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234094807 | Bacterial chemical reactions, such as urea hydrolysis can induce calcium carbonate precipitation. The induced production of calcium carbonate formed by microorganisms has been widely used in environmental and engineering applications. The present study aimed to isolate, identify and optimize growth conditions of urease positive bacteria from urea rich soil in Gaza Strip. Bacterial isolates, which tolerated ≥10% urea concentration, were selected for the investigation. Eight isolates recovered and identified to be spore forming, urease positive, alkaliphile, halotolerant, and presumptively belonged to Bacillus species. All isolates showed best growth at temperature 37°C, and pH 9-9.5. After exposure to UV irradiation, most isolates showed improved tolerance to urea concentration, however, other strains showed a decline in their adaption to urea concentrations. The mutant form of isolate in soil sample #3 showed the highest tolerance to urea concentrations at all exposure intervals, when compared with wild type. Moreover, all isolates precipitated calcium carbonate. The locally recovered isolates are promising contributors in the process of calcite Biomineralizaion and may be utilized in the remediation of concrete cracks, increase of compressive strength of concrete, decrease water permeability, and solve the problems of soil erosions. | 1 |
234094807 | INTRODUCTION Biological precipitation of minerals (Bio-mineralization) is a widespread phenomenon in the microorganism's world, and is mediated by bacteria, fungi, protists, and even by plants. Calcium carbonate (Calcite) is one of those minerals that naturally precipitate as a by-product of microbial metabolic activities (Seifan and Berenjian, 2019). Microbial metabolic activities facilitate calcium carbonate (calcite) precipitation, in a well-studied process called microbial induced calcium carbonate precipitation (MICP) (Zambare et al., 2019). MICP usually occurs due to the chemical alteration of the environment induced by the microbial activity (Sarikaya, 1999;Stocks-Fischer et al., 1999;Warren et al., 2001;De Muynck et al., 2010a). Bacteria can be invested as a major player in the MICP phenomenon through various mechanisms. The most significant mechanism is the bacterial ureolytic activity (Stocks-Fischer et al., 1999;Warren et al., 2001;Krajewska, 2018). Urea hydrolysis can be facilitated by *Corresponding author. E-mail: [email protected]. Tel: 00970594157573. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License bacteria that can produce urease (urea amidohydrolase) enzyme and thus are able to induce CaCO 3 precipitation (Stocks-Fischer et al., 1999;Hammes and Verstraete, 2002;Phillips et al., 2013;Bhaduri et al., 2016). Calcium carbonate precipitation is a chemical process controlled mainly by four key factors: (1) calcium ions concentration, (2) dissolved inorganic carbon (DIC) levels, (3) the pH, and (4) the availability of nucleation sites (Hammes and Verstraete, 2002;Seifan and Berenjian, 2018). Over recent years, MICP has received considerable attention and has been proposed as a potent solution to address many environmental and engineering issues (Seifan and Berenjian, 2019). It has been intensely investigated in bulk systems, sand columns (Dhami et al., 2013a;Seifan et al., 2016;Tziviloglou et al., 2016), and bio-cementation processes (Seifan et al., 2016). It has been found that MICP may drive many potential applications in civil engineering such as enhancing stability of slopes and dams, reducing the liquefaction potential of soil, road construction, prevention of soil erosion, increase durability and compressive strength of concrete, as well as the repair of the cracks in concrete (De Muynck et al., 2010a;Stabnikov et al., 2011, Shashank et al., 2016. Many bacterial species have been studied to exploit their abilities in the biomineralizing of calcite (MICP). One of the most robust ureolytic bacteria is Sporosarcina pasteurii (formerly known as Bacillus pasteurii). S. pasteurii is facultative anaerobes, spore forming, bacilli bacteria. It utilizes urea as an energy source and eliminates ammonia which increases the pH in the environment and generates carbonate, causing Ca 2+ and CO 3 2to be precipitated as CaCO 3 (Clive, 1990, Stocks-Fischer et al., 1999Chahal et al., 2011). Other studies showed the role of bacteria that are mostly related to Bacillus spp. in the process of MICP (Stocks-Fischer et al., 1999;Elmanama and Alhour, 2013;Ali et al., 2020). The aim of this study is to isolate, identify, and optimize growth conditions of locally isolated urease-producing bacteria that are able to produce calcite crystals. | 2 |
234094807 | Sample sources and characteristics This study utilized soil samples varying in urea content from Gaza Strip. About 100 g of each sample were collected during June 2020 from the following sources: (1) Sea coastal sand from Rafah city beach, (2) outlet sewage water (treated sewage), (3) inlet sewage water (untreated sewage), (4) soil sample with cat`s urine from Gaza city, (5) coastal sand with dog`s urine from Rafah city beach, (6) sand with dog`s urine from Rafah city, (7) agricultural soil with dog`s urine from Gaza city, (8) agricultural soil with dog`s urine from Rafah city, (9) urea rich soil from greenhouse in Gaza city, and (10) ammonia rich soil from a greenhouse in Gaza city. | 3 |
234094807 | Sample processing and bacterial isolation Five grams of soil samples were mixed with 20 ml sterile saline (stock suspension) and dilutions 10 -1 and 10 -2 were made. A volume of 0.1 ml of the stock suspension as well as the two dilutions were cultured onto 2 and 3% urea containing Nutrient Agar (NA) plates (HiMedia, India). The media were prepared according to HiMedia manufacturer recommendations. Extra pure urea suspensions (Honeywell Riedel-de Haen, Germany) were filtered, then added to media after autoclaving and cooling to 50°C. Cultures were incubated at 37°C, and plates were examined after 24 and again after 48 h. | 4 |
234094807 | Bacterial tolerance to high urea concentration Bacterial isolates were obtained as a pure culture and then cultured on 5, 8, 10, 12, and 15% urea enriched NA media, incubated at 37°C for 48-72 h, and after the incubation period bacteria were harvested to be cultivated in nutrient broth and agar plates. Bacterial isolates tolerated ≥ 10% urea concentration were selected for further testing. | 5 |
234094807 | pH profile The isolates were inoculated into 3 ml of Nutrient Broth tubes with pH scale of 1 to 14. A bacterial suspension was made and the turbidity was adjusted to 0.5% McFarland standard, incubated for 24 h at 37°C, and growth has been measured as turbidity at O.D 600 nm using CT-2200 spectrophotometer (Chrom Tech, Taiwan). Results were recorded against a blank of bacterial suspension. 1N HCl (HiMedia, India) and 1N NaOH (Frutarom, Palestine) were used to adjust the pH. An additional nutrient broth tubes at pH 7 were inoculated with the bacterial isolates, incubated at 37°C, and the change in pH was monitored during growth, using a pH meter (Jenway pH Meter 3510 /mV, USA ) results were recorded after 30 min, 1 , 2, 4, 8, 24, and 32 h of inoculation. | 6 |
234094807 | Ultraviolet (UV) induced mutagenesis for bacterial isolates The selected isolates were grown overnight in NB + 2% urea in a shaking incubator (Boeco, Germany) at 37°C. The isolates were washed three times with sterile phosphate-buffered saline, resuspended in urea free and sterile NB. The turbidity of cell suspensions was adjusted to a 0.5% McFarland reagent and exposed to UV light using a Philips 20 W germicidal lamp for 2-20 min with 2 min intervals. From each exposure interval, a loopful of the exposed bacteria was cultured onto urea-based agar (HiMedia, India). After incubation of 24 h at 37°C, a single, well-defined colony was chosen, cultivated on NA plates, and then inoculated onto NA with varying urea concentrations; 5, 8, 10, 12 and 15% respectively. After incubation, bacterial growth was observed and compared to wild type growth on the different urea concentrations. | 8 |
234094807 | Mini-scale of calcium carbonate precipitation Bacterial isolates were subjected to calcium carbonate production test as described previously (Ghosh et al., 2019). Alive bacterial isolates were inoculated into nutrient broth (NB) containing both urea and calcium chloride (NBUC), NB with only urea (NBU), and NB with only calcium chloride (NBC). The same procedures were repeated with autoclave killed bacterial suspension (pellet and supernatant filtrate). To all tubes, a concentration of 0.012 g/L phenol red was used as a pH indicator. NBUC and NBU were prepared to contain 2% of urea. NBUC and NBC were prepared to contain 2.8 g/L of calcium chloride. Urea and calcium chloride solutions were filter sterilized and separately added to phenol red containing NB before bacterial inoculation. A urease enzyme reagent obtained from Blood urea nitrogen kit (Biosystems, Spain) was used as a positive control, while Escherichia coli ATCC 25922 (urease negative) was used as a negative control. Non-inoculated tubes were used as a validity control. All tubes were incubated at 37°C for 24 h. The trial was performed in triplicate. | 9 |
234094807 | Bacterial Identification and biochemical characterization All selected isolates were spore-forming, Gram-positive bacilli, catalase and urease positive. Table 2 shows the phenotypic characteristics of the eight isolates and indicates the biochemical tests that have been used in the identification process. ABIS online Software has been used in bacterial identification (Costin and Lonut, 2017). Table 3 shows the presumptive identification of the selected isolates according to the ABIS online Software. | 12 |
234094807 | Growth conditions The optimal pH at which all selected isolates showed the highest turbidity and rapid growth ranged from 7 to 10, with a preference to the pH 9 (Figure 1), thus all tested isolates are moderate alkaliphiles. For most isolates, the pH of media has increased during growth to reach the maximum of 9 to 9.5. All isolates showed significant growth at temperature 37°C (Table 4). Most isolates showed halophilic characteristic as they grew at NaCl concentration up to 5%. | 13 |
234094807 | Mini-scale calcium carbonate precipitation experiment NBUC tubes for all live isolates, and the pure urease enzyme showed change in pH from neutral to alkaline (yellow to pink), and a precipitate of calcium carbonate were noticed at the bottom of the tubes. NBU tubes for all live isolates and urease enzyme showed only change in pH. NBC tubes for all live isolates and urease enzyme showed neither a change in pH nor calcium carbonate precipitation. Changes in color and pH indicate ureolytic activity (Table 6). A comparison of NBUC of live bacteria versus killed isolates (both killed cells and supernatant) and E. coli, all of them were unable to change pH, so there was no urea hydrolytic activity due to the absence of the enzyme. Consequently, there was no calcium carbonate precipitation. Unchanged non-inoculated tubes suggests that results obtained are reproducible and representative (Table 6). Precipitate containing and non-containing tubes were examined under light microscope to confirm the presence of calcium carbonate crystal (Figure 2). | 15 |
234094807 | DISCUSSION The present study was conducted to isolate, characterize, and optimize locally adapted urease-releasing bacteria that inhabits urea rich soils. Microbial activity that involves the cleavage of urea into ammonia and carbon dioxide by the urease enzyme, leading to the precipitation of carbonate ions as calcium carbonates. This potentially useful application explains the need to enhance urease production by various methods among candidate microorganisms (Vempada et al., 2011). The biochemical profile of the selected isolates showed that all isolates belong to the genus Bacillus (Table 3). This is similar to a previous study that isolated and characterized urease positive bacteria from urea rich soils, in which several isolates were mostly related to the Bacillus group (Ali et al., 2020). Despite the differences in their characteristics, the obtained isolates showed similar behavior in their ureolytic capability. This is in agreement with the findings of Stocks-Fischer et al. (1999); Hammes et al. (2003) and Stabnikov et al. (2011) that reported the same ureolytic Bacillus strains that can be isolated and cultivated using the same followed protocols of isolation and cultivation. Phenotypic and biochemical profiles of the isolates were matched to those Bacillus species reported previously that proved active in MICP process (Stocks-Fischer et al., 1999;Elmanama and Alhour, 2013). Ureolysis-driven MICP is a phenomenon that has many applications for biochemical and engineering purposes (Omoregie et al., 2020). It has been widely investigated for soil stabilization, healing of concrete cracks, restoration of limestone surfaces, preventing soil erosions, and treatment of industrial wastewater and removing heavy metals (Whiffin et al., 2007;Sarda et al., 2009;Van paassen, 2009;De Muynck et al., 2010a;De Muynck et al., 2010b;Wu et al., 2019). All obtained isolates showed ureolytic activity, tolerance to high urea concentrations, as well as calcium carbonate production. This suggests that isolates are potential candidates for the applications of MICP. Isolates 10.1 that was identified as B. mycoides, has been previously isolated and showed an efficient role of increased sand consolidation and compressive strength of cement (Elmanama and Alhour, 2013). Isolate 8.3 has been identified as B. licheniformis, has been reported in a previous study that it was able to precipitate calcium carbonate by ureolysis (Helmi et al., 2016). Bacteria are previously known to breakdown urea in order to: (1) elevate the ambient pH (Burne and Marquis, 2000), (2) consume it as a nitrogen source (Burne and Chen, 2001), and (3) use it as a source of energy (Mobley and Hausinger, 1989). The amount and rate of urea that can be cleaved were influenced by the urea and calcium source (Wang et al., 2017). In this system, urea is the source of the carbonate. The more urea is supplied, the more CaCO 3 can be produced, if a sufficient amount of calcium ions is available (Wu et al., 2019). In this study, isolates that were selected tolerated and grew in the presence of 10 -15% urea concentration. This because urease activity, as well as, calcium carbonate production rate depend on urea concentration. A previous study utilized S. pasteurii and emphasized the role of urea containing cultural medium in the proliferation of bacteria. Moreover, it reported that bacteria cultivated with urea displayed a healthier cell surface and more negative surface charge for calcium ion binding than the bacteria have been cultivated without urea (Ma et al., 2020). Increasingly, it has been reported that the bacterial concentration and ureolytic activity are important contributors in the efficiency of MICP process. The urea hydrolysis is an extremely slow process, whereas the presence of urease enzyme can substantially increase the hydrolysis of urea (De Belie et al., 2018). Therefore, the selection of the bacterial isolates with higher ureolytic activity is desirable for the higher production of calcium carbonate. However, it has been shown that when the content of urea is excessive, bacterial growth and ureolytic activity are inhibited. For instance, when the urea concentration was greater than 0.75 mol/L, the amount of urea breakdown was decreased and thus appears as an inhibitory component. The reason could be due to too high urea molecule transportation over the cell | 16 |
234094807 | A B membrane into the cell, at elevated urea concentrations, inhibiting other cellular processes. Therefore, a certain amount of bacteria can only metabolize a certain amount of urea hydrolysis (Wu et al., 2019). In our study, the local isolates were halo-tolerant, and corroborate with the findings of previous studies (Stabnikov et al., 2013). The observation of the pH tolerance profile of bacterial isolates showed a common moderate alkaliphile property. The best growth was at pH range 7-10 with a preference to pH 9. This is in agreement with a previous study that showed the good alkali tolerance of B. cereus which was successfully used to heal concrete cracks (Stabnikov et al., 2013;Wu et al., 2019). Generally, the optimal pH range for bacterial growth is 7 to 8. Under higher alkaline conditions (pH 9 -12) bacteria can still grow but at a much-declined rate. Although the pH is relatively high in fresh concrete, the pH at cracks may drop to 8-11 due to carbonation, exposure, and humidity (De Muynck et al., 2010a). Above pH 11, the bacteria have a limited capacity to precipitate CaCO 3 , thus limited ability to heal cracks. This implies that bacterial spores will keep dormant after being embedded in the concrete matrix (pH > 12), and only start to become active after cracks appear and crack surface pH drops (Wang et al., 2017;Wu et al., 2019). Therefore, alkaline pH is the primary factor by which bacteria promote calcite precipitation (Castanier et al., 2000;Fujita et al., 2000). Another study showed that the calcium carbonate yield (mg calcium carbonate/CFU) in the presence of Bacillus species increases when bacteria grown at a relatively high pH in compared with those bacteria that grown at uncontrolled pH solution (Seifan et al., 2017). Another study investigated the factors affecting the S. pasteurii induced biomineralization process, reported that the rise in medium pH to 9.5 accelerate bacterial growth (Ma et al., 2020). This may be promising that Bacillus isolates in this study can be used to heal concrete cracks. Especially, in the pH range of 7-11, bacteria will have a remarkable ureolytic activity, which ensures the decomposition of urea and the precipitation of CaCO 3 . This meets also with (Phang et al., 2018) findings, It has been reported that some bacterial ureases exhibited high activity in alkaline conditions at pH of 9. In the present study, the effect of temperature on isolates growth showed a temperatures range from 25 to 40°C. Bacterial mediated urea hydrolysis is an enzymatic reaction controlled by many factors including temperature. It has been reported in the literature that temperature affects bacterial activity, urease activity, and therefore reaction rate. Hence, the rate of formation of biogenic CaCO 3 and crack healing efficiency will be affected as well. Urease activity is stable between 15 and 25°C, and an increase in temperature (until 60°C) results in increased urease activity (Whiffin, 2004;Peng and Liu, 2019). Isolates that were exposed to UV irradiation were compared with their corresponding wild type isolates for the ability to tolerate higher urea concentrations. Most isolates showed improved tolerance to urea concentration. However, other strains showed a decline in their adaption to urea concentrations. This suggests that the mutagenesis process is random and did not correlate to the time of exposure to UV light. This is similar to the findings of a previous study, which used UV irradiation on S. pasteurii in order to improve urease activity (Wu et al., 2019). The established calcium carbonate precipitation process showed that all NBUC tubes containing the viable isolates showed accompanied ureolytic and calcite precipitation activity. On the other hand, NBUC tubes containing the autoclave-killed isolates (pellet or supernatant) showed neither ureolytic nor calcite precipitation activity. This suggests that bacteria activity and urease positivity is a principal contributing to pH change due to urea cleavage, as well as calcium carbonate precipitation. In all NBC tubes inoculated with the viable isolates there was no calcium carbonate precipitation observed. This suggests that calcium carbonate production is enhanced by the change of pH. In NBC tubes (without urea), there was no difference in color change or calcium carbonate precipitation between live bacteria, killed bacteria, or E. coli. All NBU tubes inoculated with the viable isolates showed a change in pH as a proof for the ureolytic activity they possess. Negative control (E. coli) showed no change in pH or calcite production. These findings matched a previous study that reported the ability of urease producing bacteria S. pasteurii to produce calcium carbonate crystals under the same conditions (Ghosh et al., 2019). This is in agreement with the previous studies that reported that Bacillus sp. is with high respect in compared with other genus and that this might be due to their physiological ability to adapt to stressed conditions (Helmi et al., 2016). In conclusion, this study successfully and easily isolated several Bacillus species from locally collected soil samples. These strains are alkaliphile, grow well at pH 7-10, and tolerate high urea concentrations. They showed calcite biomineralizing properties and may be employed in bacterial remediation of concrete cracks, increasing the compressive strength of concrete, decreasing water permeability, and solve the problems of soil erosions. Further studies on a larger scale are recommended to confirm the findings. | 17 |
