF). For CF1, the ratio between flour was 70% mocaf flour, 15% arrowroot tuber flour, and 15% suweg flour; for CF2, 70% mocaf flour, 20% arrowroot tuber flour, and 10% suweg flour; and for CF3, 70% mocaf flour, 25% arrowroot tuber flour, and 5% suweg flour. Pancakes made from 100% wheat flour (WF) were used as controls. Each formulation  was combined with additional ingredients for making pancake products: 90 g of powdered sugar (Rose Brand by Adi Karya Gemilang), 9 g of baking powder (Koepoe Koepoe by Gunacipta Multirasa), and 26 g of powdered milk (Dancow by Nestle) [The moisture, ash, total protein, fat, and total crude fiber content were determined using standard  methods.The pasting properties of the flour samples were determined using a Rapid Visco Analyzer . In an RVA tube, 2.8 g of flour samples were added with 25 mL of aquadest (distilling water that has been cleaned of impurities and purified in a lab). The temperature was originally held at 50 °C for 1 min, then increased from 50 to 95 °C at a rate of 6 °C/min, held at 95 °C for 5 min, and then decreased to 50 °C at a rate of 6 °C/min. After that, the gel was preserved at 50 °C for 5 min.The color scale for L*, a*, and b* of flour was measured using a Spectrophotometer CM-5  with Spectra Magic software. The color measurement includes L* , a* (+a* = redness/−a* = greenness), b* (+b* = yellowness/−a* = blueness), and hue. The calibration was performed with a zero-calibration plate (CM-A124) and a white calibration plate (CM-A120) using a large target mask (CM-A203).g for 30 min. The supernatant’s amount was then calculated. The amount of water that could be absorbed by 1 gram of wheat was measured as mL as water absorption capacity.The water absorption capacity of the flour samples was determined according to Marta et al. . A centrg  after being kept in a water bath at 90 °C for 20 min and cooled in cold water. The volume of the supernatant was measured to determine the swelling volume result and then dried in an oven at 110 °C for 24 h to measure the solubility of the samples.Swelling volume and solubility were determined according to Marta et al. . The samTexture evaluation was measured using a Texture Profile Analyzer  and exponent lite express software for data collection and calculation. The product was pressed using an aluminum cylinder probe P36R with a 2 kg load cell at a speed of 5 mm/s to a strain of 50%. The product texture profile including hardness, adhesiveness, springiness, cohesiveness, chewiness, and resilience was determined from exponent lite express software.A hedonic sensory test of the pancake was conducted with 20 panelists aged between 18 and 25 years old, both male and female. Pancakes were presented as whole pieces and placed on white plastic dishes coded with random three-digit numbers. Pancakes were evaluated based on the acceptability of their color, aroma, texture, taste, and overall appearance using a 5-point hedonic scale. The scale ranged from “extremely like” to “extremely dislike,” corresponding to the highest and lowest scores of “5” and “1”, respectively .p values < 0.05.Data were analyzed using one-way ANOVA, then followed by Duncan Test to detect differences. Significance was confirmed at The proximate and crude fiber test results for wheat flour, mocaf flour, arrowroot tuber flour, suweg flour, and composite flour, respectively, are presented in No composite flours were significantly different from each other in the parameters of moisture content, ash content, fat content, protein content, and crude fiber content, but all composite flours were significantly different from wheat flour . The comAmylographic tests were carried out to show the pasting properties of the starch paste and flour produced as well as changes in starch viscosity during the cooking process, as these characteristics play an important role in flour applications in the food industry. The pasting properties of wheat flour, mocaf flour, arrowroot tuber flour, suweg flour, CF1, CF2, and CF3 are presented in All parameters of pasting properties of composite flours were significantly different from wheat flour . PastingColor is one of the main factors that determine consumer acceptance . The colTesting the color characteristics of the flours was carried out with the CM-5 Spectrophotometer which will produce L, a*, and b* data, defined by CIE  and tabulated in CF3 has the highest L* (91.19), indicating a higher lightness, whereas CF1 has the lowest L* (89.58) compared to other composite flours. Similar results were also found in the whiteness index, whereas CF1 (70:15:15) has the lowest whiteness (86.16), while CF3 (70:25:5) has the highest whiteness index (87.27) when compared to other composite flours. Whiteness index indicates the degree to which a surface was white. However, when compared to all samples, suweg flour has the lowest lightness (81.64) and whiteness index (77.46), while mocaf flour shows the highest lightness (94.17) and whiteness index (88.63). The dark color of the suweg flour causes the suweg flour to have the largest ∆E (11.94) compared to all samples, which shows a very large difference when compared to wheat flour as a control.CF1 (70:15:15) has the highest a* (1.06), which indicates a more reddish color, while CF3 (70:25:5) has the lowest a* (0.70) when compared to all composite flours. All composite flours were significantly different from one another, and significantly different from wheat flour, mocaf flour, arrowroot flour, and suweg flour. In contrast to a*, it was known that all composite flours were not significantly different in b*. All composite flours have a higher level of red color, but a lower level of yellow color compared to wheat flour.Functional properties are physicochemical properties that affect the behavior of components during the process of preparation, processing, storage, and consumption. The functional properties observed in this study include water absorption capacity (WAC), swelling volume, and solubility, which are presented in The water absorption capacity (WAC) of the composite flour ranges from 1.62 g/g to 1.65 g/g, swelling volume ranges from 21.01 mL/g to 25.33 mL/g, and solubility ranges from 12.86% to 16.20%. The WAC of CF1, CF2, and CF3 were not significantly different from each other, but were significantly different from wheat flour (1.11 g/g). The swelling volume of all composite flours was significantly different from wheat flour. The WAC and swelling volume of the composite flours were significantly higher than wheat flour. The texture is one important factor that affects the final product made from the resulting flour. The characteristics of the texture can be tested using the Texture Profile Analyzer (TPA) with the parameters of hardness, springiness, cohesiveness, adhesiveness, resilience, and chewiness .Compared to all gluten-free pancake products, P1 had the highest hardness and chewiness and was not significantly different from pancakes made from wheat flour. Apart from hardness and chewiness, P1 was also not significantly different compared to pancakes made from wheat flour in adhesiveness. All pancake samples showed no significant differences in cohesiveness and resilience. Overall, the P1 pancake product had the most texture characteristics resembling wheat flour pancakes.Color is the main attribute of the appearance of food products and is an important characteristic of their quality. Color is one of the determining indicators for consumers in purchasing a food product because it is a parameter that can be assessed directly before buying a product. The color of all pancake samples can be seen in P3 has the highest L* (49.52), which indicates a higher level of brightness, while P1 has the lowest L* (43.70) compared to all gluten-free pancakes. However, the pancake made from wheat flour still had the highest L* (52.20) when compared to all pancake products. All gluten-free pancake products have a* ranging from 9.47 to 13.74, whereas all gluten-free pancake products have a* that were significantly different from one another. P2 had the highest a* (13.74) compared to other gluten-free pancake products, which had a more reddish color and was not significantly different from wheat flour, while P3 (70:25:5) has the lowest b* (9.47). All gluten-free pancake products had lower red and yellow color levels compared to wheat flour.Analysis of the organoleptic characteristics of pancake products was carried out using a hedonic test to determine the panelists’ preference and acceptance of the products. The parameters in this hedonic test include color, aroma, texture, taste, and overall appearance. The scoring for this hedonic test was 1 (dislike very much), 2 (dislike), 3 , 4 (like), and 5 (very like). The hedonic test was carried out with 20 panelists. The results of the panelists’ preference for pancake products can be seen in Pancakes made from wheat flour still had the preferred color, texture, flavor, and overall appearance. In terms of aroma, P1 had the most preferred aroma by the panelists. Overall, all pancake products have a score above 3, which indicates the product was acceptable to the panelists.w/w), so they were safe enough to prevent the growth of mold [Moisture content of the composite flours were found to be below the maximum required level  increased. Previous research has shown that suweg flour has a high phenolic content (17.66 mg GAE/g) , while aFunctional properties testing was also carried out to determine the properties of the flour to be used in making pancakes. The WAC of the composite flours  were higher than wheat flour. High water binding capacity will increase the reconstitution ability and textural properties of the dough obtained . This waIn pancake products, texture is the most important parameter to determine its quality. It was known that all gluten-free pancake products have significantly different hardness. Of all gluten-free pancakes, the hardness of P1 and P2 were not significantly different from wheat flour pancakes. This shows that P1 and P2 have good texture characteristics in terms of hardness and resemble pancakes made from wheat flour. Hardness was affected by the difference in the ratio of suweg flour in the composite flour. The hardness of the product increases as more suweg flour is added to the composite flour, indicating that the suweg flour affects the hardness of the resulting pancake product. This could be due to the high content of crude fiber in suweg flour causing the hardness of the product to increase. In pancake products, springiness was the desired texture attribute . SpringiColor is one of the determining indicators for consumers in purchasing a food product, so it is one of the criteria that need to be considered when determining the acceptability of the resulting pancake product. Lightness was affected by the ratio of flour used. The higher the ratio of arrowroot flour added, the lighter the product that is produced. This was consistent with research by Lestari et al. , where tIn terms of panelist preferences, results showed that all pancake products were acceptable to panelists. In previous research, the addition of suweg flour produced a taste that was not liked by the panelists . The resBased on the results, all pancake products were acceptable to panelists, indicating that they had good overall characteristics which resembled a pancake made from wheat flour. The existence of positive results in the manufacture of gluten-free composite flour means that it could be an alternative that can replace wheat flour. The results of this research can also be further developed to obtain gluten free composite flour that increasingly resembles wheat flour. The ratio of mocaf flour, arrowroot flour, and suweg flour had a significant effect the characteristics of the flour, such as pasting temperature, peak viscosity, hold viscosity, breakdown viscosity, setback, L*, a*, hue, whiteness, ∆E, as well as swelling volume and solubility. The ratio of mocaf flour, arrowroot flour, and suweg flour also had a significant effect on the texture characteristics of hardness, adhesiveness, chewiness, color characteristics L*, a*, whiteness, ∆E, and preference for the aroma of the resulting gluten-free pancake product. The best formulation for the resulting pancake product was P1, which was composed of 70% mocaf flour, 15% arrowroot tuber flour, and 15% suweg flour with characteristics and organoleptics hardness texture 2698.16, springiness texture 0.98, chewiness texture 2199.06, L* 43.70, a* 12.11, b* 31.41, organoleptic taste 3.76, organoleptic color 3.33, organoleptic texture 3.71, aroma organoleptic 3.67, and overall appearance organoleptic 3.62.


However, the insufficient formation and separation/transfer of photogenerated charges in such materials restricts the efficiency of H2O2 production. Herein, we provide a strategy for the design of donor-acceptor COFs to greatly boost H2O2 photosynthesis. We demonstrate that the optimal intramolecular polarity of COFs, achieved by using suitable amounts of phenyl groups as electron donors, can maximize the free charge generation, which leads to high H2O2 yield rates (605 μmol g−1 h−1) from water, oxygen and visible light without sacrificial agents. Combining in-situ characterization with computational calculations, we describe how the triazine N-sites with optimal N 2p states play a crucial role in H2O activation and selective oxidation into H2O2. We further experimentally demonstrate that H2O2 can be efficiently produced in tap, river or sea water with natural sunlight and air for water decontamination.Solar-driven photosynthesis is a sustainable process for the production of hydrogen peroxide, the efficiency of which is plagued by side reactions. Metal-free covalent organic frameworks (COFs) that can form suitable intermediates and inhibit side reactions show great promise to photo-synthesize H 2O2 photosynthesis from water, air, and sunlight.Solar-driven photosynthesis is a green and sustainable process for hydrogen peroxide production. Here the authors report that optimizing the intramolecular polarity of COFs can greatly boost H At present, oxidation of anthraquinone, electrochemical synthesis, and noble metal catalysis are the common methods used to produce H2O23. However, these methods require high energy input and cause environmental pollution by the release of toxic by-products5. Due to the use of naturally abundant water and oxygen in air as raw materials and solar light as energy input, H2O2 photosynthesis, especially without using sacrificial agents, is regarded as one of the green and sustainable methods, which yet is plagued by the severe side reactions such as the decomposition of produced H2O2 into oxygen and water due to the metastable feature of H2O2 during the synthesis process12. As one type of novel metal-free crystalline polymers that can form suitable intermediates and inhibit side reactions for H2O2 generation, covalent organic frameworks (COFs) recently show great promise in the field of H2O2 photosynthesis14. The insufficient formation or separation of excitons (bound state of electron−hole pairs) for the free charge generation in COFs yet seriously constrains the high-efficiency of H2O2 photocatalytic generation16. Some interfacial modulating strategies such as constructing heterojunction17 and incorporating single atoms18 are beneficial for enhancing the photoexcitation and charge separation/transfer of COFs photocatalysts, which yet require multistep, complex and time/energy-consuming synthesis process. Hence, developing a facile and cost-effective strategy to improve the generation and separation of photogenerated charges without introducing severe side reactions is in great demand for H2O2 photosynthesis, yet remains a great challenge.As a versatile reagent, hydrogen peroxide  as the precursors, a class of D-A COFs with different intramolecular polarity were successfully synthesized. We find that weak intramolecular polarity in D-A COFs constrains excitons dissociation, yet too strong intramolecular polarity inhibits excitons formation via weakened π-conjugated effect as well as decreases the photo-stability of COFs. The well-designed COFs with the optimal intramolecular polarity (named as COF-N32) can facilitate excitons formation and dissociation, leading to the high and stable H2O2 yield (605 μmol g−1 h−1) with solar-to-chemical efficiency of 0.31% in water without additional sacrificial reagent. COF-N32 can also efficiently produce H2O2 in real water samples including tap water, river water and sea water even with natural solar light irradiation. Moreover, COF-N32 can be assembled into practical devices for facile consecutive uses with high photocatalytic stability under natural solar irradiation. In addition, we demonstrate that the produced H2O2 aqueous solution (without further separation) can be directly employed in water decontamination, indicating the potential application feasibility of COF-N32. Via in-situ Fourier transform infrared (FTIR) characterization and density functional theory (DFT) calculation, we reveal that the suitable N 2p states and C 2p states in COF-N32 with optimal intramolecular polarity effectively reduce the energy barrier for H2O activation and oxygen reduction, respectively, contributing to the high efficiency.Herein, we provide a facile and economical strategy for the design of metal-free donor-acceptor (D-A)-type COFs with optimal intramolecular polarity by introducing suitable amounts of phenyl groups as electron donors for excitonic regulation to boost the direct photocatalytic H13C nuclear magnetic resonance (NMR) spectra  carbon and olefin carbon, respectively19. The stretching vibration bands of C=C bond at ~1573 cm−1 and C-N bond at 1255 cm−1 in the FTIR spectra of three COFs  analysis  planes and 2θ = ~ 27° corresponding to (002) planes in the X-ray diffraction (XRD) pattern  and transmission electron microscopy (TEM) images show that the three COFs are tiny granular particles with diameter of 2–3 μm, which are assembled by numerous nanorods  spectra of three COFs under dark condition at room temperature  to 1 (COF-N32) and further to 2 (COF-N33), the intramolecular polarization of composition fragments in three COFs decrease from 0.072 e Å−1 for COF-N31 to 0.032 e Å−1 for COF-N32 and further to 0.020 e Å−1 for COF-N33 , carbonyl group with or Figs.  and S523its Fig. , which iure Fig. . The parure Fig. , indicatN33 Fig. . The dirN33 Fig. . FurtherN33 Fig. .31. Similar observation has also been previously reported for COFs with high polarity32. In contrast, for both COF-N32 and COF-N33, relatively smaller positive shifts of emission peaks (<25 nm) are observed in water relative to other organic solvents  < COF-N32 (139°) < COF-N33 (145°)  is observed in fluorescence spectra of COF-N31 dispersed in water (as a polar solvent) relative to those in methanol, dichloromethane and ethyl acetate (with weaker polarity than water), indicating the strong local dipolar nature of COF-N31 with weak electron donor in π-conjugated systemnts Fig. . The wea5°) Fig. . Meanwhi5°) Fig. .sp2-hybridized orbit structure of COFs, leading to the inhibition of electron excitation35. The O atom with high electronegativity in COF-N31 with strong polarity can attract electrons surrounding amine N atoms, the delocalization on triazine rings in the lowest unoccupied molecular orbital (LUMO) of COF-N31 thus is limited across three atoms (N and C atoms) in each direction  of three COFs in the excited state based on DFT calculation is found to follow the order of COF-N31 (0.33) < COF-N32 (0.36) < COF-N33 (0.38)  Table . The obs39, while COF-N33 with more benzene units also contains more obvious signals of π–π* transition40. The band gaps of COF-N31 (2.72 eV) > COF-N32 (2.42 eV) > COF-N33 (2.40 eV)  indicate that COF-N32 and COF-N33 have relatively smaller charge transfer resistance than COF-N31  determined by temperature-dependent photoluminescence (PL) spectra and the emission peaks intensities in steady-state PL spectra of three COFs follow the order of COF-N33 > COF-N32 > COF-N31  reveals that all three COFs exhibit n–π* transition in N atomseV) Fig.  are alsoeV) Fig. . The obsN31 Fig. , contrib31 Figs.  and S15,42. Among three COFs, photoexcited electrons in COF-N32 exhibits smaller average lifetime (16 ps) relative to those in COF-N31 (46 ps) and COF-N33 (49 ps)  by three COFs   directly to H2O2 (1.77 V vs. NHE) is thermodynamically feasible by COF-N32 (1.88 V vs. NHE) and COF-N33 (1.92 V vs. NHE) but not by COF-N31 (1.63 V vs. NHE) (further discussion is provided below).The transient absorption (TA) was performed to analyze charge carrier dynamics in three COFs. The broad and positive absorbance changes (ΔA) observed around 500–700 nm can be attributed to the photoinduced absorption of photoexcited electrons in the conduction band (CB) of three COFs Fig. 41,42. Amps) Fig. , which ips) Fig. . The obssis Fig.  shows thsis Fig.  and COF-sis Fig. . The gensis Fig. . The abo3) Figs.  and S2182O2 by COFs was evaluated in pure water without using any sacrificial agent under visible light irradiation . During 12 h reaction duration, COF-N32 with moderate intramolecular polarity in the octupolar conjugated structure exhibits significantly improved performance for H2O2 photosynthesis relative to COF-N31 (with strong intramolecular polarity) and COF-N33 (with weak intramolecular polarity). Specifically, after 12 h of visible light irradiation, H2O2 yield by COF-N32 reaches 7092 μmol g−1 (605 μmol g−1 h−1), which is greatly higher than that by COF-N31  and COF-N33   could be degraded by metal-free COF-N32 under visible light irradiation  Figs.  and S22.ion Fig. , indicatO3 Figs.  and S22.ion Fig. , which iion Fig. . In addiion Fig. . The sol2O2 yield and excellent reusability for 5 reused cycles  by COF-N31 is much lower than that by COF-N32  by COF-N32 after 12 h with visible light irradiation   was also investigated under simulated visible light irradiation. Regardless without or with using rubber plug to prevent the penetration of water through membrane in reactor under gravity, COF-N32 can efficiently produce ~30 μmol H2O2 in 2 h for the successive four cycles  Fig. . Due to ely Fig. . The H2Oles Fig. .2O2 photosynthesis by COF-N32 was further examined under natural sunlight irradiation  glass slide to further ease its recovery and reuse for practical application  under visible light irradiation  under natural sunlight irradiation  analysis shows that the number of electrons transferred from COF-N32 to O2 is estimated to be 2.17  into reaction system significantly inhibits the production of H2O2 with negligible yield, suggesting ·O2– is an intermediate product crucial for the H2O2 photosynthesis. Note that ·O2– is the reduction product of O2 by electrons (e–) photo-generated by COFs  yet can oxidize H2O to form adsorbed *OH  has negligible effect on H2O2 yield by COF-N32 (p > 0.1), indicating that diffusing ·OH does not have contribution to the photocatalytic process of H2O2 production.The generation process of Hng Figs.  and S37,.17 Fig. , indicatOFs Eq. S. As statest Fig. . The DMPOFs Fig. . This inFs Figs.  and S41,O2 Eqs. S and S6. 