213695085 | : The catalytic activity of both ZIF-8 and Zr / ZIF-8 has been investigated for the synthesis of chloromethyl ethylene carbonate (CMEC) using carbon dioxide (CO 2 ) and epichlorohydrin (ECH) under solvent-free conditions. Published results from literature have highlighted the weak thermal, chemical, and mechanical stability of ZIF-8 catalyst, which has limited its large-scale industrial applications. The synthesis of novel Zr / ZIF-8 catalyst for cycloaddition reaction of ECH and CO 2 to produce CMEC has provided a remarkable reinforcement to this weak functionality, which is a significant contribution to knowledge in the field of green and sustainable engineering. The enhancement in the catalytic activity of Zr in Zr / ZIF-8 can be attributed to the acidity / basicity characteristics of the catalyst. The comparison of the catalytic performance of the two catalysts has been drawn based on the e ff ect of di ff erent reaction conditions such as temperature, CO 2 pressure, catalyst loading, reaction time, stirring speed, and catalyst reusability studies. Zr / ZIF-8 has been assessed as a suitable heterogeneous catalyst outperforming the catalytic activities of ZIF-8 catalyst with respect to conversion of ECH, selectivity and yield of CMEC. At optimum conditions, the experimental results for direct synthesis of CMEC agree well with similar literature on Zr / MOF catalytic performance, where the conversion of ECH, selectivity and the yield of CMEC are 93%, 86%, and 76%, respectively. | 1 |
213695085 | Introduction The effective transformation and utilization of anthropogenic carbon dioxide (CO 2 ) is a subject of political and environmental debates in recent years, which have been actively pursued by the academia and energy industries in order to promote a sustainable environment [1]. The current level and accumulation of CO 2 in the atmosphere is high and requires urgent attention [2]. However, regardless of environmental regulations and discharge limits placed on greenhouse gases emitted into the atmosphere, CO 2 is believed to be environmentally benign, abundant, nontoxic, non-flammable, and a readily available C1 source for the synthesis of organic carbonate [3]. Therefore, the synthesis of cyclic organic carbonates via the cycloaddition of CO 2 and epoxides is one of the most promising reaction schemes because of its 100% atom efficiency [4]. Cyclic organic carbonates such as chloromethyl ethylene carbonate (CMEC), propylene carbonate (PC), styrene carbonate (SC), and ethylene carbonate (EC) are widely used as polar aprotic solvents, electrolytes for lithium-ion batteries, automobile, cosmetic, fuel additives materials, alkylating and carbonylating reagents, and fine chemicals for pharmaceuticals [5,6]. In the past two decades, several attempts have been made to develop greener and sustainable catalytic systems for chemical fixation of CO 2 . This includes conventional solid catalysts such as zeolites, salen Cr(III) complexes, metal oxides, quaternary ammonium salts, polymer-supported catalysts, ionic liquids (ILs), etc. However, these attempts have failed to yield satisfactory results as most of these catalysts require high temperature and/or pressure (usually around 453 K and pressure higher than 8 atm), further separation and purification steps, and low product yield [7]. This is uneconomical from a commercial point of view and hence, the research has been directed to employ a novel catalyst that provides solutions to all these shortfalls i.e., metal organic framework (MOF). Although, microporous materials such as zeolites, crystalline aluminosilicate, activated carbon, etc. have been known for their high surface area and high porosity, however, their applications have been limited especially in the field of heterogeneous catalysis due to difficulty in pore modification [7]. Metal organic framework (MOF) catalysts are identified as multidimensional porous polymetric crystalline organic-inorganic hybrid materials with exceptional characteristics including an ultrahigh specific surface area, enormous pore spaces, and ordered crystalline structure [8]. MOFs have emerged as a suitable candidate for the synthesis of organic carbonates from CO 2 and epoxide due to their unique heterogeneity and reusability requirements [9]. MOF-based catalysts often display higher catalytic activity than their corresponding homogenous catalysts as evidenced in many catalytic reactions such as ring opening, addition reactions, oxidation reactions, hydrogenation and isomerization [10]. Zeolitic imidazolate frameworks, (ZIFs), is one of the subclasses of MOFs with a similar structure to zeolites. It has attractive structural properties and intrinsically lower density. Many experiments involving ZIF-8 have shown great applications in multidisciplinary fields such as catalysis, drug deliveries, purification and gas storage [11]. Recently, the stability of MOFs for large-scale industrial applications has been questioned in many published papers [11][12][13][14][15]. This is due to their weak thermal, chemical, and mechanical stability due to the structure of inorganic bricks and the nature of the chemical bonds they form with the linker [15]. In order to improve this weak thermal functionality and gain in-depth knowledge of their catalytic activities, Cavka et al. [16] was the first group to synthesize Zr-based MOFs designated as zirconium 1,4-dicarboxybenzene, UiO-66 for photocatalysis [17]. The test conducted by the group found that the increased stability of the Zr-based MOFs is owing to the Zr-O bonds formed between the cluster and carboxylate ligands [18]. Several other groups have thereafter explored this opportunity, which has seen the increased application of Zr-based MOFs in many research activities. Demir et al. [19] utilized UiO (University of Oslo) type zirconium metal-organic frameworks in a solvent-free coupling reaction of carbon dioxide (CO 2 ) and epichlorohydrin (ECH) for the synthesis of epichlorohydrin carbonate (ECHC). The results of their experiments have increased the use of zirconium-based (Zr-based) MOFs for the catalytic synthesis of organic carbonates from CO 2 and epoxides. From our experiments, the synthesis of Zr-doped MOF (Zr/ZIF-8) for the cycloaddition reaction of CO 2 and ECH in the synthesis of chloromethyl ethylene carbonate (CMEC) has demonstrated reasonable thermal stability under relatively mild reaction conditions without using any solvent or co-catalyst. Although, the synthesis of several Zr-based MOFs have been reported in recent times (albeit in early stages), only a few were employed for catalytic studies even more rarely for the synthesis of organic carbonates from CO 2 and epoxides. Zr-based MOFs have exhibited increased structural tailorability as a result of the organic linkers in the catalyst frameworks [19]. Zirconium-based MOFs have demonstrated proof-of-concept applications in several areas such as toxic analyte, catalysis, gas storage, vivo drug delivery, and bio-sensing [20]. In this paper, a novel Zr/ZIF-8 has been successfully synthesized using the conventional solvothermal method. The prepared catalyst has been assessed as an innovative greener and sustainable heterogeneous catalyst for the direct synthesis of CMEC from CO 2 and ECH. The effect of various reaction parameters has been investigated and critically analyzed. These include the effect of reaction time, catalyst loading, temperature, CO 2 pressure, and stirring speed. Catalyst reusability studies of Zr/ZIF-8 was also investigated to establish its stability and reusability for the synthesis of CMEC. | 2 |
213695085 | Catalysts Preparation Preparation of ZIF-8 and zirconium-doped ZIF-8 (Zr/ZIF-8) were synthesized according to a method, which was previously described elsewhere [20,21]. Briefly, 8 mmol of zinc nitrate hexahydrate (Zn(NO 3 ) 2 ·6H 2 O 99.99%) and zirconium (IV) oxynitrate hydrate (ZrO(NO 3 ) 2 ·6H 2 O, 99.99%) solutions in a stoichiometric ratio of Zn:Zr = 10:0 and Zr:Zn = 9:1 (to synthesis ZIF-8 and Zr/ZIF-8 respectively) were dissolved in 6.2 mmol of methanol. A separate solution of 14.2 mmol of 2-methylimidazole and 600 mml of methanol was prepared in another flask, which was added by dropwise addition to the Zr-Zn-based solution. The mixture conducted in an ambient temperature under nitrogen flow was vigorously stirred for 6 h. The Zr-doped ZIF-8 crystals were collected and separated by centrifugation at 300 rpm for 30 min. The solution was washed thoroughly with methanol three times and then dried at room temperature conditions. The crystals were left to dry overnight at 373 K. The greyish-white powders of Zr-ZIF-8 samples were further washed with DMF for 24 h in order to remove any excess of an unreacted organic linker. The solution was then heated at a temperature of 373 K in order to activate it. The samples were allowed to cool to room temperature naturally before being capped in a vial and refrigerated, ready for use in catalytic reactions. The obtained samples were identified with a stoichiometric ratio of Zr:Zn = 10:0 and Zr:Zn = 1:9 for ZIF-8 and Zr/ZIF-8 respectively. | 3 |
213695085 | Experimental Procedure for the Synthesis of Chloromethyl Ethylene Carbonate (CMEC) In a typical cycloaddition reaction, a 25 mL stainless steel high-pressure reactor was initially charged with a specific amount of Zr/ZIF-8 catalyst and the limiting reactant, epichlorohydrin. A desired temperature was set on the reactor's panel controller; the reactor was then sealed and stirred continuously at a known stirring speed. At the desired temperature, a specific amount of liquid CO 2 was charged through a supercritical fluid (SCF) pump into the reactor. The reaction was left for the desired reaction time. After the reaction was completed, the reactor was cooled down to room temperature and the mixture was collected and filtered. The catalyst was separated, washed with acetone, and dried in a vacuum oven. A known amount of methanol (used as internal standard) was added to the product and analyzed using a gas chromatograph (GC). The effect of different reaction parameters was investigated. These include catalyst loading, stirring speed, CO 2 pressure, temperature, and reaction time. Reusability studies of both catalysts were also carried out in order to investigate the stability of the catalysts for the synthesis of chloromethyl ethylene carbonate. | 4 |
213695085 | Method of Analysis A specific quantity of internal standard, methanol added to a known sample of the product was analyzed using a gas chromatography (GC) (Model: Shimadzu GC-2014). The stationary phase was a capillary column with dimensions (30 m length, 320 µm inner diameter, and 0.25 µm film thickness). Oxygen (99.9%) and hydrogen (99.9%) were used as ignition gases. The carrier gas used for the mobile phase was a high purity helium (99.9%) with a flow rate maintained at 1 mL min −1 . A temperature program was developed for the system where both the injector port and detector temperatures were kept isothermally at 523 K. The other selected program includes split ratio of 50:1 and injection volume of 0.5 µL. The column temperature was initially maintained at 323 K for 5 min, then followed by a Energies 2020, 13, 521 4 of 25 temperature ramp at a flow rate of 50 K min −1 to a temperature of 523 K with a 12 min run for each subsequent samples. The chromatogram shows that ECH peak at~3.5 min, methanol at~3.8 min, CMEC at~11 min. | 5 |
213695085 | Proposed Reaction Mechanism The proposed reaction mechanism involves two steps: The ring-opening of epoxides by a catalyst; Incorporation of carbon dioxide into the opening to form the cyclic carbonate. The coupling reaction of CO 2 with epoxides can be initiated by activating either the epoxide or CO 2 or both at the same time [22]. This reaction, using a suitable heterogeneous catalyst, produces desired organic carbonates along with other side products. Figure 1 shows reaction pathways 1, 2, and 3 with corresponding products being chloromethyl ethylene carbonate, 3-chloropropane 1,2-diol, and 2,5-bis (chloromethyl)-1,4-dioxane respectively. The epoxide is activated when the oxygen atom interacts with the Lewis acid, this is then followed by a nucleophilic attack that provokes the opening of the epoxide ring [23] as shown in Figure 2. The activation of CO 2 can occur both through a nucleophilic attack with the oxygen atom as a nucleophile or an electrophilic attack with the carbon atom as an electrophile [24]. Figure 2 shows a proposed reaction mechanism for the synthesis of CMEC, where R is an alkyl group, A is a metal atom with a Lewis acid site, while B is an oxygen atom with a Lewis basic site. Zr/ZIF-8 is a dual-functional catalyst, which contains both the acidic and basic sites that are associated with the Lewis acid Zn 2+ ions and the basic imidazole groups, respectively. The by-products identified with the coupling reaction of CO 2 and ECH as identified by the GC analysis are 3-chloropropane 1,2-diol and 2,5-bis (chloromethyl)-1,4-dioxane (see Figure 1). Figure 3 shows the schematic representation of the reaction of CO 2 and ECH to produce CMEC. Energies 2020, 13, x FOR PEER REVIEW 4 of 26 program was developed for the system where both the injector port and detector temperatures were kept isothermally at 523 K. The other selected program includes split ratio of 50:1 and injection volume of 0.5 μL. The column temperature was initially maintained at 323 K for 5 min, then followed by a temperature ramp at a flow rate of 50 K min -1 to a temperature of 523 K with a 12 min run for each subsequent samples. The chromatogram shows that ECH peak at ~3.5 min, methanol at ~3.8 min, CMEC at ~11 min. | 6 |
213695085 | The ring-opening of epoxides by a catalyst; Incorporation of carbon dioxide into the opening to form the cyclic carbonate. The coupling reaction of CO2 with epoxides can be initiated by activating either the epoxide or CO2 or both at the same time [22]. This reaction, using a suitable heterogeneous catalyst, produces desired organic carbonates along with other side products. Figure 1 shows reaction pathways 1, 2, and 3 with corresponding products being chloromethyl ethylene carbonate, 3-chloropropane 1,2-diol, and 2,5-bis (chloromethyl)-1,4-dioxane respectively. The epoxide is activated when the oxygen atom interacts with the Lewis acid, this is then followed by a nucleophilic attack that provokes the opening of the epoxide ring [23] as shown in Figure 2. The activation of CO2 can occur both through a nucleophilic attack with the oxygen atom as a nucleophile or an electrophilic attack with the carbon atom as an electrophile [24]. Figure 2 shows a proposed reaction mechanism for the synthesis of CMEC, where R is an alkyl group, A is a metal atom with a Lewis acid site, while B is an oxygen atom with a Lewis basic site. Zr/ZIF-8 is a dual-functional catalyst, which contains both the acidic and basic sites that are associated with the Lewis acid Zn 2+ ions and the basic imidazole groups, respectively. The by-products identified with the coupling reaction of CO2 and ECH as identified by the GC analysis are 3-chloropropane 1,2-diol and 2,5-bis (chloromethyl)-1,4-dioxane (see Figure 1). Figure 3 shows the schematic representation of the reaction of CO2 and ECH to produce CMEC. | 8 |
213695085 | Catalyst Characterization The powder X-ray diffraction (XRD) patterns of the samples was analyzed at room temperature with a characteristics peaks range of 5 < 2θ < 35 at a scanning rate of 0.5° min −1 . The catalyst was placed on a zero-background silicon sample holder using a Bruker D8 advance X-ray diffractometer in transmission geometry with CuK radiation ( = 1.5406° A) at 40 kV and 40 mA. The samples were slightly grinded before measurements were taken so as to prevent preferential orientation of individual crystals during sample analysis. The Brunauer-Emmett-Teller (BET) surface area of the as-prepared catalyst was analyzed with a Micromeritics Gemini VII analyzer at room temperature (291 K). Prior to BET analysis, the samples were degreased in a turbomolecular pump vacuum at 423 K for 8 h. The surface area and nitrogen adsorption/desorption isotherm measurements were taken at liquid nitrogen temperature of 77 K (purge gas supplied by BOC, UK). In order to achieve greater degree of accuracy in the accumulation of the adsorption data, the Micromeritics Gemini analyzer was fitted with pressure transducers to cover the range of 133 Pa, 1.33 kPa, and 133 kPa. The Fourier transform infrared (FTIR) spectra (4500-600 cm −1 ) of the samples were obtained using Nicolet Magna-IR 830 spectrometer in KBr disks at room temperature with a resolution of 2 cm −1 . The specimen was mixed KBr in ratio 1:300, the mixture was ground in an agate mortar to a very fine powder. The product was oven dried for 12 h at 373 K, 250 mg of the dry samples were used to make a pallet; the pallet was analyzed, and the spectra were recorded by 32 scans with 4 cm −1 . Particle size morphologies and microstructures of the as-synthesized Zr/ZIF-8 catalyst was examined using the JEOL JSM-35C instrument operated at voltage 20 kV acceleration. Prior to imaging, the specimen was carbon-coated (5-10 nm) under a vacuum condition using Emitech K550X sputter coater, this was done to enhance material conductivity. The particle mean size of the specimen were calculated by taking a manual measurement of about 300 crystals in the SEM images using the field emission scanning electron microscope (FE-SEM). FE-SEM spectra produced were used to examine the particle size and morphology. Transmission electron microscopy (TEM) images of the catalyst were examined using a high resolution TEM (HRTEM)). A sample of the specimen was sonicated in ethanol for 15 min and was then placed by a dropwise onto a carbon film-supported copper grid. The as-prepared sample was allowed to dry at room temperature before inserting into a sample holder. X-ray photoelectron Figure 2. Proposed reaction mechanism for the cycloaddition reaction of CO 2 to ECH over an acid-base pairs. R is an alkyl group, M is a metal atom (acidic site), and O is oxygen atom (basic site). Energies 2020, 13, x FOR PEER REVIEW 5 of 26 Figure 2. Proposed reaction mechanism for the cycloaddition reaction of CO2 to ECH over an acid-base pairs. R is an alkyl group, M is a metal atom (acidic site), and O is oxygen atom (basic site). | 9 |