2O2 Eq. S. Consist2O2 Eq. S, suggest2O2 generation process. The results show that H2O adsorbed onto surfaces of COFs without light irradiation can be dissociated into C-OH– (1094 cm−1) and TzH+ (1508 cm−1) in triazine rings on COF-N32  is also generated  (1.77 V vs. NHE)52. The production of H2O2 in half-reaction  under the N2 atmosphere  shows that the amount of 16O2 and 18O2 generated from the decomposition of H2O2 is generally equivalent  can decrease the yield of H2O2 under visible light irradiation  in the in-situ FTIR spectra of COF-N32  and C = C-O (1264 cm−1) in the in-situ FTIR spectra of COF-N32 further suggest the reduction of O2 occurs at C atoms of COF-N32.In-situ FTIR spectra in sealed chamber was further employed to reveal the reactive sites on COF-N32 during the HN32 Fig. 50. To fued Figs.  and S44.ere Fig.  confirmsion Fig. . The isoent Fig. , which fion Fig. . The obs32 Figs.  and S4852O2 photosynthesis process by COFs was further revealed by theoretical calculation. The calculated PDOS plots show that the valence band top and conduction band bottom in all three COFs mainly consist of N 2p and C 2p states, respectively  among three COFs. Meanwhile, the energy barrier of oxygen reduction into H2O2 by COF-N32 is only 0.24 eV, which is also the lowest among COFs. Hence, the highest H2O2 yield is obtained by COF-N32 with light irradiation.The Hely Fig. . This im2O2 by COFs under light irradiation, we propose a strategy to facilitate the formation and dissociation of excitons in COFs through optimizing the intramolecular polarity of COFs by introducing suitable amount of phenyl group as electron donors. We fabricate a class of COFs with different intramolecular polarity by using triazine-cored triamine with different amount of phenyl group  as the precursors. We find that among all three COFs, COF-N32 with moderate intramolecular polarity in the octupolar conjugated structure can generate greatest amount of charges (i.e. electrons) for H2O2 photosynthesis under light irradiation. Without the requirement of sacrificial agent, the H2O2 yield by COF-N32 reaches 7092 μmol g−1 (605 μmol g−1 h−1) after 12 h of visible light irradiation with solar-to-chemical efficiency of 0.31% and high AQY of 6.2% at 459 nm. Moreover, we find that COF-N32 can also efficiently produce H2O2 in more available real water samples including tap water, river water and sea water. COF-N32 can be assembled into practical devices for consecutive uses with high photocatalytic stability. COF-N32 either dispersed in membrane filter reactors or immobilized onto glass slides can efficiently produce H2O2 under natural sunlight irradiation. During the H2O2 photosynthesis process, suitable N 2p states and C 2p states in COF-N32 with optimal intramolecular polarity reduce the energy barrier for H2O activation and oxygen reduction, respectively, contributing to the high efficiency. This study not only provides deep insight into the design of COFs via regulating its intramolecular polarity to boost the H2O2 photosynthesis without using sacrificial agent, but also paves the way for the practical application of COFs-based photosynthesis of H2O2.In summary, in order to enhance the yield of HN,N-dimethylacetamide (DMAc)/acetic acid following the method reported in previous literature22. Specifically, 0.3 mmol of Tp (63 mg) and 0.3 mmol of melamine (38 mg) were added into a reactor, followed by the addition of 2 mL of dimethyl sulfoxide, 1 mL of DMAc and 0.3 mL of 6 M acetic acid. After ultrasonication and degassed by three consecutive freeze-pump-thaw cycles, the reactor was sealed under vacuum condition, which was then heated at 120 °C for 72 h. The collected product was firstly rinsed by DMAc, which was then solvent exchanged with DMAc, pure water and washed with acetone for three times. The final product was dried at 120 °C under vacuum.Unlike COF-N32 and COF-N33 that can be fabricated by using one-pot solvothermal method based on the Schiff-base reaction between two types of precursors in mesitylene/dioxane/acetic acid solvent, COF-N31 yet can not be successfully synthesized by the mixture of 1,3,5-triformylphloroglucinol (Tp) and melamine in mesitylene/dioxane/acetic acid solvent due to its instability in this solvent. Instead, COF-N31 were fabricated in dimethyl sulfoxide/2 for 20 min. After that, the Teflon lining was sealed in an autoclave and heated at 120 °C in the oven for 72 h. The mixture was separated through filtration and the solid was rinsed by acetone for five times, which was finally dried at 60 °C. The precursors-to-COFs efficiency is 76% for COF-N31, 78% for COF-N32 and 75% for COF-N33, respectively.COF-N32 and COF-N33 were synthesized by mixing Tp with the precursors with triazine and triamine in mesitylene/dioxane/acetic acid solvent, respectively. Briefly, 0.9 mmol (190 mg) of Tp and 0.9 mmol of 4,4',4''--trianiline  or 4,4',4''-tris)  were added into a Teflon lining with the volume of 20 mL. Then, dioxane (4.5 mL), mesitylene (4.5 mL) and 3 M acetic acid (1.5 mL) were added. The mixture was ultrasonicated and bubbled with N2O2 was first performed in a double wall quartz reactor with 50 mL of ultrapure water and 25 mg of photocatalysts . The initial solution pH of the mixture was adjusted by NaOH or HClO4. The reaction suspension was irradiated by using a 300 W Xenon lamp . The temperature was fixed at 25.0 ± 0.2 °C by circulating water system during the photocatalytic experiments. The reaction suspension was extracted and filtered for H2O2 measurement at specific time intervals. The reusability of three COFs was investigated. The H2O2 production by COF-N32 in various real waters  was evaluated under both simulated visible light and natural solar light. COF-N32 were employed in commercial membrane reactors and further immobilized onto ITO glass with the size of 10 cm × 10 cm for the continuous H2O2 photosynthesis.The photosynthesis of H13C NMR , FTIR  were employed to reveal the chemical and structural information of COFs. In-situ FTIR spectra measurement , UV-vis DRS , steady state PL spectra, time-resolved PL decay curve, temperature-dependent PL spectra , TA spectrometer , ESR analysis , water adsorption analysis , RRDE , 18O isotopic experiment and EIS  were performed to investigate the mechanisms of H2O2 photosynthesis by prepared COFs.XRD , XPS , SEM , TEM , Supplementary InformationPeer Review File


Tylopilus neofelleus and their function during the absorption of chelated inorganic phosphorus by host pine through the ectomycorrhizal system. The results showed that the endofungal bacterial microbiota in the fruiting body of T. neofelleus might be related to the dissolution of chelated inorganic phosphorus in soil. The soluble phosphorus content in the combined system of T. neofelleus and endofungal bacteria Bacillus sp. strain B5 was five times higher than the sum of T. neofelleus-only treatment and Bacillus sp. strain B5-only treatment in the dissolution experiment of chelated inorganic phosphorus. The results showed that T. neofelleus not only promoted the proliferation of Bacillus sp. strain B5 in the combined system but also improved the expression of genes related to organic acid metabolism, as assesed by transcriptomic analysis. Lactic acid content was five times higher in the combined system than the sum of T. neofelleus-only treatment and Bacillus sp. strain B5-only treatment. Two essential genes related to lactate metabolism of Bacillus sp. strain B5, gapA and pckA, were significantly upregulated. Finally, in a pot experiment, we verified that T. neofelleus and Bacillus sp. strain B5 could synergistically promote the absorption of chelated inorganic phosphorus by Pinus sylvestris in a ternary symbiotic system.Ectomycorrhizal fungi play an irreplaceable role in phosphorus cycling. However, ectomycorrhizal fungi have a limited ability to dissolve chelated inorganic phosphorus, which is the main component of soil phosphorus. Endofungal bacteria in ectomycorrhizal fruiting bodies are always closely related to the ecological function of ectomycorrhizal fungi. In this study, we explore endofungal bacteria in the fruiting body of IMPORTANCE Ectomycorrhizal fungi (ECMF) have a limited ability to dissolve chelated inorganic phosphorus, which is the main component of soil phosphorus. In the natural environment, the extraradical hyphae of ECMF alone may not satisfy the phosphorus demand of the plant ectomycorrhizal system. In this study, our results innovatively show that the ectomycorrhizal system might be a ternary symbiont in which ectomycorrhizal fungi might recruit endofungal bacteria that could synergistically promote the mineralization of chelated inorganic phosphorus, which ultimately promotes plant phosphorus absorption by the ectomycorrhizal system. Ectomycorrhizal fungi (ECMF) are typical symbionts and play an irreplaceable role in promoting nutrient cycling in the ecosystem , 2. ECMF3–Previous studies have shown that ECMF release certain compounds, such as trehalose, to recruit specific bacteria that could promote the growth of ectomycorrhizal hyphae and ectomycorrhizal formation from soil bacterial microbiota –11, and –14–Tylopilus neofelleus  is a typical ectomycorrhizal fungus that can form ectomycorrhizae with pine trees and fruiting bodies. Here, we will explore the endofungal bacterial microbiota in the fruiting body of T. neofelleus and their function and mechanism to synergize dissolved chelated inorganic phosphorus with T. neofelleus. Furthermore, a pot experiment was used to verify the promoting effect of the combined system of ECMF and their endofungal bacteria on the absorption of phosphorus by pine through its ectomycorrhizal system.T. neofelleus fruiting body and the corresponding bacterial microbiota in mycosphere soil. The results showed that bacterial richness was significantly different between the endofungal bacterial microbiota in the T. neofelleus fruiting body and the soil bacterial microbiota in mycosphere soil. The Shannon index, abundance-based coverage estimator (ACE) index, and Chao index of the endofungal bacterial microbiota were much lower than those of mycosphere soil bacterial microbiota (P < 0.001), while the Simpson index was much higher than that in the mycosphere soil bacterial microbiota (P < 0.001) . Proteobacteria was the most abundant bacterial phylum in the T. neofelleus fruiting body and mycosphere soil, accounting for 94.2% and 40.48%, respectively . Moreover, Rhizobiales and Burkholderiales dominated in the fruiting body, whereas Rhizobiales and Rhodospirillales dominated in the mycosphere soil at the order level . Richness of the top 10 bacterial communities was also significantly different at the phyla and order levels . Nonmetric multidimensional scaling analyses (NMDS) showed that bacterial diversity was different between the endofungal bacterial microbiota and mycosphere soil bacterial microbiota at the genus, order, and phylum levels   (P < 0.01) (P < 0.05) . The T. neofelleus-only treatment also had a weak ability to dissolve chelated inorganic phosphorus . Furthermore, the synergistic phosphorus dissolution abilities of Bacillus aerius strain B3, Bacillus sp. strain B5, and Bacillus altitudinis strain B14 with T. neofelleus were significantly higher than those in the other treatments. The combined system of Bacillus sp. strain B5 and T. neofelleus had the highest soluble phosphorus content in the dissolution experiment of chelated inorganic phosphorus . Thus, we selected the endofungal bacteria Bacillus sp. strain B5 that could adhere to the mycelial surface of T. neofelleus to further study the dissolution characteristics and mechanism of chelated inorganic phosphorus in the combined system .A previous study showed that fungi may choose endofungal bacteria that can support the functional roles of these fungi in terrestrial ecosystems, so endofungal bacteria involved in the release of minerals were relatively more enriched in the ectomycorrhizal fruiting body . In thisg bodies . Moreove= 0.008) . By cont < 0.01) . In this−1 in the combined system, while it was only 53.78 mg L−1 and 4.41 mg L−1 in the Bacillus sp. strain B5-only treatment and T. neofelleus-only treatment after 10 days, respectively (P < 0.05)  (−1) compared to the Bacillus sp. strain B5-only treatment (221.248 mg L−1) and T. neofelleus-only treatment   was also collected after 4 days of different treatments. Compared with the Bacillus sp. strain B5-only treatment, the amount of Bacillus sp. strain B5 increased significantly in the combined system at different times (P < 0.001) . Furthermore, the enzyme activities of alkaline phosphatase, acid phosphatase, and phytase were measured following different treatments (Table S2). There was no significant difference in alkaline phosphatase activity or alkaline phosphatase gene expression levels between the Bacillus sp. strain B5-only treatment and the combined treatment (P > 0.05) . Moreover, genomic analysis revealed the absence of acid phosphatase and phytase genes in Bacillus sp. strain B5. Thus, there were also no significant differences in acid phosphatase and phytase activities between the T. neofelleus-only treatment and the combined treatment (P > 0.05).The results showed that the soluble phosphorus content was 274 mg L < 0.05) . The sol= 0.002) ; Fig. S7 < 0.05) . To inve< 0.001) . The resBacillus sp. strain B5 in the combined treatment , l-glutamate γ-semialdehyde dehydrogenase , aconitate hydratase , and dihydrolipoamide dehydrogenase , was significantly upregulated (Bacillus sp. strain B5 was cocultivated with T. neofelleus in the combined treatment (gapA and pckA) were increased in the combined treatment (gapA [P = 0.023] and pckA [P = 0.020]) , the expression of eight randomly selected genes was assessed and showed similar trends to the RNA-seq data (P < 0.05) .Gene Ontology (GO) enrichment analysis of differentially expressed genes was then performed . Most of the differentially expressed genes with predicted functional annotations were assigned to biological process, cellular component, and molecular function . Among the differentially expressed genes, the organic acid catabolic pathway was significantly upregulated in reatment , in whicegulated . We thenreatment . In addi 0.020]) . These rPinus sylvestris seedlings could form an ectomycorrhizal structure with T. neofelleus in the T. neofelleus-only treatment and the combined treatment, including root binary branches, mantles, and Hartig nets . There was no significant difference in the ectomycorrhizal colonization rates of P. sylvestris between the T. neofelleus-only treatment and the combined treatment (P > 0.05) (Table S3). Moreover, the phosphorus content in the plant was significantly higher in the combined treatment and T. neofelleus-only treatment than in the Bacillus sp. strain B5-only treatment and the control treatment in the pot experiment (P < 0.05) (P < 0.001) . However, there was no significant difference in the available phosphorus content among the different treatments in the mycelial compartment (P > 0.05) . The total phosphorus and available phosphorus in vermiculite showed the same trend . These results indicate that the combined treatment of ECMF and endofungal bacteria significantly promotes the uptake of chelated inorganic phosphorus by P. sylvestris through the ectomycorrhizal system. Moreover, the pH values in the mycelial compartment of the other three treatments were slightly lower than that observed in the control treatment (P < 0.05) . Pinus sylvestris seedlings under the combined treatment were significantly taller than those under the control treatment in terms of shoot length, root length, and dry weight (P < 0.05) . By contrast, the number of lateral roots was not significantly different at 90 days after planting (P > 0.05) . < 0.05) . The pla< 0.001) . We alsoRhizobiales and Burkholderiales, which had strong mineralization abilities, were dominant in the endofungal bacterial microbiota in the fruiting body (P < 0.05) (P < 0.05) . A previous study also showed that the endofungal bacterial microbiota in ectomycorrhizal fungi was associated with chelated state mineral dissolution functions  . Furtherunctions . Moreoveunctions , 22. ThuT. neofelleus and Bacillus sp. strain B5 (P < 0.05)  (Bacillus sp. strain B5 proliferation in the combined system (P < 0.05)  (gapA and pckA) associated with lactate metabolism were significantly upregulated in Bacillus sp. strain B5 in the combined system    , which m < 0.05)  and prom < 0.05) . Previou < 0.05) . The sol < 0.05) . Moreovenic acid . Our res < 0.05) . The exp= 0.002) . Previouegularis . In our  < 0.05) . Previou < 0.05) , 27. ThuBacillus sp. strain B5 and T. neofelleus promoted the absorption of chelated inorganic phosphorus by P. sylvestris through the ectomycorrhizal system. In the buffer compartment of the pot, the content of available phosphorus was lower, and the total phosphorus was significantly higher in the T. neofelleus-only treatment and the combined treatment than in the other treatments (P < 0.05) , which indicated that the phosphorus might mainly be transported to plants through ectomycorrhizal hyphae from the hyphal compartment. Previous studies have mainly shown that the synergistic interaction between AMF and PSB could also enhance phosphorous uptake in plants through arbuscular mycorrhizal hyphae . Moreover, a previous study suggested that AMF combined with trifoliate orange (Poncirus trifoliata L. Raf.) seedlings could improve plant resistance to stress by promoting the growth of plant roots . The results suggest that ECMF might promote the growth of the plant lateral roots only under low-phosphorus stress conditions . Only mature fruiting bodies were sampled, excluding immature and decaying samples. Mycosphere soil samples (~2 g) underneath and adhered to the fruiting body stalk were collected.Fungal fruiting bodies of T. neofelleus were isolated from the fruiting body and cultivated on modified Pachlewski medium  at 25°C for 4 weeks as the fungal inoculum.Mycelia of 3) were obtained using a sterile scalpel, avoiding contact with the outer surface of the fruiting body, and subsequently cultivated in 1.5-mL tubes containing liquid LB medium (1 mL) (−1 and 10−4) after 3 days of incubation and cultivated on the surface of the corresponding agar medium for 7 days. Single colonies were collected and purified based on their different morphologies from the agar medium. The 16S rRNA gene of each isolate was amplified using the universal bacterial primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-CTACGGCTACCTTGTTACGA-3′) under the following PCR conditions: 94°C for 5 min, 35 cycles of 94°C for 30 s, 50°C for 30 s, 72°C for 1 min, and a final elongation step at 72°C for 10 min  . The liqACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHGGGGTWTCTAAT-3′) were used to amplify the V3-V4 regions of the bacterial 16S rRNA gene .Total DNA was extracted from each mycosphere soil sample and