213695085 | Catalyst Characterization The powder X-ray diffraction (XRD) patterns of the samples was analyzed at room temperature with a characteristics peaks range of 5 < 2θ < 35 at a scanning rate of 0.5° min −1 . The catalyst was placed on a zero-background silicon sample holder using a Bruker D8 advance X-ray diffractometer in transmission geometry with CuK radiation ( = 1.5406° A) at 40 kV and 40 mA. The samples were slightly grinded before measurements were taken so as to prevent preferential orientation of individual crystals during sample analysis. The Brunauer-Emmett-Teller (BET) surface area of the as-prepared catalyst was analyzed with a Micromeritics Gemini VII analyzer at room temperature (291 K). Prior to BET analysis, the samples were degreased in a turbomolecular pump vacuum at 423 K for 8 h. The surface area and nitrogen adsorption/desorption isotherm measurements were taken at liquid nitrogen temperature of 77 K (purge gas supplied by BOC, UK). In order to achieve greater degree of accuracy in the accumulation of the adsorption data, the Micromeritics Gemini analyzer was fitted with pressure transducers to cover the range of 133 Pa, 1.33 kPa, and 133 kPa. The Fourier transform infrared (FTIR) spectra (4500-600 cm −1 ) of the samples were obtained using Nicolet Magna-IR 830 spectrometer in KBr disks at room temperature with a resolution of 2 cm −1 . The specimen was mixed KBr in ratio 1:300, the mixture was ground in an agate mortar to a very fine powder. The product was oven dried for 12 h at 373 K, 250 mg of the dry samples were used to make a pallet; the pallet was analyzed, and the spectra were recorded by 32 scans with 4 cm −1 . Particle size morphologies and microstructures of the as-synthesized Zr/ZIF-8 catalyst was examined using the JEOL JSM-35C instrument operated at voltage 20 kV acceleration. Prior to imaging, the specimen was carbon-coated (5-10 nm) under a vacuum condition using Emitech K550X sputter coater, this was done to enhance material conductivity. The particle mean size of the specimen were calculated by taking a manual measurement of about 300 crystals in the SEM images using the field emission scanning electron microscope (FE-SEM). FE-SEM spectra produced were used to examine the particle size and morphology. Transmission electron microscopy (TEM) images of the catalyst were examined using a high resolution TEM (HRTEM)). A sample of the specimen was sonicated in ethanol for 15 min and was then placed by a dropwise onto a carbon film-supported copper grid. The as-prepared sample was allowed to dry at room temperature before inserting into a sample holder. X-ray photoelectron | 10 |
213695085 | Catalyst Characterization The powder X-ray diffraction (XRD) patterns of the samples was analyzed at room temperature with a characteristics peaks range of 5 < 2θ < 35 at a scanning rate of 0.5 • min −1 . The catalyst was placed on a zero-background silicon sample holder using a Bruker D8 advance X-ray diffractometer in transmission geometry with CuKα radiation (λ = 1.5406 • A) at 40 kV and 40 mA. The samples were slightly grinded before measurements were taken so as to prevent preferential orientation of individual crystals during sample analysis. The Brunauer-Emmett-Teller (BET) surface area of the as-prepared catalyst was analyzed with a Micromeritics Gemini VII analyzer at room temperature (291 K). Prior to BET analysis, the samples were degreased in a turbomolecular pump vacuum at 423 K for 8 h. The surface area and nitrogen adsorption/desorption isotherm measurements were taken at liquid nitrogen temperature of 77 K (purge gas supplied by BOC, UK). In order to achieve greater degree of accuracy in the accumulation of the adsorption data, the Micromeritics Gemini analyzer was fitted with pressure transducers to cover the range of 133 Pa, 1.33 kPa, and 133 kPa. The Fourier transform infrared (FTIR) spectra (4500-600 cm −1 ) of the samples were obtained using Nicolet Magna-IR 830 spectrometer in KBr disks at room temperature with a resolution of 2 cm −1 . The specimen was mixed KBr in ratio 1:300, the mixture was ground in an agate mortar to a very fine powder. The product was oven dried for 12 h at 373 K, 250 mg of the dry samples were used to make a pallet; the pallet was analyzed, and the spectra were recorded by 32 scans with 4 cm −1 . Particle size morphologies and microstructures of the as-synthesized Zr/ZIF-8 catalyst was examined using the JEOL JSM-35C instrument operated at voltage 20 kV acceleration. Prior to imaging, the specimen was carbon-coated (5-10 nm) under a vacuum condition using Emitech K550X sputter coater, this was done to enhance material conductivity. The particle mean size of the specimen were calculated by taking a manual measurement of about 300 crystals in the SEM images using the field emission scanning electron microscope (FE-SEM). FE-SEM spectra produced were used to examine the particle size and morphology. Transmission electron microscopy (TEM) images of the catalyst were examined using a high resolution TEM (HRTEM). A sample of the specimen was sonicated in ethanol for 15 min and was then placed by a dropwise onto a carbon film-supported copper grid. The as-prepared sample was allowed to dry at room temperature before inserting into a sample holder. X-ray photoelectron spectroscopy (XPS) of the samples was recorded on the krato axis ultra DLD photoelectron spectrometer, a surface Energies 2020, 13, 521 6 of 25 science instrument SSx-100 using a monochromatic Al KR X-ray source operating at 144 W. Raman spectroscopy measurements of the specimen were taken at room temperature with the Horiba Jobin Yvon LabRAM spectrometer equipped with an aHeNe laser operating at a wavelength of 633 nm (E ex = 1.96 eV) and Coherent Innova 70 ion laser at a wavelength of 458 nm, 488 nm, and 514 nm. | 11 |
213695085 | Catalyst Characterization The X-ray diffraction patterns of ZIF-8, Zr/ZIF-8 and recycled Zr/ZIF-8 are shown in Figure 4, confirming that Zr/ZIF-8 has high crystal stability under the normal reaction conditions. These results are in agreement with simulated patterns reported in other literature [25][26][27][28]. The decrease in peak intensity of these diffractions was also observed at (2θ = 28-35 • ) indicating the effect of excess doping of Zr into the ZIF-8 framework. The XRD pattern of Zr/ZIF-8 also show a characteristic peak of ZIF-8 with no diffraction peak of zirconium nitrate. temperature with the Horiba Jobin Yvon LabRAM spectrometer equipped with an aHeNe laser operating at a wavelength of 633 nm (Eex = 1.96 eV) and Coherent Innova 70 ion laser at a wavelength of 458 nm, 488 nm, and 514 nm. | 12 |
213695085 | Catalytic Activity After catalyst characterization, the catalytic activity of the novel materials was compared with ZIF-8 for the synthesis of chloromethyl ethylene carbonate from CO2 and epichlorohydrin under solvent-free conditions. It is interesting to note that the combination of acid and basic sites (Lewis and Brönsted site) existing in the MOF catalyst may improve the catalytic activity of both samples. The reactions were carried out under the same conditions of 353 K reaction temperature, 8 bar CO2 pressure, 10% (w/w) catalyst loading, 8 h reaction time, and 350 rpm of stirring speed. From the Table 2, it follows that at optimum CO2 pressure of 8 bar, reaction time of 8 h, catalyst loading of 10 % w/w, and variable temperature, Zr/ZIF-8 exhibits a higher catalytic activity than ZIF- | 14 |
213695085 | Catalytic Activity After catalyst characterization, the catalytic activity of the novel materials was compared with ZIF-8 for the synthesis of chloromethyl ethylene carbonate from CO 2 and epichlorohydrin under solvent-free conditions. It is interesting to note that the combination of acid and basic sites (Lewis and Brönsted site) existing in the MOF catalyst may improve the catalytic activity of both samples. The reactions were carried out under the same conditions of 353 K reaction temperature, 8 bar CO 2 pressure, 10% (w/w) catalyst loading, 8 h reaction time, and 350 rpm of stirring speed. From the Table 2, it follows that at optimum CO 2 pressure of 8 bar, reaction time of 8 h, catalyst loading of 10 % w/w, and variable temperature, Zr/ZIF-8 exhibits a higher catalytic activity than ZIF-8 (Zr/ZIF-8: 93%, 86%, 76%; and ZIF-8: 77%, 74%, 52%) for conversion, selectivity, and yield respectively at the same reaction conditions. The presence of acid and/or basic site in heterogeneous catalyst has significantly catalyzed the reaction of CO 2 and ECH to produce CMEC [42]. | 15 |
213695085 | Effect of Different Heterogeneous Catalysts Catalysts are very important parts of any chemical reaction; they contain active sites, which are able to speed up the kinetics of chemical reaction by reducing the activation energy. Different types of homogenous and heterogeneous catalysts have been synthesized to catalyze the reaction of CO 2 and epoxide to produce corresponding organic carbonates. In order to assess the stability and effectiveness of the samples, the catalytic activity of both ZIF-8 and Zr/ZIF-8 was investigated in the synthesis of chloromethyl ethylene carbonate from CO 2 and epichlorohydrin. Table 2 shows the effects of the two catalysts for the conversion of epichlorohydrin, selectivity and yield of chloromethyl ethylene carbonate. The catalysts were synthesized using solvothermal method as per standard procedures. The samples were heat-treated at about 373 K in order to enhance an improved catalytic activity and were labelled as ZIF-8 and Zr/ZIF-8 for pure and doped samples, respectively. The reaction of CO 2 and ECH to produce CMEC was carried out in a 25 mL high-pressure reactor at 353 K reaction temperature, 8 bar CO 2 pressure, 10% catalyst loading, and 8 h reaction time. It can be seen from Table 2 that when ZIF-8 was used to catalyze the reaction of CO 2 and ECH, the conversion of ECH, selectivity, and the yield of CMEC were 77%, 74%, and 52% respectively. However, incorporating zirconium into ZIF-8 has significantly increased catalytic performance of Zr/ZIF-8 with the conversion of ECH, selectivity and the yield of CMEC being 93%, 86%, and 76% respectively, although, the presence of side products were reported in both reactions by GC analysis. These side products include 3-chloropropane 1,2-diol and 2,5-bis (chloromethyl)-1,4-dioxane. With similar pore spaces and same embedded Lewis acid metal sites in both ZIF-8 and Zr/ZIF-8 catalysts, the increase in the catalytic activity of Zr/ZIF-8 as shown in Figure 12, may be ascribed to high CO 2 affinity via the introduction of zirconium into MOF, which has significantly increased those pore spaces of ZIF-8 [55]. A fine balance of proximity between pure and Zr-doped MOF was critically examined by Demir and research group. Their experimental results in the solvent-free coupling reaction of ECH and CO 2 to produce epichlorohydrin carbonate (ECHC) concluded that 79.6% yield of ECHC and 97.3% selectivity were achieved after 2 h using Zr-MOF catalyst (Zr/MOF-53). It is however | 16 |
213695085 | Effect of Temperature The cycloaddition reaction of CO2 and epoxide can be referred to as exothermic in nature. The influence of temperature on the cycloaddition of CO2 to ECH to produce CMEC was investigated between temperature ranges of 323 to 373 K. All experiments were conducted with optimized reaction conditions, which were determined during our previous studies with a 10% catalyst loading and 8 bar CO2 pressure for 8 h and a stirring speed of 350 rpm. Table 2 shows the catalytic performance of Zr/ZIF-8 and ZIF-8 as a function of temperature, CO2 pressure, reaction time, and catalyst loading. It can be depicted from Figure 13 that the conversion of epichlorohydrin, selectivity and yield of CMEC were temperature-dependent. Generally speaking, variation in temperature has similar trends in the catalytic activity of both frameworks; the conversion of epichlorohydrin, the selectivity and yield of CMEC increases as temperature increases from 323 to 353 K. However, incorporating zirconium into ZIF-8 has significantly improved the performance of Zr/ZIF-8 with the conversion of ECH, selectivity and yield of CMEC as 93%, 86%, and 76% respectively, while ZIF-8 gave a conversion of ECH, selectivity and yield of CMEC as 77%, 74%, and 52%, respectively, under the same optimum reaction temperature. Further increase in reaction temperature beyond 353 K was unfavorable to selectivity and yield of CMEC in both systems. A slight decrease of the CMEC yield (from 76% to 75%; Zr/ZIF-8 and 52% to 51%; ZIF-8) was observed upon an increase in temperature. This may be due to the formation of diols and dimers of epichlorohydrin [57] and a small amount of by-products such as polymerized CMEC could also affect the yield. Adeleye et al. [58] reported that the increase in the reaction temperature caused a decrease in carbonate yield, due to the decomposition of the catalyst at a higher temperature. Kim et al. [59] also concluded that the reaction temperature for optimal performance is dependent on the nature of the catalyst employed. Therefore, for this set of experiments, the optimized reaction temperature for both frameworks in the synthesis of chloromethyl ethylene carbonate was 353 K. All the subsequent experiments for the chloromethyl ethylene carbonate were conducted at 353 K. To affirm the superior catalytic performance of Zr/ZIF-8 over ZIF-8, nitrogen adsorption and desorption isotherms of the two frameworks were collected and presented in Table 1. Zr/ZIF-8 showed higher CO 2 adsorption capacity which explains in part the improved catalytic performance. | 17 |
213695085 | Effect of Temperature The cycloaddition reaction of CO 2 and epoxide can be referred to as exothermic in nature. The influence of temperature on the cycloaddition of CO 2 to ECH to produce CMEC was investigated between temperature ranges of 323 to 373 K. All experiments were conducted with optimized reaction conditions, which were determined during our previous studies with a 10% catalyst loading and 8 bar CO 2 pressure for 8 h and a stirring speed of 350 rpm. Table 2 shows the catalytic performance of Zr/ZIF-8 and ZIF-8 as a function of temperature, CO 2 pressure, reaction time, and catalyst loading. It can be depicted from Figure 13 that the conversion of epichlorohydrin, selectivity and yield of CMEC were temperature-dependent. Generally speaking, variation in temperature has similar trends in the catalytic activity of both frameworks; the conversion of epichlorohydrin, the selectivity and yield of CMEC increases as temperature increases from 323 to 353 K. However, incorporating zirconium into ZIF-8 has significantly improved the performance of Zr/ZIF-8 with the conversion of ECH, selectivity and yield of CMEC as 93%, 86%, and 76% respectively, while ZIF-8 gave a conversion of ECH, selectivity and yield of CMEC as 77%, 74%, and 52%, respectively, under the same optimum reaction temperature. Further increase in reaction temperature beyond 353 K was unfavorable to selectivity and yield of CMEC in both systems. A slight decrease of the CMEC yield (from 76% to 75%; Zr/ZIF-8 and 52% to 51%; ZIF-8) was observed upon an increase in temperature. This may be due to the formation of diols and dimers of epichlorohydrin [56] and a small amount of by-products such as polymerized CMEC could also affect the yield. Adeleye et al. [57] reported that the increase in the reaction temperature caused a decrease in carbonate yield, due to the decomposition of the catalyst at a higher temperature. Kim et al. [58] also concluded that the reaction temperature for optimal performance is dependent on the nature of the catalyst employed. Therefore, for this set of experiments, the optimized reaction temperature for both frameworks in the synthesis of chloromethyl ethylene carbonate was 353 K. All the subsequent experiments for the chloromethyl ethylene carbonate were conducted at 353 K. | 18 |
213695085 | Effect of CO 2 Pressure CO 2 pressure is another important factor influencing the cycloaddition of CO 2 to epoxides. The pressure of carbon dioxide has been established as one of the most crucial factors affecting the conversion, yield, and selectivity of cyclic carbonate [59]. The reaction of epichlorohydrin and CO 2 to produce chloromethyl ethylene carbonate was examined by varying the CO 2 pressures. For this study, the experiments were carried out at 353 K, 10% catalyst loading, and 350 rpm for 8 h. The selectivity and yield of CMEC was found to increase steadily from 67% and 58% to 86% and 76%, respectively, as the CO 2 pressure increases from 2 to 8 bar. These results indicate that the catalytic performance of the Zr/ZIF-8 depends on the concentration of available CO 2 at the reactive sites. Similar variation was observed in the catalytic activity of the two frameworks with changing CO 2 pressure where the selectivity and yield of CMEC increased from 57% and 37% to 77% and 52%, respectively, at the same pressure of 8 bar of CO 2 as in the case of Zr/ZIF-8. Figure 14 demonstrates the dependence of CO 2 pressure on the yield of CMEC. It can be observed from the graph that the CMEC yield increased with increasing pressure, the maximum of the CMEC yield was reached at 8 bar. By increasing the CO 2 pressure more than 8 bar, a negative effect was observed on both reaction systems, where both yield and conversion experience a slight drop. Wang et al. [60] observed that the introduction of too much CO 2 dissolves in epoxide may result in the formation of CO 2 -epoxide complex, and retards the interaction resulting in a lower conversion. Similar results were also reported by Onyenkeadi et al. [61], where the introduction of higher pressure of CO 2 dissolved in the epoxide and becomes an unfavorable factor due to the difficulty of separating CO 2 and ECH. This condition inhibits the reaction between ECH and catalyst, thus resulting in lower yield [62]. Liang et al. [63] also reported that many diols and dimers of epichlorohydrin were produced as side products at high pressure. Based on the experimental results and theoretical study, it can be concluded that 8 bar CO 2 pressure was the optimum and all subsequent experiments for the CMEC synthesis were carried out at a CO 2 pressure of 8 bar. | 19 |
213695085 | Effect of CO2 Pressure CO2 pressure is another important factor influencing the cycloaddition of CO2 to epoxides. The pressure of carbon dioxide has been established as one of the most crucial factors affecting the conversion, yield, and selectivity of cyclic carbonate [60]. The reaction of epichlorohydrin and CO2 to produce chloromethyl ethylene carbonate was examined by varying the CO2 pressures. For this study, the experiments were carried out at 353 K, 10% catalyst loading, and 350 rpm for 8 h. The selectivity and yield of CMEC was found to increase steadily from 67% and 58% to 86% and 76%, respectively, as the CO2 pressure increases from 2 to 8 bar. These results indicate that the catalytic performance of the Zr/ZIF-8 depends on the concentration of available CO2 at the reactive sites. Similar variation was observed in the catalytic activity of the two frameworks with changing CO2 pressure where the selectivity and yield of CMEC increased from 57% and 37% to 77% and 52%, respectively, at the same pressure of 8 bar of CO2 as in the case of Zr/ZIF-8. Figure 14 demonstrates the dependence of CO2 pressure on the yield of CMEC. It can be observed from the graph that the CMEC yield increased with increasing pressure, the maximum of the CMEC yield was reached at 8 bar. By increasing the CO2 pressure more than 8 bar, a negative effect was observed on both reaction systems, where both yield and conversion experience a slight drop. Wang et al. [61] observed that the introduction of too much CO2 dissolves in epoxide may result in the formation of CO2-epoxide complex, and retards the interaction resulting in a lower conversion. Similar results were also reported by Onyenkeadi et al. [62], where the introduction of higher pressure of CO2 dissolved in the epoxide and becomes an unfavorable factor due to the difficulty of separating CO2 and ECH. This condition inhibits the reaction between ECH and catalyst, thus resulting in lower yield [63]. Liang et al. [64] also reported that many diols and dimers of epichlorohydrin were produced as side products at high pressure. Based on the experimental results and theoretical study, it can be concluded that 8 bar CO2 pressure was the optimum and all subsequent experiments for the CMEC synthesis were carried out at a CO2 pressure of 8 bar. | 20 |