fungal inner tissue with the commercial FastDNA spin kit for soil  according to the manufacturer’s protocols. Illumina MiSeq sequencing was performed for the detection of endofungal bacterial microbiota in fruiting bodies and mycosphere soil using an Illumina MiSeq PE300 platform at Majorbio Bio-pharm Technology Co., Ltd. . Primers 338F , and low-quality reads were removed by Trimmomatic. Qualified sequences were clustered into operational taxonomic units (OTUs) at the 97% identity threshold using the Usearch program (version 7.1). The taxonomic classification of OTUs was annotated with the Ribosomal Database Project (RDP) classifier (http://rdp.cme.msu.edu/), and the obtained data were used for subsequent analyses. NMDS was implemented using the vegan package in the R version 3.20 environment . The molecular functions of microbiota from each sample were predicted by PICRUSt based on KEGG databases. All raw reads were archived in the NCBI Sequence Read Archive database (accession number PRJNA882605).Sequences were quality filtered using Quantitative Insights into Microbial Ecology (QIIME) pipeline software  at 5,000 × g for 5 min and resuspended in saline to the final inoculum concentration. The dissolution ability of seven endofungal bacterial strains on chelated inorganic phosphorus was determined in the four treatments as follows: bacteria-only treatment, 10 μL of different bacterial inocula (106 CFU mL−1) was added to 50 mL of modified National Botanical Research Institute’s phosphate (NBRIP) medium ; T. neofelleus-only treatment, 5-mm clumps of T. neofelleus inoculum were added to 50 mL of NBRIP medium; combined treatment, 10 μL of different bacteria inocula and 5-mm clumps of T. neofelleus inoculum were added to 50 mL of NBRIP medium; control treatment, 50 mL of NBRIP medium without bacteria and T. neofelleus inoculum. All treatments were incubated on a rotary shaker (180 rpm and 30°C).Seven endofungal bacterial strains were cultured in liquid LB medium for 12 h at 200 rpm and 37°C and centrifuged at 5,000 × Bacillus sp. strain B5 to further study the dissolution characteristics and mechanism of chelated inorganic phosphorus in the combined system of endofungal bacteria and T. neofelleus because it could adhere to the mycelial surface of T. neofelleus to establish a combined system, which had the highest soluble phosphorus content in the dissolution experiment of chelated inorganic phosphorus . Samples of Bacillus sp. strain B5-only treatment, T. neofelleus-only treatment, and the combined treatment of Bacillus sp. strain B5 and T. neofelleus were taken every 2 days to determine the content of soluble phosphorus using the molybdenum blue method  and ultraviolet absorption detector (UVD). The mobile phases were A (0.05 M H2SO4 [adjusted pH to 2.75 with H2SO4]) and B (methanol). HPLC conditions were as follows: 95% A and 5% B at a flow rate of 0.5 mL min−1 and detection at 210 nm with an oven temperature of 30°C. The solutions were filtered with a 0.22-μm filter before HPLC injection.Organic acids in different treatments were analyzed by HPLC as described by Xu et al. . HPLC anThe activities of acid phosphatase (ACP), alkaline phosphatase (ALP), and phytase were determined in the culture after filtration through a 0.22-μm membrane (Merck Millipore) –35.–Bacillus sp. strain B5 and T. neofelleus and Bacillus sp. strain B5-only treatment was sampled at 4 days according to the dissolution assay of chelated inorganic phosphorus. Harvested bacterial cells were obtained after removing the fungal clump , immediately frozen with liquid nitrogen, and stored at −80°C. The methods for RNA extraction, sequencing, and annotation are described in the Supplementary Appendix. The experiment was performed with three individual replicates. The RNA-seq reads were deposited in the Sequence Read Archive of the GenBank database under accession number PRJNA880718.For RNA-seq, the combined treatment of Bacillus sp. strain B5 cells was extracted using an RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. To eliminate genomic DNA contamination, the extracted RNA was treated with DNase at 42°C for 60 s. The integrity and concentration of RNA were determined by gel electrophoresis and a NanoDrop 2000 spectrophotometer (Thermo), respectively. Approximately 500 ng of RNA was used to generate the first-strand cDNA using a HiScript II first strand cDNA synthesis kit . RT-qPCR was performed on a 7500 real-time PCR system (Applied Biosystems) with AceQ qPCR SYBR green master mix . The PCR conditions were as follows: 95°C for 5 min and 40 cycles of 95°C for 10 s, 60°C for 30 s, and 72°C for 20 s. A melting curve was recorded at the end of each run to verify the specificity of the reaction. The 16S rRNA gene of Bacillus sp. strain B5 was used as a reference gene to normalize the expression of selected genes using the cycling threshold (2−△△CT) method.Total RNA from frozen −1 Ca3(PO4)2, respectively. Pinus sylvestris seeds were surface sterilized with 70% (vol/vol) ethanol for 3 min and 3% (vol/vol) sodium hypochlorite for 3 min and washed three times with sterilized distilled water (repeated three times). The surface-sterilized seeds germinated in the dark at 28°C on sterilized vermiculite, and five seedlings were transplanted to the root compartment after being cultivated for 28 days. Furthermore, T. neofelleus inoculum (5 g) was added into the plant compartment when needed in different treatments, while the autoclaved T. neofelleus inoculum (5 g) served as a control. The Bacillus sp. strain B5 inoculum  was added into the hyphal compartment when needed in different treatments, while the autoclaved Bacillus sp. strain B5 inoculum served as a control. Thus, there were four treatments as follows: control treatment, no inoculation of T. neofelleus and Bacillus sp. strain B5; T. neofelleus-only treatment, inoculation with T. neofelleus and sterilized Bacillus sp. strain B5; Bacillus sp. strain B5-only treatment, inoculation with Bacillus sp. strain B5 and sterilized T. neofelleus; combined treatment, inoculation with T. neofelleus and Bacillus sp. strain B5. All treatments were cultivated in a controlled environment greenhouse  at Nanjing Normal University  for 90 days. Fresh sterilized phosphorus-free Hoagland nutrient solution  was added every 2 weeks. The ectomycorrhizal structure was observed using the toluidine blue method after 3 months  separated by 30-μm nylon mesh and a 1-cm buffer compartment from the mesh toward the hyphal compartment . There were 340 g, 40 g, and 130 g of vermiculite in the plant compartment, buffer compartment, and hyphal compartment with 2.5 μg g3 months .2SO4-H2O2 solution. Inorganic phosphorus concentrations in the digestions and extracts were determined using the molybdenum blue method. Moreover, the lateral roots of P. sylvestris seedlings randomly selected from each treatment were embedded in paraffin, and the cross sections were taken for the determination of the ectomycorrhizal colonization rate based on the presence of mantles or Hartig net structures.Plant growth parameters, including root length, shoot length, dry weight, and the number of lateral roots, were measured at 90 days after planting. The dried plant sample (0.1 g) was ground and digested with H2SO4-H2O2 solution and extracted with 0.5 mol L−1 NaHCO3 for Olsen P. Inorganic phosphorus concentrations in the digestions and extracts were determined using the molybdenum blue method (The vermiculite from the three compartments was collected and air dried. The dried vermiculite (0.2 g) was digested with He method . Soil pHpost hoc Tukey and least significant difference (LSD) multiple range test, Mann-Whitney U test, independent sample t test, and one-way analysis of variance via Duncan’s honestly significant difference, as appropriate.The data are expressed as mean ± standard error (SE). Statistical analyses were performed with SPSS v. 18.0  using Kruskal-Wallis analysis of variance, PRJNA882605). The RNA-seq reads were deposited in the Sequence Read Archive of the GenBank database (accession number PRJNA880718).All raw reads of high-throughput sequencing were archived in the NCBI Sequence Read Archive database (accession number


UBE3A gene in neurons. The paternally inherited UBE3A allele is unable to compensate because it is silenced by the expression of an antisense transcript (UBE3A-ATS) on the paternal chromosome. UBE3A, encoding enigmatic E3 ubiquitin ligase variants, regulates target proteins by either modifying their properties/functions or leading them to degradation through the proteasome. Over time, animal models, particularly the Ube3amat−/pat+ Knock-Out (KO) mice, have significantly contributed to our understanding of the molecular mechanisms underlying AS. However, a shift toward human pluripotent stem cell models (PSCs), such as human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), has gained momentum. These stem cell models accurately capture human genetic and cellular characteristics, offering an alternative or a complement to animal experimentation. Human stem cells possess the remarkable ability to recapitulate neurogenesis and generate “brain-in-a-dish” models, making them valuable tools for studying neurodevelopmental disorders like AS. In this review, we provide an overview of the current state-of-the-art human stem cell models of AS and explore their potential to become the preclinical models of choice for drug screening and development, thus propelling AS therapeutic advancements and improving the lives of affected individuals.Angelman syndrome (AS) is an imprinted neurodevelopmental disorder that lacks a cure, characterized by developmental delay, intellectual impairment, seizures, ataxia, and paroxysmal laughter. The condition arises due to the loss of the maternally inherited copy of the  By integrating the ubiquitin-proteasome protein degradation pathway and possibly other regulatory processes, UBE3A regulates protein function and/or degradation of several specific targets through its ubiquitination activity. As a result, disruption of normal UBE3A expression is thought to affect several key neuronal processes necessary for normal synaptic function and plasticity. While printing . The lacPostmortem analysis of human AS tissues, animal models such as mouse, rat, or Drosophila, and in vitro cellular studies, have all furthered the knowledge of this disease and its mechanisms (Ube3amat−/pat+ Knock-Out (KO) mouse (Our current understanding of AS has been built upon studies using different models. chanisms . The mosO) mouse  which adO) mouse . Here, w‘Puppet’ children. A report on three cases . The mystery was later solved by the discovery that the parental origin of the chr15q11-q13 MD dictated the disease presentation: inheritance of the paternal deletion results in PWS, while maternal deletion results in AS (“D15S63 (PW71) and SNRPN” thanks to microdeletions found in AS and PWS individuals and further fine-tuned to two regions now known as AS and PWS ICs (AS-IC and PWS-IC). PWS-IC, also known as SNURF TSS-DMR, was then confirmed to hold a DMR inheriting the methylation mark only from the maternal germline (It was not until the late 1980s that the first genetic aberration was linked to AS. Two studies reported megabase deletions (herein named megadeletions or MDs) within the chr15q11-q13 region present in individuals with AS . At the ts in AS  Figure 76270. Thts in AS . This wats in AS , while mts in AS . Once thts in AS , severalts in AS . Unusualts in AS . This regermline .UBE3A as the gene implicated in AS  were yet to be identified. In 1997, two back-to-back publications undoubtedly pinpointed ed in AS  , soon followed by another one also using oligonucleotide-based therapeutics (NCT04428281). Other attempts to perturb UBE3A-ATS transcript, by taking advantage of the CRISPR/Cas9 genetic editing technology, are under development with promising results achieved in the mouse model , early infantile epileptic encephalopathy , FOXG1 syndrome (OMIM#613454), Christianson syndrome , or Pitt-Hopkins syndrome   . Given tUBE3A gene is located, is regulated by genomic imprinting , exclusively expressed from the paternal allele. SNHG14 encodes the bicistronic SNURF/SNRPN gene pair and several long and small RNAs. These include IPW, PWAR1 long noncoding RNAs (lncRNAs), and tandem-repeated C/D snoRNA genes clustered in two domains, known as SNORD116, which is ubiquitously expressed, and SNORD115. At the 3’ end tip of the SNHG14 transcript unit sits the UBE3A-ATS lncRNA which overlaps with the UBE3A gene , conserved sites, and CTCF (CCCTC-binding factor) binding motifs, results in the extension of the SNHG14 transcript genetic defects (UBE3A gene—AS Mut (10%); paternal uniparental disomy of chromosome 15—AS UPD (10%); imprinting defects on the maternal PWS-IC—AS ID (3%–5%). Some cases (<10%), despite having AS-like clinical diagnosis, are not attributed to any of the four known (epi)genetic causes and may arise from genetic abnormalities in other genes , which are genes implicated in neuronal development, synaptic function, and epilepsy   involved in brain development and function (Significant heterogeneity in disease severity is observed across the different (epi)genetic origins of AS . AS MD pB3 genes   . Class Ifunction . The diffunction . Howeverfunction . FurtherUBE3A, and results in overexpression of paternally expressed imprinted genes, such as SNURF/SNRPN, IPW, SNORD115/116, and UBE3A-ATS , most of which are nonsense mutations that lead to frameshifts and premature stop codons genetic causes, with less pronounced development delay , and if negative, AS-like syndromes should be investigated. Instead, if DNA methylation is anomalous, the second step is to determine whether or not the patient has a large deletion of the chr15q11-13 region, by MPLA, fluorescence in situ hybridization (FISH) for chr15q11-q13, or a comparative genomic hybridization array (array CGH). If a maternal MD is detected, the patient has AS MD. If instead, no megadeletion is present, microsatellites or nucleotide polymorphism markers for chr15 are used to assess if the patient has AS UPD  or AS ID .Molecular diagnosis of AS is important not only to rule out other clinically similar diseases but also to understand the (epi)genetic cause underlying the clinical AS diagnosis, which could impact disease management. Given the distinct (epi)genetic causes of AS, more than one molecular diagnostic test is needed . If an iUBE3A, also known as E6-associated protein (E6AP), is a 100 kDa protein that tags proteins for proteasomal degradation or for acquiring novel properties through target-specific ubiquitination  Figure . UbiquitGiven the pivotal role of UBE3A loss-of-function in AS, its downstream targets may be relevant to uncover pathophysiological mechanisms and potential therapeutic targets for the disease. Examples of these ubiquitination targets include tumor suppressor p53 , synaptiUbe3a showed severe behavior impairments and synaptic defects, while KO mice for the cytoplasmic isoform were asymptomatic  templates to discern the relative contribution of each isoform for the building up of the AS phenotype  or against UBE3-ATS (to increase expression) and repeated their proteomic workflow. A range from 70 to 225 proteins were identified with the main outcome being the discovery of the secreted retrovirus-like GAG-domain-containing protein PEG10 and associated proteins as being upregulated in AS neurons  testing inhibitors of UBE3A targets or 2) using molecules targeting the molecular cause of the disease. To exemplify the first case,  miR-134 . An antiUBE3A-ATS RNA and can be achieved in multiple ways . A similar trial from Roche using the same technology also reached phase I clinical trials . This is illustrative of the power of stem cell-based research to study diseases such as AS.Targeting the se model . This pr neurons . More re effects . These fIn this article, we provide a comprehensive overview of stem cell-based research in AS and contextualize it within the historical advancements made over the past ∼50/60 years in understanding this disease. Although the use of human stem cell models of AS is relatively recent, it already provided significant scientific progress in uncovering novel pathophysiological aspects of the disorder, as well as in identifying new proteins affected by UBE3A that could be targets for future therapeutic interventions. Additionally, stem cell models are serving as valuable preclinical tools for evaluating human sequence-specific genetic therapies such as ASOs or CRISPR-based gene editing. In the forthcoming years, the AS stem cell model portfolio will likely expand, hopefully encompassing the full spectrum of (epi)genetic variability observed in this condition. Current concerns in the epigenetic and genetic fidelity of human stem cells are being tackled  and hope


The adsorption of industrial pollutants over C6N6 occurs through physisorption, with adsorption energies ranging from −9.36 kcal/mol to −16.46 kcal/mol. The non-covalent interactions of analyte@C6N6 complexes are quantified by symmetry adapted perturbation theory (SAPT0), quantum theory of atoms in molecules (QTAIM) and non-covalent interaction (NCI) analyses. SAPT0 analyses show that electrostatic and dispersion forces play a dominant role in the stabilization of analytes over C6N6 sheets. Similarly, NCI and QTAIM analyses also verified the results of SAPT0 and interaction energy analyses. The electronic properties of analyte@C6N6 complexes are investigated by electron density difference (EDD), natural bond orbital analyses (NBO) and frontier molecular orbital analyses (FMO). Charge is transferred from the C6N6 sheet to HCN, H2S, NH3 and PH3. The highest exchange of charge is noted for H2S (−0.026 e−). The results of FMO analyses show that the interaction of all analytes results in changes in the EH-L gap of the C6N6 sheet. However, the highest decrease in the EH-L gap (2.58 eV) is observed for the NH3@C6N6 complex among all studied analyte@C6N6 complexes. The orbital density pattern shows that the HOMO density is completely concentrated on NH3, while the LUMO density is centred on the C6N6 surface. Such a type of electronic transition results in a significant change in the EH-L gap. Thus, it is concluded that C6N6 is highly selective towards NH3 compared to the other studied analytes.Industrial pollutants pose a serious threat to ecosystems. Hence, there is a need to search for new efficient sensor materials for the detection of pollutants. In the current study, we explored the electrochemical sensing potential of a C Another industrially demanding chemical is NH3, which is a colourless gas with a pungent smell. It is most widely used in the pharmaceutical and fertilizer industries [3 is applied in silicon processing [In the last few decades, industrial pollutants have become serious threats to living beings. Industrial pollutants are adversely deteriorating our ecosystem. It is reported that every year, 4.2 million people are affected by direct exposure to industrial pollutants at levels beyond the safety limits . Among io humans . Hydrogeo humans . In caseaterials . It rapidustries ,9. Phospocessing .2 nanoparticles [2N) [The above-mentioned facts demonstrate the need to design an effective electrochemical sensor for environmental monitoring of these hazardous gases. In the recent past, numerous materials such as ZrOarticles , carbon articles , metal oarticles ,12, poroarticles , carbon les [2N) , hexagonles [2N) , metal oles [2N)  and zeolles [2N)  have beeles [2N) . In addiles [2N) , boron nles [2N) , carbon les [2N) , metal cles [2N)  