213695085 | Influence of Reaction Time The effect of varying the reaction time on the yield of CMEC was investigated by carrying out a set of coupling reaction of CO2 and epichlorohydrin using both ZIF-8 and Zr/ZIF-8 catalysts. For this study, all experiments were conducted at 353 K and 8 bar CO2 pressure with 10% (w/w) catalyst loading of ZIF-8 and Zr/ZIF-8. Figure 15 demonstrates the influence of reaction time on CMEC yield and selectivity. The results shown on the graph illustrates that the yield increased continuously at the beginning and reached 76% and 52% within 8 h for Zr/ZIF-8 and ZIF-8, then decreased to 75% and 51% respectively, indicating that a slight change in the reaction condition can influence the product formation in a reaction. Similarly, the conversion of ECH was observed to increase from 353 to 366 K when the reaction time was increased from 2 to 8 h. However, when the reaction time was increased further to 10 h and above, a progressive decrease in conversion of ECH was recorded. A similar observation was previously reported in the conversion of ECH to chloropropene carbonate with Zn-ZIF-67 by Adeleye et al. [65]. According to him, conversion of epoxides reaches an equilibrium plateau at optimum reaction time. This phenomenon is referred to as induction period. The induction period is attained when the CO2 and epoxides sufficiently diffuse into the catalytic frameworks of the ZIF-material to reach the active sites of the catalyst and then be converted to the organic carbonate. Beyond the induction period, low conversion of epoxides as well organic carbonates may be observed. From Figure 15, it can be concluded that prolonged reaction time produces lesser ECH conversion and consequently lesser CMEC yield and selectivity. Based on the experimental results and theoretical study, the reaction time of 8 h was considered the optimum for ZIF-8 and Zr/ZIF-8. | 21 |
213695085 | Influence of Reaction Time The effect of varying the reaction time on the yield of CMEC was investigated by carrying out a set of coupling reaction of CO 2 and epichlorohydrin using both ZIF-8 and Zr/ZIF-8 catalysts. For this study, all experiments were conducted at 353 K and 8 bar CO 2 pressure with 10% (w/w) catalyst loading of ZIF-8 and Zr/ZIF-8. Figure 15 demonstrates the influence of reaction time on CMEC yield and selectivity. The results shown on the graph illustrates that the yield increased continuously at the beginning and reached 76% and 52% within 8 h for Zr/ZIF-8 and ZIF-8, then decreased to 75% and 51% respectively, indicating that a slight change in the reaction condition can influence the product formation in a reaction. Similarly, the conversion of ECH was observed to increase from 353 to 366 K when the reaction time was increased from 2 to 8 h. However, when the reaction time was increased further to 10 h and above, a progressive decrease in conversion of ECH was recorded. A similar observation was previously reported in the conversion of ECH to chloropropene carbonate with Zn-ZIF-67 by Adeleye et al. [64]. According to him, conversion of epoxides reaches an equilibrium plateau at optimum reaction time. This phenomenon is referred to as induction period. The induction period is attained when the CO 2 and epoxides sufficiently diffuse into the catalytic frameworks of the ZIF-material to reach the active sites of the catalyst and then be converted to the organic carbonate. Beyond the induction period, low conversion of epoxides as well organic carbonates may be observed. From Figure 15, it can be concluded that prolonged reaction time produces lesser ECH conversion and consequently lesser CMEC yield and selectivity. Based on the experimental results and theoretical study, the reaction time of 8 h was considered the optimum for ZIF-8 and Zr/ZIF-8. | 22 |
213695085 | Effect of External Mass Transfer in Heterogeneous Catalytic Processes Mass transfer limitations play significant roles in chemical reactions by controlling the rate of reaction towards the desired product. In homogenous catalytic reaction, the effect of mass transfer between the phases is mostly negligible. However, in a heterogeneous catalytic reaction, the reaction rate significantly relies on the mass or diffusion between these phases. Mass transfer is typically higher in porous solid or fine particles of nanoscale than large nonporous catalyst [66], transfer of material from the exterior to the interior of a particle happens through pores that open to the external surface, which provides access to the interior of the crystallite material [66]. A typical example is zeolitic imidazolate framework (ZIF-8). In the heterogeneous catalytic conversion of CO2 and epoxide, the internal and external gradient of transport materials between system phases lowers the activity and selectivity of the catalyst towards the desired product [67]. It is important to know that when designing a new catalyst and directing such a catalyst to be selective towards a particular desired product mass transfer resistance and the kinetics are key functions [67]. In cycloaddition reaction of CO2 with ECH, the physicochemical properties of the catalyst and the operating conditions all have a direct effect on the activity of the catalyst as well as the quality of CMEC formed [68]. When a chemical reaction occurs on an active surface, intraparticle diffusion takes place through the pores and the film surrounding the solid catalyst [68]. The coupling reaction of ECH with CO2 to produce chloromethyl ethylene carbonate is an exothermic reaction. In order to reduce or eliminate the effects of mass transfer resistance, it is recommended to employ a highly porous heterogeneous catalyst [68]. The influence of mass transfer on the reaction of ECH and CO2 to synthesize CMEC at 353 K reaction temperature for 8 h with a | 23 |
213695085 | Effect of External Mass Transfer in Heterogeneous Catalytic Processes Mass transfer limitations play significant roles in chemical reactions by controlling the rate of reaction towards the desired product. In homogenous catalytic reaction, the effect of mass transfer between the phases is mostly negligible. However, in a heterogeneous catalytic reaction, the reaction rate significantly relies on the mass or diffusion between these phases. Mass transfer is typically higher in porous solid or fine particles of nanoscale than large nonporous catalyst [65], transfer of material from the exterior to the interior of a particle happens through pores that open to the external surface, which provides access to the interior of the crystallite material [65]. A typical example is zeolitic imidazolate framework (ZIF-8). In the heterogeneous catalytic conversion of CO 2 and epoxide, the internal and external gradient of transport materials between system phases lowers the activity and selectivity of the catalyst towards the desired product [66]. It is important to know that when designing a new catalyst and directing such a catalyst to be selective towards a particular desired product mass transfer resistance and the kinetics are key functions [66]. In cycloaddition reaction of CO 2 with ECH, the physicochemical properties of the catalyst and the operating conditions all have a direct effect on the activity of the catalyst as well as the quality of CMEC formed [67]. When a chemical reaction occurs on an active surface, intraparticle diffusion takes place through the pores and the film surrounding the solid catalyst [67]. The coupling reaction of ECH with CO 2 to produce chloromethyl ethylene carbonate is an exothermic reaction. In order to reduce or eliminate the effects of mass transfer resistance, it is recommended to employ a highly porous heterogeneous catalyst [68]. The influence of mass transfer on the reaction of ECH and CO 2 to synthesize CMEC at 353 K reaction temperature for 8 h with a range of stirring speed between 320 and 550 rpm in an autoclave reactor. It was observed that there was no significant change in the conversion of ECH (~93), selectivity (~86), and the yield of CMEC (~76) when the stirrer speed was maintained above 330 rpm. Therefore, it was concluded that there was no effect of external mass transfer resistance on the experimental conditions. | 24 |
213695085 | Effect of Catalyst Loading To investigate the influence of catalyst loading on the CMEC synthesis, several number of experiments were performed by varying the molar ratio of both ZIF-8 and Zr/ZIF-8 catalyst to ECH. For this study, all experiments were conducted at 353 K and 8 bar CO 2 pressure for 8 h. The results of varying catalyst loading are presented in Figure 16. It can be observed from the graph that by increasing the catalyst loading, there was a corresponding increase in ECH conversion, yield, and selectivity of CMEC. For example, for the experiments conducted with catalyst loadings from 2.5%-7.5%, there was a significant increase in ECH conversion, yield, and selectivity of CMEC. Also, for the experiment conducted at 10% (w/w) of catalyst loading, there was a sharp increase of ECH conversion, yield, and selectivity of CMEC from 90%-96%, 45%-56%, and 73%-79%, respectively. According to Maeda et al. [68], the decrease in epoxide conversion may be ascribed to a decrease in the substrate concentration around the pore cavities of the catalyst at higher catalyst loading. This effect neutralizes the Brönsted acid centers of the catalyst, thereby preventing the interaction between the acidic sites of the catalyst and the oxygen atom of epoxide from the ring opening. This consequently reduces the epoxides conversion to organic carbonates. Considering the percentage error of ±2%, it can be concluded that the number of active sites for ECH and CO 2 to react and produce CMEC was large enough at 10% (w/w) catalyst loading. From the results obtained with respect to catalyst loading, 10% (w/w) was the optimum. From the experimental results for both ZIF-8 and Zr/ZIF-8 catalysts, it is satisfactory to conclude that 10% (w/w) catalyst loading was considered the optimum and further experiments were carried out at 10% (w/w) catalyst loading. Energies 2020, 13, x FOR PEER REVIEW 20 of 26 range of stirring speed between 320 and 550 rpm in an autoclave reactor. It was observed that there was no significant change in the conversion of ECH (~93), selectivity (~86), and the yield of CMEC (~76) when the stirrer speed was maintained above 330 rpm. Therefore, it was concluded that there was no effect of external mass transfer resistance on the experimental conditions. | 25 |
213695085 | Effect of Catalyst Loading To investigate the influence of catalyst loading on the CMEC synthesis, several number of experiments were performed by varying the molar ratio of both ZIF-8 and Zr/ZIF-8 catalyst to ECH. For this study, all experiments were conducted at 353 K and 8 bar CO2 pressure for 8 h. The results of varying catalyst loading are presented in Figure 16. It can be observed from the graph that by increasing the catalyst loading, there was a corresponding increase in ECH conversion, yield, and selectivity of CMEC. For example, for the experiments conducted with catalyst loadings from 2.5%-7.5%, there was a significant increase in ECH conversion, yield, and selectivity of CMEC. Also, for the experiment conducted at 10% (w/w) of catalyst loading, there was a sharp increase of ECH conversion, yield, and selectivity of CMEC from 90%-96%, 45%-56%, and 73%-79%, respectively. According to Maeda et al. [69], the decrease in epoxide conversion may be ascribed to a decrease in the substrate concentration around the pore cavities of the catalyst at higher catalyst loading. This effect neutralizes the Brönsted acid centers of the catalyst, thereby preventing the interaction between the acidic sites of the catalyst and the oxygen atom of epoxide from the ring opening. This consequently reduces the epoxides conversion to organic carbonates. Considering the percentage error of ±2%, it can be concluded that the number of active sites for ECH and CO2 to react and produce CMEC was large enough at 10% (w/w) catalyst loading. From the results obtained with respect to catalyst loading, 10% (w/w) was the optimum. From the experimental results for both ZIF-8 and Zr/ZIF-8 catalysts, it is satisfactory to conclude that 10% (w/w) catalyst loading was considered the optimum and further experiments were carried out at 10% (w/w) catalyst loading. Figure 13a,b shows the effect of varying reaction temperature on catalysts' selectivities towards CMEC. For example, it can be observed that when the temperature was increased from 50 to 80 • C, both catalysts show a corresponding increase in selectivities from 68% and 50% to 86% and 74%, respectively. However, when the temperature was increased beyond the 353 K, a marginal decrease in selectivities was observed in both frameworks, demonstrating that the 353 K was the optimum temperature for the reaction. Meanwhile, the gas chromatography-mass spectroscopy (GC-MS) analysis of the samples shows that 17.3% of 2,5-bis (chloromethyl)-1,4-dioxane (by-product) formed at 353 K, this may explain in part why a drop in catalysts' selectivities was recorded for both samples. Similar results and by-product have been previously reported with ZIF-8 by Carron et al. [69]. They also agree that almost 100% selectivity of ZIF-8 to chloropropene carbonate was achieved at a temperature of 393 K, but decreased to 78.6% when the temperature was increased to 403 K. | 26 |
213695085 | Effect of Reaction Conditions on Catalysts Selectivity to Chloromethyl Ethylene Carbonate In addition to the effect of temperature on catalysts' selectivities, the influence of varying CO 2 pressure on catalysts' selectivities was also investigated. According to Figure 14a,b, the selectivity of the catalysts towards CMEC was found to increase steadily from 67% and 58% to 86% and 76%, respectively, as the CO 2 pressure was increased from 2 to 8 bar. These results indicates that the activity and selectivity of both catalysts were influenced by the concentration of available CO 2 at the reactive sites. Although, similar effect was observed in the responses of both catalysts to variation in CO 2 pressure, however, the results show that Zr/ZIF-8 has higher selectivity than the ZIF-8 catalyst, where the selectivity of both catalysts increased from 69% and 60% to 87% and 77%, respectively, for Zr/ZIF-8 and ZIF-8catalysts. Concersely, both samples experienced decline in selectivities from 87% and 77% to 85% and 70% for ZIF-8 and Zr/ZIF-8, respectively, when the CO 2 pressure was increased beyond the optimum level of 8 Bar. Miralda et al. [70], further argues that ZIF-8 is a dual-functional catalyst with both acidic and basic sites that have been associated with the Lewis acid Zn 2+ ions and the basic imidazole groups, respectively. This bifunctional characteristic enhances the catalyst selectivity for cycloaddition reaction. In a separate report, Miralda et al. [70], also ascertained that it is likely that Lewis acid sites associated with Zn 2+ ions in the ZIF-8 framework play the vital role in catalyzing the reaction of epichlorohydrin and CO 2 to chloropropene carbonate. They further explained that the presence of basic nitrogen atoms of the imidazole ligand, probably, favours the adsorption and binding of CO 2 as well as activation of the carbon-oxygen bonds in CO 2 . In agreement with other similar doped ZIF-8, the open metal centers in the Zr/ZIF-8 has the potential to easily activate the epoxides and the basic sites present in the frameworks. This could be the reason for the higher selectivity that were observed in the solvent-free ECH-CO 2 cycloaddition reactions under mild conditions. Comparatively, the higher selectivity of Zr/ZIF-8 than ZIF-8 towards CMEC may be attributed to the presence of zirconium (Zr). According to a 2019 publication by de Caro et al. [71], the effect of Zr doping on Mg-Al hydrotalcite, the catalyst has significantly increased its selectivity from 90% to >99% towards glycerol carbonate (GC). | 27 |
213695085 | Reusability of ZIF-8 Catalysts Reusability is an important and essential feature of any heterogeneous catalyst in order to be considered useful in industrial applications [71]. The influence of catalyst reusability on the catalytic properties of ZIF-8 and Zr/ZIF-8 in the cycloaddition reaction was investigated. The experiments were carried out in a high-pressure reactor at optimum reaction conditions, i.e., at 353 K, 8 bar with fresh 10% (w/w) ZIF-8 catalyst loading, for 8 h, and at a stirring speed of 350 rpm. The catalysts after Run 1 in the cycloaddition reaction were washed with ethanol and acetone, centrifuged, and oven-dried at 343 K for 12 h before reuse. The recovered catalysts were reused for up to 7 subsequent experiments following the same experimental procedure. ZIF-8 showed a progressive loss in catalytic activity after each run as shown in Figure 17, while Zr/ZIF-8 exhibited no loss of activity indicating the catalyst stability for cycloaddition reaction of CO 2 epichlorohydrin. Yuan et al. [72] stated that the presence of dopant in ZIF-8 show that zirconium is more stable and resilient during the reaction. There was no significant change in the conversion of ECH, selectivity, and yield of CMEC using Zr/ZIF-8. Although, a very slight decrease in the yield of CMEC from 70% (fresh) to 69% (recycled) was observed. The low catalytic activity of the recycled Zr/ZIF-8 catalyst may be ascribed to formation of carbonaceous materials during the cycloaddition reaction as previously reported by Yuan et al. [72]. Furthermore, the XRD and FT-IR analyses results confirmed that Zr/ZIF-8 maintained its crystallinity throughout the reaction process. Zr/ZIF-8. Although, a very slight decrease in the yield of CMEC from 70% (fresh) to 69% (recycled) was observed. The low catalytic activity of the recycled Zr/ZIF-8 catalyst may be ascribed to formation of carbonaceous materials during the cycloaddition reaction as previously reported by Yuan et al. [73]. Furthermore, the XRD and FT-IR analyses results confirmed that Zr/ZIF-8 maintained its crystallinity throughout the reaction process. | 28 |
94087844 | INTRODUCTION As one of the most important raw materials, aldehydes are widely used in organic synthesis for the preparation of fine chemicals both in laboratory and industry. [1] Traditional methods for the synthesis of aldehydes mainly rely on the oxidation of alcohols, [2] organic halides, [3] amines, [4] alkenes, [5] and the methyl group on the aromatic ring. [6] Among these methods, the oxidation of alcohols is convenient and effective because of atom economy, available raw materials, and relative greater yields. [7] Many transition-metal compounds have been used for this purpose, such as vanadium (V), [8] cobalt (Co), [9] copper (Cu), [10] manganese (Mn), [11] ruthenium (Ru), [12] rhodium (Rh), [13] palladium (Pd), [14] iron (Fe), [15] and chromium (Cr). [16] In contrast to other metals, Cu is an abundant metal with less toxicity. [17] There are several reported methods in which Cu has been used as a cheap and "green" catalyst for the oxidation of alcohols. Copper catalytic systems employing 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO) or dialkylazodicarboxylate as cocatalysts have emerged as some of the most effective methods in the oxidation of alcohols to aldehydes. [18] The firstgeneration copper-catalyzed aerobic oxidation protocol, which was developed by Riviere and Jallabert, [19] employed equivalents of CuCl and 1,10-phenanthroline (Phen). Subsequently, Marko and coworkers [20] developed a series of catalytic systems for aerobic alcohol oxidation using the catalytic amount of CuCl and Phen as the catalyst in combination with dialkylazodicarboxylates as redox-active cocatalysts, which exhibited the broadest scope of alcohol oxidation. On the other hand, the Cu=TEMPO system was also widely used for the oxidation of alcohols. [21] The related catalytic system, which consists of a copper salt with 2,2'-bipyridine as a ligand and TEMPO as a cocatalyst, could also enable the mild aerobic oxidation of primary alcohols to aldehydes. [22] Meanwhile, salen-type Schiff bases, including the salen and salophen Schiff bases, can be prepared by condensation between aldehydes and amines in different reaction conditions. [23] They are able to stabilize many different metals in various oxidation states with four coordinating sites and control the performance of metals in a large variety of useful catalytic transformations. [24] Schiff base metal complexes are of great importance for catalysis, [25] such as the asymmetric epoxidation of alkenes [26] and oxidation of alcohols. [27] Transition-metal Schiff base complexes that catalyzed oxidation of alcohols were attractive, especially for their more accessible synthesis conditions and versatile coordination structures. [16b] Salen-Cu(II) could be used in the oxidation of primary alcohols to the carboxylic acids in good yields. [27] However, when combined with TEMPO, the salen-Cu(II) complex could selectively oxidized the alcohols to the corresponding aldehydes at reflux temperature. [28] Because the system described requires the toxic TEMPO and high temperature, a mild, "green," and efficient catalytic system is still desirable. Herein, we developed an efficient method for the oxidation of benzyl alcohols to the corresponding aldehydes using a salophen copper(II) complex (Scheme 1) as the catalyst and tert-butyl hydroperoxide (TBHP) as the source of oxygen in the presence of base. To our delight, excellent yields and good selectivity were achieved for a variety of benzyl alcohols with no trace of corresponding carboxylic acid. | 2 |