and phosles [2N)  have beeles [2N) ,24. Mateles [2N) ,27,28,29les [2N) ; photoniles [2N) ; chemicales [2N) , mechaniles [2N)  and DNA les [2N) ; electroles [2N) ; and eleles [2N)  applicatles [2N) , transitles [2N) , silicenles [2N) ,35,36, ples [2N) , etc., ales [2N) . Engineeles [2N) ,40. Manyles [2N) . CertainAnother newly developed class of 2D and 3D materials are covalent organic frameworks, which consist of a variety of organic units as monomers. In 2005, Yaghi et al. synthesized COF1 through a mechanism based on reticular chemistry . Covalen6N6), which contains six nitrogens in its cavity. Recently, C6N6 has been used in many fields such as catalysis [6N6 surface is more comparable to other available COFs for the detection of H-containing industrial pollutants such as HCN, H2S, NH3 and PH3. The high amount of nitrogen in the C6N6 unit also leads to a high surface area and a cavity size of 5.46 Å, which can efficiently interact with various analytes through H bonding, π–π stacking or other types of chemical interactions. According to the best of our knowledge, C6N6 has not yet been tested as a sensor for the above-mentioned analytes. In the designed study, we used density functional theory simulations to investigate C6N6 as a sensor surface. Changes in geometrical properties are explored through NCI, QTAIM and SAPT0 analyses, while changes in the electronic properties of analytes in C6N6 complexes are determined by NBO, EDD and FMO analyses.Keeping in view the above-mentioned characteristics of covalent triazine frameworks (CTFs), we selected a recently designed covalent triazine framework  level of theory. The Ꞷb97XD functional was adopted in the current study because it is a range-separated functional which can effectively capture non-localized intermolecular interactions [6N6 before and after adsorption of analytes were explored by NBO, EDD and FMO analyses at the B3LYP/6-31G(d) level of theory. B3LYP/6-31G(d) is considered as the best to study the electronic changes of interacted systems [6N6 complexes were investigated by adsorption of analytes through different orientations of C6N6. A frequency analysis was performed to confirm that the geometries of the complexes were true minima on the potential energy surface. The energies of adsorbed analytes in C6N6 are determined by Equation (1): is the interaction energy of the complexes, EC6N6 is the energy for bare C6N6 and Eanalytes is the energy of the industrial pollutants used as analytes.Gaussian 09 software was used to carry out the simulations in the current study. The selected geometries of bare Cractions . In Ꞷb97ractions . Additioractions . The cha systems ,63,64,656N6 complexes [ind), dispersion (∆Edisp), electrostatic (∆Eelstat) and exchange (∆Eexch). Hence, the SAPT0 energy is calculated through Equation (2):A SAPT0 analysis was applied to determine the role of the individual components of the interaction energy in the stabilization of the analyte@Complexes . The tot6N6, was examined by an NCI analysis. The nature and strength of non-covalent interactions is given by the reduced density (s), Equation (3) [The nature of adsorption, such as the electrostatic, dispersive and repulsive forces between the analyte and Ction (3) .(3)s=12(ρ) for non-covalent interactions is generally very small. However, small changes in (ρ) result in prominent changes in RDG values, which appear in the form of spikes in a 2D-RGD plot. An NCI analysis characterizes non-covalent interactions through 3D-isosurfaces and 2D-RGD plots. These plots use ρ and the second eigen value (λ2) to differentiate between the nature of interactions [The value of electron density ractions ,69,70.6N6 was further studied through a QTAIM analysis. Through a QTAIM analysis, we can examine the nature of the interactions, which cannot be explored through any other method. The major factor through which non-covalent interactions can be studied through QTAIM analysis is the bond critical point (BCP). BCPs are based on various parameters such as the Laplacian (ρ), electron density (ρ), total energy density (H), potential energy density (V) and the Lagrangian kinetic energy density (G) [The interaction between the analytes and Csity (G) ,72,73. Msity (G) ,75,76.6N6 is presented in 6N6 are 1.53 Å and 1.33 Å, respectively, which are consistent with previously reported theoretical and experimental values [6N6 comprises of six C3N3 rings which are connected with each other through C-N bonds [6N6 has a high electron density due to the centring of the nitrogen atoms towards the cavity. The diameter of the C6N6 cavity (between two nitrogen atoms) is 5.46 Å [6N6 can act as a potential surface for the detection of hydrogen-containing industrial pollutants. The topologies of selected industrial pollutants  were also simulated at the Ꞷb97XD/6-31G  level.The optimized structure of the selected model of Cl values . Each un-N bonds . The cavs 5.46 Å . The nit6N6, several orientations of each analyte in C6N6 were considered. 6N6 complexes. The most stable geometries of each analyte@C6N6 complex are reported in 6N6 > H2S@C6N6 > NH3@C6N6 > PH3@C6N6.In order to obtain stable complexes of analyte@C6N6 complexes, the most stable complex is formed between HCN and C6N6, with an adsorption energy value of −16.46 kcal/mol. In this complex, HCN is projected to be perpendicular to the C6N6 cavity . This happened because of the repulsion between the N atom of HCN and the N atoms of C6N6.Among the analyte@Cvity see . The H a2S@C6N6 is obtained with an adsorption energy of −13.64 kcal/mol, which is the 2nd most stable complex among all studied analyte@C6N6 complexes. The optimized topology of the H2S@C6N6 complex shows that both H atoms of H2S are positioned towards the C6N6 cavity. Interaction distances of 2.90 Å (N1---H7), 2.29 Å (N2---H7) and 2.68 Å N2---H7 are noted between N atoms  of C6N6 and the H atom (H7) of H2S  compared to the H2S@C6N6 complex  is attributed to longer interaction distances between the H atoms of NH3 and the N atoms of C6N6 compared to H2S.The stable complex of NH3@C6N6, which is the lowest amongst all studied analyte@C6N6 complexes. The stable structure of the PH3@C6N6 complex consists of an inward pointing H atom of PH3, while the other two H atoms point upwards. The inward pointing H atom of PH3 is almost at the centre of the C6N6 cavity. The bond distances between the H atoms of PH3 and the N atoms of C6N6 range from 2.70 Å to 3.09 Å. The low adsorption energy  of the PH3@C6N6 complex is due to the low polarity of the H atoms of PH3 and the longer interaction distances. The results of adsorption energies  show all analytes  physically absorb on the C6N6 sheet.An adsorption energy of −9.36 kcal/mol is observed for the stable complex of PH6N6. The SAPT0 results of all the studied complexes are presented in 6N6 sheet.A SAPT0 analysis is the most effective method for quantifying non-covalent interactions. The SAPT0 interaction energy is the sum of four factors, including induction, exchange, electrostatic and dispersion. Therefore, in SAPT0, the role of each factor is investigated in the stabilization of analytes in C6N6), −12.26 kcal/mol (H2S@C6N6), −10.06 kcal/mol (NH3@C6N6) and −9.22 kcal/mol (PH3@C6N6). Among the studied H-containing industrial pollutants over C6N6, the highest SAPT0 value  is obtained for the HCN@C6N6 complex, which is consistent with the results of the interaction energy analysis. The SAPT0 attractive component values for the HCN@C6N6 complex are Eelst , Eind  and Edisp . These values indicate that Eelst is the major stabilizing factor compared to Eind and Edisp. The highest value of Eelst results from the strong interactions of the H atom of HCN with the N atoms of C6N6 through a shorter interaction distance (vide supra).The SAPT0 energies of all the complexes obtained through PSI4 are −16.88 kcal/mol , −4.27 kcal/mol (15%) and −12.34 kcal/mol (42%), respectively. These values of the attractive components show that Eelst (43%) and Edisp (42%) play a major role in the stabilization of the H2S@C6N6 complex, while a lower contribution is observed for Eind (15%). The SAPT0 values for the NH3@C6N6 and PH3@C6N6 complexes are −10.06 kcal/mol and −9.22 kcal/mol, respectively. The SAPT0 component analysis for the NH3@C6N6 complex reveals that Eelst (47%) and Edisp (40%) are dominant in the stabilization of NH3 over C6N6. Among the studied pollutant complexes, the lowest SAPT0 value is observed for the PH3@C6N6 complex . The lowest SAPT0 value for the PH3@C6N6 complex results from the lower polarity of the interacting H atoms of PH3 with the N atoms of C6N6. Among the SAPT0 energy, a key role is noted for Edisp (55%) in the stabilization of the PH3@C6N6 complex, while Eelst (33%) and Eind (12%) contribute less towards the total SAPT0 energy.The second highest SAPT0 value is seen for the Helst is the dominant contributing factor in the case of HCN@C6N6, whereas a good balance between Eelst and Edisp is observed for H2S and NH3. In the case of PH3, the dispersion interaction played a dominant role in the stabilization of PH3 over C6N6. The SAPT0 values of all the studied complexes are consistent with the interaction energy analysis.The SAPT0 analysis reveals that E6N6 was further explored through non-covalent interaction analyses. Through an NCI analysis, the van der Waals, electrostatic and repulsive interactions can be differentiated based on a colour scheme. An NCI analysis comprises 3D isosurfaces and 2D reduced density gradient (RDG) plots. The 3D isosurface topological analysis is based on three types of colours: red (repulsive), green  and blue (electrostatic interactions). These colours appear in the form of patches between the analytes and C6N6 through which the nature of interactions can be differentiated. A 2D RDG plot is obtained by taking the reduced density on the Y-axis and product of 2nd value of the Laplacian (sign(λ2)) and density gradient on the X-axis. A 2D RGD plot gives information about the strength of each type of interaction. In 2D RDG plots, non-covalent interactions appear as blue, green and red spikes in the low density gradient region. The vdWs interactions are indicated by the green spikes which form the when product of sign(λ2)2)2)The nature of interactions between analytes and C6N6. The strength of the vdWs interaction in each complex is different, indicated by the variation in the thickness of 3D isosurface patches and a projection of green spikes in the 2D-RDG plots on the X-axis. In the case of the HCN@C6N6 complex, a ring on the green isosurface develops between the H atom of HCN and the six N atoms of C6N6. This shows that all six N atoms of C6N6 strongly interact with the H atom of HCN. Similarly, in the 2D-RDG plot, a mixture of bluish-green spikes arises at a low-density gradient between −0.01 and −0.02 a.u., which indicates that the complex is stabilized through electrostatic interactions. In the case of the H2S@C6N6 complex, a stronger interaction is observed, where the H atoms of H2S closely interact with the N atoms of C6N6 , (ρ), (H), (G) and (V). The values of the BCP parameters of all complexes are given in QTAIM analysis is a useful tool to investigate all non-covalent interactions which are impossible to capture through any other analysis. Through QTAIM analysis, inter- and intra-molecular interactions such as ionic interactions, hydrogen bonding, van der Waals interactions and covalent bonds can be studied. These inter- and intra-molecular interactions are studied by various parameters such as cage critical points (CCP), nuclear critical points (NCP), ring critical points (NCP) and bond critical points (BCPs). In QTAIM analyses, non-covalent interactions are best explained by BCPs. BCPs between two interacting systems are represented by a brown line see . The natρ(r) < 0, whereas for close shell interactions, the value of ρ(r) > 0. For electrostatic interactions (H bonding), the values of (ρ) and (ρ) at BCPs are between 0.024 and 0.139 a.u. and 0.002 to 0.034 a.u., respectively [The nature of interaction is shared if ectively ,80. In aectively . The strectively . For H bectively .(4)Eintρ and H. The values of ρ and H are less than zero for shared shell interactions and greater than zero for close shell interactions. If the values of ρ are greater than zero and H is less than zero, it indicates H bonding. Another parameter used to explain the BCP is the ratio -V/G. If the ratio of -V/G is >2, it indicates covalent bonding, and a ratio of −V/G of < 1 represents non-covalent interactions [In addition, the BCP can further be investigated by Equation (5):ractions ,85.6N6 cavity with the H atoms of interacting analytes. However, the strengths and types of bonding in all complexes are different due to the different values of their BCP parameters  are in the strong electrostatic region, while the rest of the BCPs indicate dispersion interactions , ρ (.01586 a.u), H (0.00 a.u.) and Eint  are observed for H7---N2 and H8---N5, respectively. The BCP parameter such as ρ, ρ and Eint of H7---N2 and H8---N5 are in the range of H bonding, but no such types of blue isosurface and spikes are seen in the 3D isosurfaces and 2D-RGD plots of NCI analysis. The interaction distances between H7 and N2 and H8 and N5 are 2.29 Å each, which is greater than the 1.8 Å required for interacting system to demonstrate H bonding [A topological analysis of the Hions see . Again,  bonding .3@C6N6 complex are in the range of medium to strong interactions between NH3 and C6N6. The strongest interactions are observed between H7 and N2 and H9 and N6 of NH3 and C6N6, respectively. The values of these two BCP parameters ρ, ρ and Eint are almost comparable , −0.026 e− (H2S), −0.015 e− (NH3) and −0.008 e− (PH3). These values of charges show that the analytes extracted charge from C6N6. In all studied analytes, the H atoms of analytes interacted with the electron rich cavity of C6N6, due to which charge is transferred from C6N6 to the analytes. The highest charge value is extracted by H2S (−0.026 e−) due to a close interaction of the H atoms of H2S with the N atoms of C6N6. Similarly, the accumulation and depletion of electron density in the interacting sites of C6N6 and H2S clearly indicate that the nitrogen of C6N6 transferred more charge towards H2S. In the case of HCN, a charge of −0.019 e− is determined, which is contrary to its interaction energies. The EDD analysis of HCN adsorption over C6N6 shows that charge is transferred from the N atoms of C6N6 to the H atom of HCN. However, the lowest values of charge are observed in NH3 (−0.015 e−) and PH3 (−0.008 e−) due to the weak interactions of these analytes with the C6N6 sheet.The charge transfer values from C6N6 and analyte@C6N6 complexes are shown in 6N6 are −7.16 eV and −3.30 eV, respectively, with a HOMO–LUMO energy gap (EH-L gap) of 3.86 eV. The interaction of selected industrial pollutants with C6N6 causes prominent changes in the energy levels of the HOMO, LUMO and EH-L gap. The computed values of the EH-L gaps of HCN@C6N6, H2S@C6N6, NH3@C6N6 and PH3@C6N6 complexes are 4.02 eV, 2.76 eV, 2.58 eV and 3.35 eV, respectively.The electrical signal produced by an electrochemical sensor mainly depends on changes in its electronic properties after interaction with an analyte. To explore these changes in electronic properties of sensors, a frontier molecular orbital analysis was performed. The energy values determined from an FMO analysis of bare C2S, NH3 and PH3 on C6N6 causes a pronounced increase in the HOMO energy, while the LUMO energy of these complexes remains almost unchanged. However, adsorption of HCN shows the inverse behaviour, where a significant decrease in the HOMO energy (−7.43 eV) is noticed compared to C6N6 (−7.16 eV). A more pronounced decrease in the HOMO energy causes an increase in the EH-L gap (4.02 eV) of the HCN@C6N6 complex compared to bare C6N6 (3.86 eV). This increase in the EH-L gap of the HCN@C6N6 complex indicates a decrease in the conductivity of C6N6. As a result, C6N6 cannot act as a good sensor for HCN.The results presented in 2S onto C6N6 resulted in a prominent increase in the energy of the HOMO (−6.07 eV), while the LUMO energy (−3.31 eV) is almost comparable to bare C6N6 (−3.30 eV). This increase in the HOMO energy resulted in a pronounced reduction in the EH-L gap (2.76 eV) of the H2S@C6N6 complex. Among the different industrial pollutants adsorbed on C6N6, the most significant increase in electrical conductivity is observed for the NH3@C6N6 complex due to an appreciable decrease in the EH-L gap. The energies of the HOMO and LUMO for the NH3@C6N6 complex are 5.85 eV and −3.27 eV, respectively. The interaction of PH3 changes the HOMO (−6.62 eV) and LUMO (−3.27 eV) energies in such a way that a negligible decrease in the EH-L gap (3.35 eV) is observed for the PH3@C6N6 complex. Among the studied analyte@C6N6 complexes, the highest decrease in the EH-L gap is observed for NH3@C6N6. This indicates that C6N6 can act as potential surface for this analyte. Thus, it is concluded that C6N6 is highly selective towards NH3 compared to the other studied analytes.Adsorption of H2S, NH3 and PH3) onto C6N6, the dispersal of orbitals densities was also explored in order to comprehend the sensing abilities of C6N6. We observed three types of orbital distribution patterns. In the case of the HCN@C6N6 complex, the orbital densities are localized on the triazine ring. In the HOMO, the orbital densities are observed on the carbon and nitrogen of the triazine ring, while in the LUMO, the orbital densities are shifted to the carbon of the triazine ring. This shows that analytes do not participate in the electronic shift from the HOMO to the LUMO  level of theory. The adsorption of industrial pollutants over C6N6 occurred through physisorption, with adsorption energies ranging from −9.36 kcal/mol to −16.46 kcal/mol. Among the considered analytes, the maximum energy is observed for HCN@C6N6 . The non-covalent interactions of analyte@C6N6 complexes are explored through symmetry-adapted perturbation theory (SAPT0), quantum theory of atoms in molecules (QTAIM) analyses and non-covalent interaction (NCI) analyses. The attractive components in SAPT0 analyses show that electrostatic and dispersive interactions play a dominant role in the stabilization of analytes in the C6N6 cavity. Similarly, NCI and QTAIM analyses also verified the findings of SAPT0 and interaction energy analyses. In addition, the electron density (ρBCP) and Laplacian of electron density (∇2ρbcp) remained high for the HCN@C6N6 complex, which is consistent with the NCI and SAPT0 results. The electronic properties of analyte@C6N6 complexes are investigated by electron density difference (EDD), natural bond orbital analyses (NBO) and frontier molecular orbital analyses (FMO). Charge is transferred from the C6N6 sheet to HCN, H2S, NH3 and PH3. The highest exchange of charge is noted for H2S (−0.026 e−). The results of FMO analyses show that the interaction of all analytes results in changes in the EH-L gap of the C6N6 sheet. However, the highest decrease in the EH-L gap (2.58 eV) is observed for the NH3@C6N6 complex compared to other studied analyte@C6N6 complexes. The orbital density pattern shows that the HOMO density is completely concentrated on NH3, while the LUMO density is transferred towards C6N6. This indicates that C6N6 can act as potential surface for these analytes. Hence, which analyte is more selective towards C6N6 depends on its electrical conductivity and not its adsorption. Thus, it is concluded that C6N6 is highly selective towards NH3 compared to the other studied analytes.The electrochemical sensing application of C