94087844 | SELECTIVE OXIDATION OF BENZYL ALCOHOLS no catalyst or base was used ( Table 1, entries 1 and 2), which indicated that both of them are necessary for the oxidation. To optimize the reaction conditions, the oxidant amount was varied from 0.6 to 2 equiv with other conditions remaining constant (Table 1, entries 6 and 10-18). A trend of increasing yield with oxidant amount was observed up to 1.1 equiv TBHP. There was still no carboxylic acid formed even when the oxidant amount was up to 2.0 equiv (Table 1, entry 6). It was worth noting that even when 0.6 equiv of TBHP was used for the oxidation (Table1, entry 10), the reaction provided the product in 91% yield. It is possible that oxygen may participate in the reaction as the reaction was conducted in the open air. The assumption was proved by the results from the controlled experiments (Table 1, entries 11 and 12). Under the same reaction conditions, O 2 was used as the oxidant but only 36% yield was obtained (Table 1, entry 13). The loading amount of catalyst was also examined ( Table 1, entries 6 and 19-23). The best yield was achieved when the amount of the catalyst was 2 mol%. The type and amount of the bases also played important influences on the reaction and 0.6 equiv NaOH resulted in the best yield ( To determine the application scope of this catalytic system, a wide range of benzyl alcohols were oxidized under the optimized conditions ( Table 2). All the benzyl alcohols employed were converted into the corresponding aldehydes with excellent yields and selectivity, and no carboxylic acids were detected with Reaction conditions: alcohols (0.5 mmol), salophen copper(II) complex (2 mol%), TBHP (1.1 equiv), and NaOH (0.6 equiv) in acetonitrile were stirred under room temperature in air overnight. b Isolated yield. c GC yield. | 4 |
94087844 | SELECTIVE OXIDATION OF BENZYL ALCOHOLS high-performance liquid chromatography (HPLC) after the reaction completed. No obvious influence of the electronic effects was found in the reaction, and both benzyl alcohols containing the electron-withdrawing and electron-donating groups could produce the reaction with satisfactory yields (Table 2, entries 1-7). 2-Iodobenzyl alcohol provided a poor yield, which may be explained by the steric effects of the iodo substituent (Table 2, entry 10). In the case of 2-aminobenzyl alcohol ( Table 2, entry 11), an undesired by-product of imine was afforded, leading to a poor yield. The aliphatic primary alcohol exhibited low conversion and poor reactivity (Table 2, entries 20 and 21), but there was still no trace of carboxylic acid. Finally, a probable mechanism for this reaction has been proposed (Fig. 1). The role of the base is to deprotonate the alcohol and accelerate the formation of the benzyloxy-Cu(II) complex 1 by favoring the coordination of the resulting alcoholate to the salophen copper(II) complex. [22b,29c] The aldehydes were then obtained by the reaction of the benzyloxy-Cu(II) complex with the oxidant TBHP with release of the by-product H 2 O and the tert-butoxy-Cu(II) complex 2. Finally, the tert-butoxy-Cu(II) complex exchanged with the starting alcohol to release the tert-butyl alcohol and completed the catalytic cycle. [29] CONCLUSION In summary, a mild and selective oxidation method of benzyl alcohols to the corresponding aldehydes has been established when using the TBHP as the oxidant and a salophen copper(II) complex as the catalyst in the presence of NaOH at room temperature. In this protocol, a variety of benzyl alcohols are oxidized to the corresponding aldehydes in moderate to excellent yields and no overoxidation takes place. | 5 |
94087844 | EXPERIMENTAL All reagents were purchased from commercial sources and used without treatment; 70% TBHP in water was used. The products were purified by column chromatography over silica gel. 1 H NMR spectra were recorded on a Bruker AMX500 (500 MHz) spectrometer and tetramethylsilane (TMS) was used as a reference. A Nicolet IS-10 spectrometer was recorded for IR spectroscopy. | 7 |
94087844 | Preparation of Salophen H 2 O-Phenylenediamine (108 mg, 1 mmol) in 5 mL MeOH was added to a stirred mixture of salicylaldehyde (244 mg, 2 mmol) in 10 mL MeOH. The resulting orange mixture was stirred overnight at room temperature. The solid product was collected by filtration, washed with cool alcohol, and dried in vacuo (256 mg, yield: 81%); 1 | 8 |
94087844 | Preparation of Salophen Copper(II) Complex Solution of salophen H 2 ligand (189 mg, 0.5 mmol) in EtOH (10 mL) and Cu (OAc) 2 . H 2 O (99 mg, 0.5 mmol) in water (1 mL) were mixed and refluxed with vigorous stirring for 2 h. The resulting solution was then cooled to room temperature and filtered. After filtration, the solid product was washed with H 2 O, MeOH, and Et 2 O subsequently, then dried in vacuo to afford the desired copper complex (139 mg, yield 71%). IR: v (cm -1 ) ¼1602, 1519, 1334. Typical Procedure for the Oxidation of Alcohols Alcohol (0.5 mmol), salophen copper(II) complex (2 mol%), NaOH (0.6 equiv), and 70% TBHP in water (1.1 equiv) were dissolved in acetonitrile (5 mL), and the homogeneous solution was stirred at room temperature in air overnight. After completion of the reaction, the solvent was evaporated under reduced pressure. The residue was purified over silica gel by column chromatography (10-25% EtOAc in hexane). All the products were known compounds and were identified by comparison of their physical and spectra data with those of authentic samples. | 9 |
235502386 | In this study, the toxic effects of melittin on Madin-Darby Bovine Kidney cells (MDBK) were analyzed with respect to mitochondrial functionality by reduction of MTT and flow cytometry, apoptosis potential, necrosis, oxygen reactive species (ROS) production, lipid peroxidation, and DNA fragmentation using flow cytometry and cell membrane destabilization by confocal microscopy. The toxicity presented dosedependent characteristics and mitochondrial activity was inhibited by up to 78.24 ±3.59% (P<0.01, n = 6) in MDBK cells exposed to melittin (10μg/mL). Flow cytometry analysis revealed that melittin at 2μg/mL had the highest necrosis rate (P<0.05) for the cells. The lipoperoxidation of the membranes was also higher at 2μg/mL of melittin (P<0.05), which was further confirmed by the microphotographs obtained by confocal microscopy. The highest ROS production occurred when the cells were exposed to 2.5μg/mL melittin (P<0.05), and this concentration also increased DNA fragmentation (P<0.05). There was a significative and positive correlation between the lipoperoxidation of membranes with ROS (R=0.4158), mitochondrial functionality (R=0.4149), and apoptosis (R=0.4978). Thus, the oxidative stress generated by melittin culminates in the elevation of intracellular ROS that initiates a cascade of toxic events in MDBK cells. | 1 |
235502386 | INTRODUCTION Apitoxin or bee venom is secreted by a specialized gland present in the worker bees and confined in a vesicle until the moment of stinging (Benton et al., 1963). It is a complex mixture of nitrogenous compounds, containing several biologically active components, including enzymes, peptides, and biogenic amines, conferring a wide variety of allergic and pharmacological properties, such as anti-inflammatory, antimicrobial, and antitumor activities (Cardoso et al., 2003;Abreu et al., 2010). Melittin is a highly active water-soluble toxic peptide, with only 26 amino acids in its conformation, present in the venom of honeybees and contributes to about 50% of its dry weight (Cruz-Landim and Abdalla, 2002). In the venous vesicle, melittin is arranged in a tetrameric form, which gives it a low toxicity (Cardoso et al., 2003). However, after being released, it dissociates into a highly toxic monomeric form. In addition to it, phospholipase A2 is also present in the venom, which further amplifies the catalytic actions of melittin (Cardoso et al., 2003;Koumanov et al., 2003). Melittin exerts a rapid cytolytic action by destabilizing the membranes and releasing the cytoplasmic content of various cell types (Dempsey, 1990). The hemolytic activity, being a characteristic biological effect, is used to detect the peptide in poisonous extracts (Tosteson et al., 1985). Its lithic capacity is not only restricted to animal cells as it also exerts antibacterial and antifungal activities (Ashthana et al., 2004). The objective of this study was to examine the effects of melittin in MDBK (Madin-Darby Bovine Kidney) cells by analysis of mitochondrial functionality, apoptosis potential, necrosis, ROS production, lipid peroxidation, DNA fragmentation and cell membrane destabilization. (KASVI®, Brazil). Fetal bovine serum (FBS) was obtained from Gibco (Grand Island, NY, USA) and added to E-MEM (10%) when the need for cell multiplication. Dimethyl sulfoxide (DMSO), MTT (3-(4,5-dimethylthiazol-2yl)-2-5-diphenyl-2H-tetrazolate reagent), as well as other reagents used in flow cytometry and confocal microscopy, were commercially purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). | 2 |
235502386 | Melittin Melittin was dissolved in sterile E-MEM at a concentration of 1mg/mL and stored at -20°C. The different concentrations used in the experiments were made by diluting the stock solution in E-MEM. MDBK cells were maintained in a humid incubator at 37°C and 5% CO2 in cell culture bottles with E-MEM supplemented with 10% FBS. After the establishment of the monolayer and attaining approximately 80% confluency, the cells were transferred to 96 well plates (100μL/well) at a concentration of 3 × 10 4 cells/mL. After 24h in the incubator, the medium was carefully aspirated and melittin was added to the wells (100μL/well) at different concentrations (1 to 10μg/mL) in six replicates. All plates were incubated for 72h under the same conditions until the time of reading. MDBK cells maintained in E-MEM without exposure to melittin were used as controls. The MTT reduction assay is used to determine cell viability through mitochondrial functionality (Mosmann, 1983). After an exposure to melittin, the supernatant was carefully aspirated and 50μL/well of 1mg/mL MTT solution was transferred. Then, the plates were incubated for another 4h under the same condition, the supernatant was removed and 100μL/well of DMSO was added to solubilize the generated formazan crystals. After 15min of constant stirring, the plates were read spectrophotometrically at 540nm. The 50% and 90% cytotoxic concentrations (CC) were calculated by the formula (AT/AC) × 100, where AT indicates the mean absorbance of the wells where cells received treatment and AC the mean of the control wells where cells did not receive melittin. For flow cytometry, MDBK cells, cultured and exposed to melittin at different concentrations (1.0 to 2.5μg/mL) for 72h, were subjected to the specific reagent for each analysis and then trypsinized, resuspended in 100μL of E-MEM, and stored under refrigeration. The analysis was performed on Attune Acoustic Focusing Cytometer® (Applied Biosystems) and the results were evaluated using the Attune Cytometric Software version 2.1. Hoechst 33342 fluorescent probe (2mM) was used to separate MDBK (Hoechst positive) cells from the cell debris (negative hoechst). Cell populations were detected by a VL1 photomultiplier (450/40 filter). Twenty-thousand events were analyzed per sample with a flow rate of 50μL/s. For the detection of MDBK cell population, an FSC x SSC scatter plot was constructed. | 3 |
235502386 | Arq About apoptosis/necrosis: after exposure to melittin, 2μL of fluorescein isothiocyanateconjugated Annexin V antibody (FITC) was added and the cells were incubated for 1h. After, propidium iodide (PI, 50μg/mL) was added with further incubation for 10min (Masango et al., 2015). The cells were removed from the plate and placed under refrigeration for readings in the flow cytometer. The viable cells were not labeled with fluorophores (FITC-, PI-), while apoptotic cells outsource phosphatidylserine which is recognized by Annexin V (FITC+, PI-), and the necrotic cells due to the ruptured nuclear membrane, show binding with PI (FITC-, PI+ and FITC+, PI+). The fluorescence was read through the photomultiplier BL3 (640 LP filter). The results were expressed as the percentage of the cell populations as calculated by the formula: (number of positive events/total number of events) × 100. The Mitochondrial membrane potential (MMP) analysis was performed using rhodamine 123 fluorescent dye which concentrates on the active mitochondrial membranes when electrons are donated to the respiratory chain. After exposure of the MDBK cells to melittin, rhodamine 123 (100nM) was added into the plates and maintained for 1h, and the excess reagent was aspirated. The cells were analyzed for the fluorescence intensity emitted in more active (higher concentration of fluorescence, greater accumulation of rhodamine) and less active mitochondria (less fluorescence, less accumulation of rhodamine) (Gillan et al., 2005). Rhodamine 123 fluorescence was read through the BL1 photomultiplier (530/30 filter). The expressed data refer to the percentages of cells with low MMP, calculated by the formula: (number of cells with low MMP/total cell number) × 100. In order to analyze the intracellular production of reactive oxygen species (ROS) the oxidation of the fluorescent dye 2', 7' dichlorofluorescein diacetate (H2DCF-DA) by the intracellular ROS was monitored. h2DCF-DA (1 mM) was added and retained for 1h to the cells after exposure to melittin. The fluorescence emitted was read by the photomultiplier BL1 (530/30 filter) and the data were expressed by the mean green fluorescence intensity ±standard error (Domínguez-Rebolledo et al., 2011). The lipid peroxidation of cell membranes was evaluated by the lipophilic fluorophore probe C11-BODIPY581/591, which is analogous to unsaturated fatty acids. The fluorescence of this probe changes after the lipidic peroxidation, as it emits red fluorescence when present on intact membranes but emits orange to green shades when membranes are attacked by oxidative radicals (Aitken et al., 2007). C11-BODIPY581/591 was added to the cells for 2h, and it was aspirated and the concentrations of melittin were added. After incubation for 72h, the cells were trypsinized and subjected to read fluorescently. The results are expressed as the percentage of cell shaving peroxidized lipids at membranes. The results were obtained using the formula: (number of events with red fluorescence/total number of events) × 100. For DNA fragmentation, after exposure to melittin, the cells were treated for 5min with the fluorescent probe acridine orange (AO) (Ojeda et al., 1992). AO is inserted into the double-stranded DNA as a monomer but can also aggregate to single-stranded DNA. The monomeric AO bound to the intact DNA emits green fluorescence, while the aggregated AO emits orange to red fluorescence (Hoshi et al., 1996). The results were expressed as percentages of cells with DNA fragmentation, calculated by the formula: (number of cells with orange fluorescence/total number of cells) × 100. The use of confocal microscopy: MDBK cells were grown on 0.13mm thick glass cover plates in 24-well plates. After the establishment of the monolayer, the cells were exposed to different concentrations of melittin ranging from 1.0 to 2.5μg/mL for 72h. The specific reagents were then added to the wells for the given time. The supernatants were aspirated, and the coverslips removed from the wells of the plate with the aid of forceps and placed on a slide for analysis under confocal microscopy (Inverted Spectral Laser Scanning Confocal Microscope, Leica, TCS SP8). The destabilization of the plasma membranes was evaluated by the fluorescent and hydrophobic probe merocyanine-540 that presents tropism and intensely blends the membranes that have a high disorder of their components and that have lost their typical asymmetry (translocation of the phospholipids in lipid bilayer) (Langner and Hui, 1993). The analysis was combined with the addition of YO-PRO-1, a semipermeable probe that binds to cellular DNA, enabling a viability analysis associated with the membrane state (Thomas et al., 2006). For this analysis, after culturing the cells and exposing to melittin, merocyanine-540 (2.7 μM) and YO-PRO-1 (25 nM) were added to the wells for 10 min before visualizing under confocal microscope (the method was adapted from Peña et al., 2004). In addition, Hoechst 33342 was added in the same manner as already described. The intensity of red fluorescence emitted by merocyanine-540 indicates the levels of membrane destabilization and green fluorescence emitted by YO-PRO-1 indicates the cells with the labeled DNA due to the high degree of membrane disorganization. The data obtained by the MTT assay are represented by the mean ± standard deviation (SD). Analysis of variance (ANOVA, Tukey's test) was performed to evaluate the differences between the treatment groups and the control group. The flow cytometry data were subjected to analysis of variance (ANOVA, LSD test) for comparison of the groups. Pearson's correlations were performed to evaluate associations between the analyzed variables. In all cases, Statistix statistical software version 10.0® was used and significant results were considered when P<0.05. | 4 |
235502386 | RESULTS AND DISCUSSION The MTT assay revealed a decrease in mitochondrial functionality of up to 78.24 ±3.59% (P<0.01, n = 6) for MDBK cells at a concentration of 10μg/mL of melittin in a dose-dependent manner ( Table 1). The lowest concentrations (1 and 2μg/mL) showed the highest mitochondrial activity rates (0.97% ±0.04% and 0.94% ±0.03%, respectively) and did not differ from the control, whereas all other concentrations of melittin decreased the mitochondrial activity of exposed cells (P<0.05, n = 6). In this context, an active state of mitochondria does not always reflect cellular health, since their activity is also increased in cases of cellular injury. This organelle may act as a pro-apoptotic signal after a damage to its membrane with consequent permeabilization and release of pro-apoptotic molecules present therein (Grivicich et al., 2007). Our results are in agreement with previous findings reported by Zhou et al. (2013), who described only 6.46% ±1.83% (P<0.05, n = 6) inhepG2 cells exposed to melittin at 10.0μg/mL. Results obtained by flow cytometry analysis indicated damage to cells, which were not detectable by the MTT test, as the latter estimates cell viability only by mitochondrial activity. The rates of apoptosis and necrosis, as analyzed by flow cytometry, are given in Figure 1. Other physiological changes in the cells were demonstrated by flow cytometry analysis (Table 2). When the cells were exposed to higher concentrations of melittin, the necrotic cell rates were increased. Regarding the cell necrosis, the control cells showed 20.9% ±0.8% of cellular necrosis. Zhou et al. (2013), by a flow cytometric analysis after an exposure of hepG2 cells (derived from hepatocarcinoma) to melittin (0, 1, 2, and 5μg/mL), found necrosis rates of 0%, 6.8%, 1.6%, and 0.8%, respectively. The treatments with 1 and 1.5μg/mL of melittin did not show difference from the control. However, there was statistically significant difference (P<0.05) at the highest concentrations of melittin, as 39.4% ±1.5% necrotic cells were seen at 2μg/mL of melittin (non-toxic to MTT reduction) and 69.7% ±1.8% at 2.5μg/mL of melittin. Zhou et al. (2013) results demonstrated lower rates of cellular necrosis than those found in this study, but it should be considered that in their study, the necrosis rate of the control group was subtracted from all other groups. Furthermore, the cells studied by these authors are derived from hepatocarcinoma and were exposed to melittin for 24h while in our study MDBK cells were exposed to melittin for 72h. DNA fragmentation was also evaluated by flow cytometry which revealed that MDBK cells exposed to 2.5μg/mL of melittin (4.8% ±0.4%) showed the maximum impact of melittin toxicity. The difference was statistically significant when compared to the cell control (2.57% ±0.77%) (P<0.05) (Table B.2); however, interestingly, the DNA fragmentation rate (0.6% ±0.06%) at 1μg/mL of melittin was lower than cell control. An exposure to melittin caused oxidative stress in MDBK cells by increasing the intracellular production of ROS. The cells exposed to 2.5μg/mL of melittin presented a mean of 43360 ±12289 LI and differed statistically (P<0.05) from the control (17237 ±5017.3) and from other concentrations. The elevated level of intracellular ROS indicates a biochemical imbalance to the point of altering the natural balance between prooxidants and antioxidants. The changes in the redox balance of biological systems such as cells, organelles, and tissues can cause oxidative stress (Schafer and Buettner, 2001). There were no other changes in mitochondrial membrane potential (MMP) patterns. All treatments resulted in an increase in MMP of MDBK cells, but there was a statistical difference (P<0.05) between control (16.07%±0.69%) and treatment with 2μg/mL (17.8%±0.67%). Also, LPO showed a significant correlation with MMP (r = 0.4149, P<0.05), suggesting an increase in mitochondrial functions in an attempt to revert the stress caused to the cells by melittin. The rate of apoptosis could still be influenced by the release of pro-apoptotic mitochondrial messengers but there was a low and non-significant correlation between MMP and apoptosis (r = 0.1238, P>0.05). The action of melittin occurs mainly on phospholipid membranes of the cells as described by Terwilliger et al. (1982), causing permeabilization, artificial pore formation, disruption, and lysis (Lee et al., 2008;Zhang et al., 2011). The mechanisms of action proposed on the membranes were explained by a "barrel model" and "carpet model". In the first model, the aggregates of melittin are perpendicularly formed on the surface of the membrane, resulting in membrane rupture in the form of toroidal pores (or in the form of a barrel). In the carpet model, melittin is distributed on the surface of the membrane in a parallel fashion, disorganizing the lipid bilayer and causes permeabilization (Bechinger, 1999;Gordon-Grossman et al., 2012). Both proposed models suggest the formation of pores or permeabilization of the membranes, explaining the appearance of necrotic cells after exposure to melittin and amplified in a proportion to the concentration of it. While analyzing the destabilization of the phospholipid membranes of MDBK cells exposed to melittin with the aid of confocal microscopy, the microphotographs revealed greater destabilization concomitant with the increase in the concentration of melittin (Figure 2), according to the results obtained by flow cytometric analysis for lipid peroxidation. As shown in Table 2, the increase in the LPO of membranes was directly proportional to the concentration of melittin, which showed statistically significant difference from the control (P<0.05). In addition to the already known mechanisms of action of melittin directly on the lipid membranes (barrel and carpet models), the LPO may also be caused by ahigh production intracellular ROS. Lipid peroxidation is a chain reaction that starts by attacking lipids by an ROS that has sufficient reactivity to sequester a hydrogen atom from a methylene (CH-2) group. The termination of this process is marked by the propagation of lipid and peroxyl radicals produced until they destroy themselves (Ferreira and Matsubara, 1997). Basically, LPO involves incorporating molecular oxygen into a fatty acid to produce a lipid hydroperoxide as the starting primary product. The LPO process occurs in several stages and with numerous possibilities of chemical reactions, which makes it difficult to understand and evaluate the process as a whole (Lima and Abdalla, 2001). Figure 2. Membrane destabilization. Microphotographs were obtained by confocal microscopy of MDBK cells exposed to melittin (0, 1.0, 1.5, 2.0 and 2.5μg/mL) for 72h. An exposure to melittin elevates cellular metabolism. The increase in the concentration of melittin resulted in a concomitant increase in the levels of ROS, LPO, MMP, and cellular necrosis but the rate of apoptosis was decreased, which was inversely proportional to the rate of necrosis (r = -0.7049, P<0.001). The free radicals attack the cell itself and the lipids in the cell membrane are peroxidized, which explains the correlation between ROS and LPO (r = 0.4158, P<0.05). The peroxidation of lipids resulted in the destabilization of membranes, as shown in Figure 2, making possible the cyclization of phosphatidylserine, characterizing an apoptotic process. This mechanism explains the observed correlation between LPO and the rate of apoptosis (r = 0.4978, p<0.05). The correlations between the analyzed variables are shown in Figure3, which may contribute for the elucidation on the mechanism of action of melittin on MDBK cells. Figure 3. Pearson's correlations between the cellular function parameters as evaluated in MDBK cells exposed to melittin for 72h. | 5 |
50989490 | Introduction Effective implementation of semiconductor metal oxide sensors (SMOS) for air and gas media analysis may find logical extension.There are good options to use these sensors for analysis of various gas components dissolved in polar liquids (for instance, in water or liquid phase biological systems).Such possibility results from an increase of electron transfer rate between adsorbed gas molecules and semiconductor surface, caused by polar media.That is why theoretic options exist for development of a method for gas analysis at a temperature of a liquid system.Such method could not only simplify a measurement process, but also improve sensor sensitivity as a result of increase of gas molecules adsorption rate under the conditions of temperature decrease. Though advantages of semiconductor metal oxide sensors are explicit (i.e.high sensitivity, operating speed, small sizes, options for real time measurements, comparatively low cost), only few papers were thitherto published on the implementation of this method for gas analysis in liquid media. Experimental approach was presented in the series of papers of Miasnikov and co-authors [1][2][3][4], in which the semiconductor sensors technique for gas analysis in polar liquids was developed.According to these papers, the trend has been toward intensification of the processes of adsorption and desorption of gases and radical particles on the semiconductor surfaces (ZnO, TiO 2 ) in polar liquids without additional heating, which is necessary in the case of gas media.Equations are proposed [3], which interlink sensor electrophysical parameters with oxygen concentration in a liquid. It should be emphasised that the results in [1][2][3][4] were obtained in the sells, separated from environment, and in the low concentration range of dissolved oxygen (2,24E -03 -4,80E -7 µg/l).But the most important, as a practical matter, liquid phase systems, i. e biochemical liquids, such as blood, cytoplasm and others, constitute open systems with wide range of oxygen concentrations, as compared to concentrations investigated in the above papers.That is why our understanding is that SMOS-based biosensors have good prospects of implementation for open systems at high concentrations of dissolved oxygen.We should also like to emphasise that direct extrapolation would be unfounded of the results, obtained in [1][2][3][4], to the open liquid phase systems.Special investigations should be carried out for reconciliation of the conclusions, made in [1][2][3][4], with the results, which would be obtained for the open systems at high concentration of dissolved oxygen.Bio-sensors seem to be the efficient in this direction, and special research should be carried out with the use of bio-system models for biosensors development on the basis of SMOS. When choosing test model for our investigations we were guided by one of the most important problem of medical diagnostics of virus infections, which is connected to early virus recognition in human organism.This problem could be solved by means of assessment of the quantity of gas phase vital function products of the microorganisms (including oxygen) in a nutrient medium at a stage, that forerun active growth of the above microorganisms.With this purpose we have developed sensitive semiconductor metal oxide sensors for evaluation of micro alterations of oxygen concentrations in a nutrient medium, when it contains pathogen bacteria. In our paper the results are presented of implementation of In 2 O 3 -based semiconductor sensors for oxygen concentration evaluation in the LB-nutrient media (15.5 g/l Luria Broth Base, Miller (Sigma, Lot-1900) and NaCl) without bacteria and with .colibacteria before and after UV-irradiation. | 2 |
50989490 | Experimental procedure Semiconductor sensors for measurement of dissolved oxygen concentration in the water represent insulating substrate with measurement Pt-electrodes, applied by cathodic sputtering method, and In 2 O 3 sensitive semiconductor layer (width 1 m) applied according to special technology, providing good adhesion and water resistance. .coli 600-lux culture was used, which contains multy-copy plasmid with luciferase gene (luminosity range from 400 to 480 nm).The cells were cultivated at 33 0 in LB-nutrient media, produced with the following contents: 15.5 g/l Luria Broth Base, Miller (Sigma, Lot-1900) with addition of N Cl..coliC600-lux culture, containing multy-copy plasmid with luciferase gene with LEX-A promotor, was chosen in such a way that produced system provide oxygen self-sufficiency. Sensor calibration was carried out in the isolated calibration cell for all water systems.Nitrogen, helium or argon was used as carrier gas for extrinsic oxygen.Gas mixtures with predetermined concentrations by means of dynamic generator of standard mixtures ( -3 brand).Gas mixture was delivered to calibration cell by bubbling through glass tube with Shott filter.Output sensor response was transferred from the secondary actuator to PC through L-card interface. | 3 |
50989490 | Results and Discussion Results of sensor calibration in pure water, in NaCl (0,5g/l) water solution and in LB-nutrient media are presented in Fig 1 .As it was revealed in [3], under stationary operation condition of a sensor in separated sell at low oxygen concentration, oxygen concentration dependence of electrocunductivity is as follows: where σ 0 and σ m -initial (before oxygen adsorption) and stationary (after achievement of oxygen adsorption-desorption process) sensor electroconductivity, -constant, which is proportional to the ratio of constants of oxygen adsorption-desorption ( 1 and 2 ); -concentration of dissolved oxygen; -partial oxygen pressure over liquid surface; α -Bunzen factor.Calibration curves for all three systems (Fig. 1) are described by formula1 (1) within the range of experimental error.Observed sensor sensitivity in NaCl solution is higher as compared to pure water (Fig. 1).The same effect, though at less extent (Fig. 1), is observed in LB-nutrient media containing 0,5 g/l NaCl and organic admixtures.We have carried out a series of experiments on low NaCl concentration influence on oxygen sensitivity of the sensor in an open water system.Obtained results are presented in Fig. 2. As it is shown in Fig. 2, the sensitivity of the sensor increases with NaCl concentration increase, which is in good agreement with the data of [4], which was obtained for closed cell.As it was already mentioned, the major objective of our investigation is to show that semiconductor metal oxide sensors can be successfully implemented in biological liquids for detection of micro changes of dissolved oxygen concentration, resulted from bacteria vital function at various stages of its growth.With the help of sensor we intended to reveal the dependence between dissolved oxygen concentration changes and state of bacteria, i.e. their viability level.With the purpose of changes in bacteria state they were subjected to UV-irradiation, which influenced upon their viability.Beforehand the influence of UV-irradiation upon LB-nutrient media was investigated (Fig. 3).It is shown that during first minutes of UV-irradiation, the kinetic of sensor response changes strongly.In the inset in Fig. 3 the result of subtraction of sensor responses before and after UVirradiation is presented.The result of subtraction may be explained as the rise of new donor signal (after UV-irradiation of LB-media) on the background of unchanged oxygen sensor response.The above donor signal completely disappears 10-15 minutes after UV-irradiation.The only possible explanation of this fact is the appearance of fragments of organic molecules with short life period in LB-media, as a result of UV-irradiation. It is well known that .colicells are characterised by complicated mode of damage repair, which was elaborated during evolution process.In the case of damage in DNA molecules, resulted from various chemical or physical factors, including UV-irradiation, the SOS-lux system is being launched.This system is based upon luminescent bacteria ability to radiate visible light as a result of luciferase catalysis.This process is accompanied by oxygen absoption and is described by the below scheme: where FNMH 2 -unsaturated flavionic mononucleotid; RCHO -long oil polymer aldehyde; RCOOHlong oil polymer acid.So, after irradiation in the culture methabolic processes are launched, which are accompanied by decrease of oxygen absorption.But simultaneously the system is launched of DNA repairing in .colibacteria cells, which increases oxygen absorption. With the purpose of investigation UV-irradiation influence upon change of dissolved oxygen concentration in LB-media, containing .colibacteria, the test specimens of bacteria culture were prepared and stored at room temperature (20 ).Metabolism rate substantially depends upon temperature.Optimal temperature value is higher than 30 .That's why at 20 the rate of cells division process is low or equals to zero.If the division process takes place, it should influence upon dissolved oxygen concentration.Slow oxygen concentration decrease was indicated by means of sensor in the test specimens of the bacteria culture (Fig. 4). Then test specimen was placed into UV-irradiation chamber (wave length 10-400 nm, irradiation time 3 minutes, integral flow density 250 watt-second/m 2 ).During the period of specimen irradiation, the sensor was placed into pure nutrient medium.5 seconds after the completion of test specimen irradiation, the measurements of concentration of dissolved oxygen were started. Fig. 4 presents kinetic curves concentration changes of dissolved oxygen in the test specimen after irradiation, which was obtained by means of semiconductor sensor.The comparison of the curves reveals that sensor response in the specimen after UV-irradiation decreases 3 times quicker than sensor response in the test specimen of the same culture before irradiation.Just this result was should be realised in case of validity of scheme 2. The curve of the irradiated specimen (bottom curve, Fig. 4) also presents a some peculiarity, which reveals at the same time interval as additional donor signal in the LB-pure nutrient medium (Fig. 3).So, we can assume that to parallel processes take place, and their influence upon the sensor response kinetics differs one from another.First of all, it is quick decrease of dissolved oxygen concentration in the specimen after irradiation, which results from increase of oxygen absorption by .colibacteria through the launch of SOS-reparation mechanism.Secondly, it is creation of high molecular radicals LB-media and their further recombination and disappearance. | 4 |
92981822 | : The different shapes and sizes of wine glass are claimed to balance the different wine aromas in the headspace, enhancing the olfactory perception and providing an adequate level of oxygenation. Although the measurement of dissolved oxygen in winemaking has recently received much focus, the role of oxygen in wine tasting needs to be further disclosed. This preliminary study aims to explore, for the first time, the effect of swirling glasses of different shapes and sizes on the oxygen content of wine. Experimental trials were designed to simulate real wine tasting conditions. The O 2 content after glass swirling was affected to a considerable extent by both the type of wine and the glass shape. A lack of correlation between the shape parameters of five glasses and the O 2 content in wine was found which suggests that the nonequilibrium condition can occur during wine tasting. The International Standard Organisation (ISO) glass—considered to be optimal for the wine tasting—allowed less wine oxygenation than any other glass shapes; and the apparent superiority of the ISO glass is tentatively attributed to the more stable oxygen content with time; i.e., less variability in oxygen content than any other glass shape. | 1 |
92981822 | Introduction Although the critical role of oxygen in enology has recently been disclosed from a chemistry [1] and winemaking point of view [2][3][4], little information is available from the sensory perspective. Before tasting, the glass of wine is usually "swirled" by holding the glass by the stem and gently rotating it. This action, technically called 'orbital shaking', increases the surface area of the wine by spreading it over the inner part of the glass and consequently enabling some evaporation to take place [5]. Moreover, it is also expected to draw in some oxygen from the air. The ingress estimated on the undisturbed surface of a wine is about 200 mg/h/m 2 [6]. The physics of wine swirling was recently investigated with an elegant fluid dynamic approach, which modelled the pumping mechanism induced by the wave propagation along the glass wall [7]. Three factors seemed to determine whether the team spotted one big wave in the wine or several smaller ripples: (i) the ratio of the level of wine poured in to the diameter of the glass; (ii) the ratio of the diameter of the glass to the width of the circular shaking; (iii) and the ratio of the forces acting on the wine. From a practical point of view, these findings suggest that the mixing and oxygenation may be optimized with an appropriate choice of shaking diameter (d) and rotation speed (rpm). In this view, the glass shape parameters ( Figure S1) can play a key role as they may influence the perceived volume of wine [8], and the perception of wine odors [9][10][11], and color [12][13][14], and therefore the consumer's preference [15,16] as well. Moreover, with time, the glass shape affects the change of headspace chemical composition of wine poured inside, and the D ratio (i.e., maximum diameter divided by opening diameter) seems to be the most important parameter relating glass shape to headspace composition [17]. Considering that both consumers and professional wine tasters usually swirl the glass of wine for approx. 10 to 20 s to unlock odors, there is a need for information to disclose the effect of glass shape on the oxygen content of wines during simulated tasting condition. This preliminary trial aimed to study whether the glass shape can affect the oxygen content in wine under both static and dynamic conditions (i.e., swirling), the latter to simulate the standard procedure of sensory evaluation of wine. The use of optical oxygen sensors (also called minisensors) allowed for the first time the on-line non-invasive and non-destructive oxygen measurements in a glass under dynamic conditions. | 2 |
92981822 | Samples Both red and white wines were selected for this study, including (i) a Rebola 'Nita' white wine Colli di Rimini DOP 2013, (ii) a Sangiovese red wine Carlo Leo Romagna DOP 2013, and (iii) a Cabernet Sauvignon 'Tano' Colli di Rimini DOP 2013 (Az. Agric. Le Calastre, Rimini, Italy). Sangiovese is the main red grape in Italy, Cabernet Sauvignon is a well know international grape variety, and Rebola is an emerging local white grape variety of great interest as well. The bottled wines were provided by the producer and were stored at room temperature (20 ± 1 • C) until the swirling trials. Preliminary characterization of wine composition (Table S1) was carried out according to endorsed methods (RESOLUTION OIV/OENO 390/2010). | 3 |
92981822 | Oxygen Measurement Non-invasive dissolved oxygen (DO) in wine was measured using an OXY-4 oxygen meter (PreSens GmbH, Regensburg, Germany) equipped with a polymer optical fiber and PSt3 spot, also called minisensor (Presens GmbH). The PSt3 spot had a thickness of 1 mm, a diameter of 4 mm, and a response time (t 90 , the time for 90% of the change in signal to occur) of 10 s. In each glass, one minisensor was glued 5 mm below the wine level and calibrated with water at a controlled temperature according to the manufacturer's instructions ( Figure S2). In each bottle, once uncorked, the dissolved O 2 content of wine was directly measured with an oxygen-dipping probe (Presens GmbH) placed in the middle of the bottle in a dynamic regime (i.e., with stirring). Before bottling the wines, three glass bottles (750 mL) were equipped with two minisensors each to ascertain both the headspace and the dissolved O 2 content in static regime (i.e., bottled wines). | 4 |
92981822 | Swirling Trials To simulate the gesture of hand swirling during wine tasting, each glass of wine was placed onto an orbital shaker (model 709/R ASAL, Cernusco s/N, Italy) with a shaking diameter of 3 cm and a shaking speed of 150 rpm (this value was optimized with preliminary screening trails from 100 to 250 rpm). Each glass of wine was swirled for 10, 20 and 40 s, using independent wine samples. Table 1 for details). | 6 |
92981822 | Results and Discussion The oxygen content was measured (i) before and after opening the bottle of wine and (ii) before and during the glass swirling trails, for which protocol was designed to simulate the usual wine tasting by consumers and experts. As expected, the concentration of oxygen in bottled wine was very low with an average range from 4 to 14 µg/L for headspace O2, and in the range of 3 to 29 µg/L for dissolved O2 in wine. In bottled wine, the rate of O2 dissolution was less than the consumption; however, once the bottles are opened the wine comes into contact with air and the oxygen content is expected to rise. In fact, soon after opening the O2 content in bottled wine increased regularly up to 0.99 mg/L in 15 min (Figure 2). These findings are consistent with the initial oxygen absorption capacity of Madiran red wines with pH 3.78 [19]. The O2 accumulation in wine implied that the rate of oxygen dissolution was higher than the rate of its uptake. The latter can be (indirectly) measured by the drop in SO2. According to Boulton [20], the oxygen consumption reactions in wine involve the phenolic compounds as the main substrates and the oxygen consumption is the rate-limiting reaction. The rates of this reaction are first order in oxygen concentration and catalyzed by ferrous ion; therefore, the rate constant would be related to the ferrous ion concentration, but the rate law would depend only on the oxygen concentration. The amount of oxygen found after 15 min most likely increases the wine redox value of ca. 25 mV and will theoretically consume ca. 4 mg/L of sulfur dioxide [21]; both parameters could affect the sensory properties of wines to some extent [4,13]. Table 1 for details). | 8 |
56377194 | Small-angle X-ray scattering (SAXS) patterns from breast tissue samples are compared with their histology. Formalin fixed human breast tissue specimens containing ductal and lobular carcinoma were studied. Histo-pathological information is compared with the scattering data, and there is a clear spatial correlation. Supra-molecular organisation of collagen fibrils is modelled and the model is used to create scattering maps. The model parameters include the axial periodicity (d-spacing), radius and packing of the fibrils, and these are derived from comparison with the experimental scattering patterns. The d-spacing is to 0.5% larger in malignant zones of the tumours than in the healthy zones. There are also characteristic differences in the fibril diameter and packing. | 1 |
56377194 | Introduction Breast cancer is one of the principal causes of death among women in developed countries [5].The mortality of the disease is considerably reduced, if tumours are found at an early stage of growth [9].Frequently the disease may be fully symptomless, and therefore mammographic screening is carried out among women over 50 years old in many countries.Nevertheless, in young dense breast, the cases of false positives and/or missed tumours, false negatives, are frequent [12], especially in the case of the lobular carcinoma, and/or in the absence of micro-calcifications or masses of different densities. Mammography is based on the absorption of X-rays in the tissues, and it reveals changes in density and morphology.The absorption contrast is weak, and the mammographic signs of cancer are subtle, and new methods for imaging of breast cancer are being developed [13].In particular, the contrast in soft tissues such as mammary gland is much more enhanced with phase contrast methods. The nature of the breast tumours is indicated either by morphology (in the core needle biopsies) or radiographic signs (in the mammograms).In the case of malignant diagnosis an increasingly employed treatment is conserving surgery in combination with chemotherapy, hormone treatment or postoperative radiotherapy.However, before proceeding to definitive treatment, the confirmation of diagnosis is always done by the histological biopsy.In a core needle biopsy, the needle is introduced into the tissue and a little piece of the tumour is extracted.Then the biopsy is examined by histo-pathological methods in order to determine the nature of the tumour. Breast tumour growth (hyperplasia) is closely related to collagenosis, and connective tissue may wrap the tumour and encapsulate it in the healthy tissue.Fibrillar collagen is newly formed in the tumours, and it has a supra-molecular structure different from the one found in healthy tissues [10,11].Smallangle X-ray scattering (SAXS) is an efficient technique for retrieving the supra-molecular structure of the breast tissues, especially those rich in collagen [4].Different "signatures" of the SAXS patterns of the tissues can be systematically studied and the scattering signals can be mapped and compared with histo-pathology of the samples. | 2 |
56377194 | Small-angle X-ray scattering SAXS is a powerful method to determine structural features of molecular systems in soft tissue and their different degrees of organisation.The scattering vector k is defined by two unit vectors that indicate the incident (s o ) and the scattered beam (s), respectively (Fig. 1), The modulus of the scattering vector is or alternatively, Here θ is half of the scattering angle.Both k and s will be used in the following.The Bragg Law gives the condition for constructive interference of scattering from a structure that has a periodicity d in the direction of the scattering vector, where n = 1, 2, 3, . . .are the different orders.Combining Eqs (3) and ( 4), This equation is particularly useful, because it gives the real space periodicity d in terms of the positions of the diffraction maxima, s. The scattering amplitude in the units of the electron scattering length (electron classical radius) is the so-called structure factor, and ρ is the electron density.Basically, the scattering amplitude is the Fourier transform of the electron density of the scatterer.The observable quantity is the intensity, which is the Fourier transform of the autocorrelation function P (r) of the electron density, The intensity distribution of X-ray scattering contains information about the structure of the object on many different levels.Periodicity in the atomic scale produces maxima at large values of s, while the intensity modulations due to macromolecules and their assemblies are seen at small s, i.e., in the SAXS regime.Typical length scales for these objects are from a few nanometers to several hundred nanometers, so that with 0.1 nm radiation the SAXS pattern of a tissue sample is observed at scattering angles of one degree and less.Specific formulae for SAXS can be given only for isolated, randomly oriented independent objects, but some of the results are valid under quite general conditions [6].At the limit of forward scattering the intensity is proportional to the square of the Fourier transform of the shape function of the object, i.e., the object size can be deduced from the intensity at very small k.Another important general result is the Porod law, which gives the intensity of scattering at the large values of k in the SAXS pattern.For a 3-dimensional object with a smooth surface the asymptotic form of intensity is Here ∆ρ is the density difference between the object and its surroundings, and S is the surface area of the scatterers per unit mass.Similar results are obtained for thin disks and long rods, for which the power law exponents are −2 and −1, respectively.For the present case, particularly relevant are polymer chains, which are locally rod-like but become coils over large distances.For these the power law exponent is −2 or −5/3.Tissues are made of hierarchical molecular and supra-molecular structures, so that the requirement of independent scatterers is not met.However, realistic models can be constructed and the corresponding diffraction patterns can be calculated.Model parameters are obtained from comparison with the experimental data, so that the tissues may be characterised by these parameters.In the following, a model is presented for one of the most ordered tissue components, namely fibrillar collagen. | 3 |
56377194 | Collagen Collagen is a protein, a polypeptide chain of amino acids, where every 3rd residue is Glycine (Gly-Xxx-Yyy).Collagen type I and III are found in the connective tissue of breast, and both types are fibrillar [8].The molecule of fibrillar collagen consists of a triple α-helix, coiled up as a rope [7].Collagen type I has two identical chains, and the third one is different.Collagen III has all three identical α-chains.In fibrils, collagen molecules pack laterally to each other with hydrogen bonds, in an approximately hexagonal close-packed (hcp) structure [14].Longitudinally, collagen molecules bind to each other in a staggered arrangement.This is a periodic structure, repeated along the fibril axis.The fibrils eventually pack together also in a near-hcp arrangement.This is the supra-molecular structure that gives rise to a characteristic SAXS pattern of collagen. Collagen fibrils in healthy breast tissue have a radius of about 90 nm, have an inter-fibrillar distance of approximately 100 nm, and the axial periodicity (d-spacing) is approximately 65 nm. | 4 |
56377194 | SAXS from collagen Scattering from a single collagen fibril is well known [14], and also scattering from closely packed fibrils [2,3].For scattering from a single fibril one must consider two cases: well-oriented (referred to the beam direction) and randomly oriented fibrils.The SAXS pattern is obtained as a linear combination of the contributions from the orientation and size distributions of the fibrils.The contribution from welloriented fibrils can be divided into equatorial and meridional directions.In the equatorial direction there is intensity modulation, which can be described by Bessel functions, and these provide estimates for the distribution of the fibril radii.In the meridional direction many orders of distinct Bragg reflections are observed, and these give the axial d-spacing.The contribution of the randomly oriented fibrils is that of scattering from long rods, which falls off as k −1 .The fibrils are closely packed, and the interference gives rise to a broad maximum at small k-values in the equatorial direction.In spite of its simplicity, this model gives a good description of the observed SAXS pattern [4]. | 5 |
56377194 | Collagen degradation SAXS patterns give information about the supra-molecular arrangement of collagen, so can be used to identify modifications of such structures.Tissues containing collagen can be classified by the fibril diameter, their packing, and by their axial periodicity.It has been demonstrated that collagen fibrils in healthy tissues have molecular characteristics different from those in malignant tumours [10,11].The SAXS patterns from collagen reflect these differences.For instance, the radii of the fibrils and distances between them are larger in the benign case than in the malignant case, while the axial periodicity in the malignant lesions is about 1% bigger than in healthy tissue [4]. Another important indication that changes take place in the collagen when cancer develops is the change in the average scattered intensity in certain regions of the SAXS pattern.For instance, the background intensity between the 5th and 6th collagen peaks is significantly higher in the case of invaded collagen1 than in the case of totally healthy collagen from the same tumour.Other degradation indicators are also possible to measure, but those are not so evident.For instance, there is a loss of sharpness of the collagen peaks, which reduce their maximum intensity with respect to the background. | 6 |
56377194 | Experiment The experiment was carried out at ID02 High Brilliance beamline of the European Synchrotron Radiation Facility (ESRF) in Grenoble, France.The ESRF is a 6 GeV third generation synchrotron radiation source, which offers a wide range of possibilities to X-ray research.In our case, an undulator was used as the source of radiation, which was monochromatized using Bragg reflections from perfect silicon crystals and focused with a toroidal mirror.A two-dimensional position sensitive detector2 was used to acquire the scattering patterns.The integration time was typically of 20-50 milliseconds.The set-up is shown schematically in Fig. 2. The samples were formalin fixed, and they were sealed hermetically in a holder between Kapton3 foils.The diameter of the samples was 20 mm, and the thickness 1 mm.The samples were scanned through a 200 µm beam in steps of 1 mm to 0.25 mm. | 7 |
56377194 | Sample preparation The samples used in this work are human breast tumours and tissues from surgically excised specimens.These excised specimens were flash frozen in liquid nitrogen and stored in freezer at −80 • C, until their use.Cylindrical pieces, containing tumour and non-tumour tissue were cut off from the specimens while frozen.Several transversal slices were cut from each cylinder.Three holes parallel to the cylinder axis were made using a hollow needle.Surgical marking dyes were introduced in the holes, which were used as reference marks.Immediately after, a thin slice was cut off, let defrost and submerged in formalin 4 .As soon as the sample was formalin fixed, it was processed for histological examination5 by a pathologist.The histology of this slice was used as guide to select SAXS measurement points during the experiment.The actual SAXS samples were cut off from the cylindrical pieces right next to the one used for histology, and submerged in formalin.The two slices, guide and sample, are adjacent, so that the histology provides a map of the sample.The colour marks were present in both guide and sample slices, which helped orienting them correctly. After the SAXS measurement, the samples (already formalin fixed) were introduced in pathological processing cassettes and prepared for the final histo-pathological examination.In this way, a one-to-one correspondence between the classifications by pathology and SAXS patterns was obtained. This research work was performed in accordance with the ethical regulations of the hospital. | 8 |
56377194 | Data acquisition and mapping The scattering patterns were recorded with the CCD and corrected to spatial distortions and normalised to correspond to the same sample thickness.One-dimensional scattering curves were extracted after azimuthal integration of the two-dimensional patterns. Some of these scattering patterns are shown in Fig. 3.The differences in the SAXS patterns are clear enough to easily identify tissues. In order to compare the histology with the scattering patterns, some of the indicators of collagen degradation obtained from the SAXS patterns were used to construct maps of these indicators.The coordinates of measurement spots were retrieved from the positions of the motors, calibrated to the sample holder frame before the experiment started.The colour marks provided reference for sample position in the holder frame, so that the exact location of the measurement point was known. The d-spacing was one of the indicators used for mapping.In this experiment the samples were formalin fixed, and the fixation process removes water from the samples, which may change the axial period.The position of the fifth order collagen peak, in terms of the scattering vector s, was retrieved from the Fig. 3. Tissue characterisation using SAXS patterns.The featureless curve of lowest intensity corresponds to the healthy adipose tissue (fat).Adipose tissue invaded by cancer has diffraction peaks due to the presence of newly formed collagen among the cancer cells (invaded fat).Healthy collagen scattering curve shows the typical collagen peaks, as well as features related to the size and packing of the collagen fibrils.The intensity of scattering from collagen invaded by cancer cells is clearly higher.It preserves, though, the collagen structures in general, but somewhat different.These differences arise from changes in the supra-molecular structure of the collagen.The highest intensity is observed from a necrotic region of a tumour, and the pattern is structureless with a k −2 fall-off of intensity, suggesting disintegrated polypeptide coils with a large specific surface.scattering patterns of all the measurement spots in every sample (see Fig. 4).From these positions the axial d-spacing of the collagen fibrils was determined (cf.Eq. ( 5)), In general, the values of d were 0.3 to 0.4 nm smaller than those measured from fresh samples [4]. The second indicator of collagen degradation is the background intensity between collagen peaks (see Fig. 4).It is seen from Eq. ( 8) that the intensity increases with the surface area of scatterers per unit of volume, and with an increase of the contrast of electron density.Collagen fibrils may suffer of "peelingoff" when they degrade, which increases their surface per unit volume.An observed effect of degradation is the reduction of the fibril diameter.The average intensity in a certain range of the SAXS pattern is plotted in a selected region of the sample.The range is chosen to be the background between two collagen peaks in the regime where the Porod law applies.The range chosen was between the 5th and the 6th collagen peaks (or s in the range 0.08-0.09nm −1 ).In this case, the lower intensities correspond to the adipose tissue, the medium intensities to the healthy collagen, slightly higher intensities to the invaded collagen and the highest intensities to the necrotic tissues.Using the motors positions as reference and the histology, it is possible to build maps of the scattered intensity along certain regions (cf.Figs 5a and 5b). The axial period of collagen fibrils was measured from all the SAXS patterns obtained from all the samples used in this work.All of them, but two, were histologically classified as carcinoma, either lobular or ductal.However, depending on the way the specimens were prepared, one can find more or less healthy tissue from the tumor surroundings.For this reason, samples marked as malignant in a general pathological diagnosis might be locally benign and, therefore, they present general characteristics of benign tissues.Two examples of this can be found in samples E/16C and G/61B, Table 2.These samples are both parts of the same tumor, a ductal carcinoma.Their histology shows a majority of benign tissue, with some islands of in situ6 carcinoma and sporadic invasive cells.Therefore, the axial period from these samples is about 0.3 nm shorter than in the rest of the sample. The samples used for this experiment were formalin fixed.It was already noted that formalin fixation removes water from the tissues, and reduces the axial period of the collagen fibrils.However, the differences in the axial period are systematically the same as in fresh samples: the period is longer in invaded collagen than in the healthy one.In the earlier work with fresh samples [4], the difference in the axial period was 0.3 nm, while in the present case the difference is 0.2 nm on the average (cf.Tables 1 and 2). | 9 |
56377194 | Fig. 5 . Fig. 5. (a) Mapping of a sample containing islands of necrotic tissue encapsulated in collagen (ductal carcinoma).Top panel, the area under the 3rd collagen peak (cf.Fig. 4).Step size is 0.25 mm.The bottom panel shows the corresponding variation of the intensity of between the 5rd and 6th collagen peaks.Collagen peaks are small or disappear when the background scattered intensity is high, and vice versa.(b): Histology of the sample.The area where the SAXS patterns were recorded is shown by the rectangle, and the scan numbers are indicated (57 to 89).Pink stain indicates collagen, and yellowish stained tissues are necrotic. | 14 |
104266470 | In this study, we present a biomimetic approach to improve the stability and reproducibility of droplet generation processes and to reduce the adhesion of aqueous droplets to channel surfaces of microfluidic polymer chips. The hierarchical structure of the lotus leaf was used as a template for a partial laser structuring of the moulds that were used for casting the polymer chips. The hydrophobic wax layer of the lotus leaf was technologically replicated by coating the polymer chips using a plasma deposition process. The resulting microfluidic polymer chip surfaces reveal a topography and a surface free energy similar to those of the lotus leaf. Subsequent droplet-based microfluidic experiments were performed using a 2D flow focussing set-up. Droplets from both, serum-supplemented cell culture medium and anticoagulated human whole blood, could be generated stably and reproducibly using a fluorocarbon as continuous phase. The presented results illustrate the application potential of the lotus-leaf-like polymer chips in life sciences, e.g. in the field of personalised medicine. | 1 |
104266470 | Introduction Microfluidic chips are used in droplet-based microfluidics to prepare serially arranged micro-reactors based on the immiscibility of at least two fluids (Chong et al. 2016). An increasing number of research groups use such micro-reactors for biological applications, e.g. for screening of microorganisms regarding their potential to produce pharmaceuticals (Zang et al. 2013) and to discover novel enzymes (Beneyton et al. 2016) or for investigating their resistance against heavy metals (Cao et al. 2013). Other groups reported about the cultivation of single cells in microfluidic droplets to detect their protein expression quantitatively (Huebner et al. 2007) or about screening and development studies on multicellular spheroids (McMillan et al. 2016) and even on embryos of multicellular organisms like the zebrafish (Funfak et al. 2007). Our group has recently reported about the dropletbased cultivation of embryoid bodies (EBs) formed from murine embryonic stem cells [mESCs (Lemke et al. 2015)]. These mESCs experiments were performed using the modularly constructed technological platform "pipe based bioreactors" (pbb) which has the potential to serve as long-term cultivation system for 3D cell cultures in the volume scale from 100 nL up to 10 µL (Spitkovsky et al. 2016). Furthermore, droplet-based processes can be used to encapsulate pancreatic islets (Wiedemeier et al. 2011) and to analyse the quality of food (Schemberg et al. 2009(Schemberg et al. , 2010. An important prerequisite for the establishment of droplet-based applications is the availability of special devices for manipulating the droplets. One example is devices for the rapid and targeted sorting of droplets (Xi et al. 2017). Furthermore, new droplet generation concepts using different pressure conditions allow varying droplet sizes (Teo et al. 2017). However, despite the huge potential of droplet-based microfluidics and the continuously growing number of publications in this field (Dressler et al. 2014), most studies only present proof-of-concepts (Casadevall i Solvas and deMello 2011; Volpatti and Yetisen 2014). One reason for this limited transferability to commercial/industrial applications is the restricted availability of disposable polymer chips that allow for a stable and reproducible droplet generation of challenging biologically relevant fluids with a high protein content (Shembekar et al. 2016). Cyclic olefin copolymer (COC) (Mair et al. 2006) and polycarbonate (PC) (Sun et al. 2005) are well-established thermoplastic materials for microfluidic applications since they are optically transparent, sterilisable, easy to handle and economically priced. However, micro-channel surfaces of fluidic chips moulded from these polymers by standard injection moulding processes do not support a stable and reproducible droplet generation process. Especially in the case of generating droplets from biologically relevant fluids with a high protein concentration like cell culture media supplemented with serum, an adhesion of the droplets to the micro-channel surface and consequently droplet pinning resulting in cross-contamination are frequently observed. To avoid droplet pinning effects, micro-channel surfaces have to be modified to achieve superhydrophobic properties. There are a lot of papers describing surface modifications to achieve superhydrophobic properties on polymers, e.g. (Shirtcliffe et al. 2011;Xue et al. 2010). Usually, the surface will be microstructured and hydrophobically coated. Well-established procedures are hot embossing (Wang et al. 2017), sol-gel coating (Wu et al. 2016) and deposition of soot (Esmeryan et al. 2017) or nanoparticles (Saarikoski et al. 2009). Furthermore, laser ablation procedures are used to structure the surfaces (Bachus et al. 2017;Rowthu et al. 2015). However, all these procedures are more expensive than producing microstructured chips by moulding or not suitable for droplet-based microfluidic applications. The final hydrophobisation is usually carried out by means of a wet chemical or plasma-supported coating (Jankowski et al. 2011). In this study, we present a biomimetic approach to prepare anti-adhesive micro-channel surfaces on PC-and COC-chips with superhydrophobic lotus-leaf-like properties (Barthlott and Neinhuis 1997) for droplet-based microfluidic applications. The lotus effect is supposed to result from the combination of a hierarchical microtopography (i.e. papillae with a defined microstructure and nanostructure) and a hydrophobic epicuticular wax layer (Andreas et al. 2007;Darmanin and Guittard 2015;Quéré 2008). In order to replicate the lotus structure (Gornik 2004;Kim et al. 2013;Oh et al. 2011;Tuvshindorj et al. 2014), the surfaces of the moulds were equipped with a microtopography (Fig. 1) employing femtosecond pulsed laser ablation as described by Groenendijk (Groenendijk 2008). After injection moulding, the chips were treated with a plasma coating procedure. The resulting surfaces were characterised topographically as well as physico-chemically. In order to analyse these microstructured and plasma-coated fluidic chips regarding their behaviour during and after droplet generation from aqueous media with a high protein concentration, fluidic experiments were performed. The fluid micro systems (FMS chips) described in this paper were developed to handle cells and 3D cell structures like spheroids with diameters up to 500 µm. For this reason, the diameters of the droplet guiding channels were determined to be 1000 µm resulting in droplet volumes not less than 523 nL to guarantee droplet contact to the circular channel walls. Droplet contact to the walls prevents merging of droplets. The smallest channel diameter for a reasonable manufacture by milling is proven to be 200 µm resulting in droplet volumes of about 4 nL. However, using moulding techniques, smaller channel diameters could be realised. | 2 |
104266470 | Preparation of microstructured moulds by laser ablation To investigate the chips surface properties on the one hand and their microfluidic properties on the other hand, two different chip types were moulded: (1) test elements (MST chips) without microfluidic channels but with a microstructured planar surface that were primarily employed for topographical and physico-chemical analyses (Fig. 1a) and (2) FMS chips with microstructured half-channels for droplet generation experiments (Fig. 4b, c). For these experiments, two of the half-channel possessing chips have to be assembled face-to-face resulting in circular cross sections of the microfluidic channels. For the characterisation of unstructured surfaces, the reverse surfaces of the MST chips were used. The FMS chips were designed to perform 2D flow focussing experiments, i.e. each face-to-face assembled microfluidic chip possesses one main channel (diameter: 1000 µm) and two side channels (diameter: 300 µm), perpendicularly arranged to the main channel, see Fig. 4b. The size of a FMS chip is 24 × 24 × 4 mm 3 . The respective moulds were manufactured from hot working steel (1.2343). The semi-circular negative structures for moulding the half-channels were manufactured by electrical discharge machining. Subsequently, surface microstructuring was performed by laser ablation using an ultrashort pulse laser (Hyper Rapid, Coherent GmbH, Germany) equipped with a Galvanometer scanner. The pulse width was 8 ps at a wavelength of 355 nm. The laser power was 0.5 W. For the mould of the MST chips, the microstructured area was 20 × 20 mm 2 . For the FMS chip mould, only the negative structure for moulding the main channel and also the parts of the side channel that are in close proximity to the droplet generation zone were microstructured. A home-made tilting stage (ifw Jena, Germany) was employed to rotate the mould during the laser ablation process in order to obtain an angle of incidence of close to 90° with respect to the curved surface of the semicircular negative structures of the mould. Both the tilting and the positioning of the samples demanded a high degree of precision (µm scale). Inspired by the microstructure of the anti-adhesive lotus leaves, micro-cavities with a depth of 10-15 µm and a spacing of 10-15 µm were created by laser ablation (Groenendijk and Meijer 2006). The diameter of the laser focus was 10 µm. The depths of the micro-cavities were adjusted by varying the density of the laser pulses per area (repetitions or pulse count). To achieve a high surface density, the microcavities were arranged in a hexagonal pattern (Andreas et al. 2007;Feng et al. 2008). | 3 |
104266470 | Injection moulding and coating COC (COC615) and two PC materials (PC2400 and PC2805) with different viscosities were used as chip moulding polymers. The viscosity increased from PC2400 < PC2805 < COC615. The chips were manufactured by injection moulding (Allrounder 320 s, Arburg, Germany) with the following process parameters (Table 1). After injection moulding, the chips were cleaned by treatment with isopropanol for 15 min in an ultrasonic bath (Sonorex super RK 100 H, Bandelin electronic GmbH & Co. KG, Germany) and successive rinsing with ethanol [80%(v/v)] and deionised water. Scheme of the three-phase system: microstructured, plasma-coated channel surface/hydrophobic phase (perfluorodecalin)/hydrophilic phase (droplet) and c scheme of the Cassie-Baxter wetting regime Subsequently, the surfaces of the chips were coated with a hydrophobic layer employing a low-pressure plasma system (Pico 110265, Type F, Diener electronic GmbH + Co. KG, Germany). After evacuating the reaction chamber down to a pressure of 9 Pa, the octafluorocyclobutane precursor (C 4 F 8 , Air Liquide, Germany) was injected with 15 sccm, and the plasma was initiated at 13.56 MHz. Plasma coating was performed for 30 min. The coated chips were stored until use in a closed chamber. | 4 |
104266470 | Scanning electron microscopy (SEM) After sputter coating with an approx. 9 nm gold layer (K550X Sputter Coater, Quorum Technologies Ltd, UK), the microstructured surface topography of the MST chips was characterised by means of stereo scanning electron microscopy (Evo LS10, Carl Zeiss Microscopy GmbH, Germany). SEM images from three tilt angels (0°-7°-15°) were recorded with a 250 fold magnification, 9 mm working distance and 1024 pixel × 768 pixel resolution. From these SEM images, a three-dimensional, digital surface model (3D-DSM) was composed employing the MeX ® software (Alicona Imaging GmbH, Austria). The analysis tool of the same software was used to determine the depths of the micro-cavities. | 5 |
104266470 | Atomic force microscopy (AFM) AFM investigations on MST chips were performed using the atomic force microscope Nanowizard ® equipped with a 100 µm z-scan module CellHesion ® (both jpk instruments AG, Germany). All scans were performed in contact mode at ambient conditions with cantilevers ARROW-NC (NanoWorld AG, Switzerland) having a nominal spring constant of 42 N/m and a tip radius less than 20 nm. Due to the high aspect ratio of the micro-cavities, the scans were performed with a low scan rate of 0.1 Hz. In addition to the analyses of the microstructured chips, the hierarchical topography of a lotus leave (Nelumbo nucifera) was investigated, as well. The lotus leaf was stabilised with glycerol to avoid drying artefacts during the scans. Post-processing and 3D images of data were realised using the background correction feature and a Gaussian smoothing filter of the software SPIP™ (Image Metrology A/S, Denmark). | 6 |
104266470 | Physico-chemical characterisation Surface free energy determinations (including polar and dispersive components) were performed on the microstructured MST chips before and after the plasma coating procedure according the OWRK approach (Owens, Wendt, Rabel und Kaelble). For this approach, the contact angles of deionised water, formamide, ethylene glycol (predominantly polar) and diiodomethane (dispersive) were recorded using the OCA System (sessile drop, 3 droplets of 3 µL, DataPhysics Instruments GmbH, Germany). | 7 |
104266470 | Fluidic experiments Fluidic experiments were performed with a highly versatile microfluidic platform comprising the following functional modules: • Droplet generation FMS, • Detection module (equipped with two optical sensors), • Tube storage disc, • Stirring unit (only for experiments with blood), • Syringe pump system (neMESYS, cetoni GmbH, Germany). The droplet generation FMS is composed of two plasmacoated chip halves assembled reversibly face-to-face and pressed together with screws guaranteeing leak tightness . Pins and drillings serve as positioning elements to guarantee for a reproducible assembly and disassembly, e.g. for refreshing of the chip coating. After its assembling, the FMS is mounted into a frame that serves for the fluidic connections between the tubes and the FMS (Fig. 4a). All functional modules were connected with tubes made from polytetrafluoroethylene (PTFE) and fluorinated ethylene propylene (FEP), respectively. For both, the transport of droplets as well as the mutual separation of the droplets, perfluorodecalin (PFD) served as continuous phase. Additionally, reference experiments with Novec 7500 and Pico-Surf TM 2 as continuous phase were performed. Two aqueous fluids with a high biotechnological and biomedical relevancy were used for the experiments performed in this study: (1) cell culture medium Dulbecco's Modified Eagle's Medium (DMEM), product number D5523 (Sigma-Aldrich Chemie GmbH, Germany) supplemented with 4.5 g/L d-glucose, 2 mmol/l l-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 10%v/v foetal calf serum and 0.01%w/v phenol red and (2) anticoagulated human blood (supplemented with trisodium citrate 0.106 mol/L in S-Monovette ® , order number 04.1955.100, Sarstedt AG & Co., Germany). For droplet-based microfluidic experiments, those fluids are often challenging due to their high concentration of surface active compounds like proteins. In order to characterise the influence of the FMS surface microstructure and coating on the droplet generation process, most experiments were performed without surfactants. However, as reference to usual droplet-based applications, also experiments with surfactants were performed. Prior to the experiments, the droplet generation FMS and the tubes were intensively rinsed and filled with PFD. For each parameter, five experiments were performed with a PFD flow rate (Q c ) of 500 µL/min, an aqueous fluid flow rate (Q d ) of 100 µL/min and a droplet generation time of 6 min. To evaluate the droplet generation reproducibility, the volume of each droplet and its standard deviation were assessed. Each droplet was considered as a sphere (sphere diameter ≤ main channel diameter of the droplet generation FMS) or as cylinder with hemispherical ends. In the latter case, the cylinder diameter corresponds to the main channel diameter of the droplet generation FMS. For the cylinder volume calculation, the droplet length was measured photometric at 525 nm by recording the absorption shift between PFD and the droplet. These measurements were realised using a detection chip with two optical sensors. The droplet volume calculation bases on the precondition of an idealised droplet shape. However, depending on the droplet velocity, deviations from these idealised shapes were observed. Compared to a non-moving (nondeformed) droplet, the shape deviation became more significant with increasing velocity. Taking these shape deviations into account, a correction factor was introduced. This factor T s/s represents the ratio of the droplet length average s D,SP , calculated from the sample volume delivered by the syringes pump (SP) divided by the number of generated droplets and the droplet length average s D,OS , determined by the optical sensors (OS). Subsequently, the volume of each droplet was calculated employing this correction factor. To evaluate the droplet generation reproducibility, the following parameters were determined for each experiment: (1) the mean droplet volume, (2) the standard deviation and (3) the coefficient of variation (CV). For the final analysis of the droplet generation stability, two further experiments were performed. Within the scope of these experiments, interactions of the sample medium with the FMS channel surface should be investigated. For this, droplets were continuously generated for 6 h using the FMS chips to be examined. The evaluation was based on the pure optical observation of the droplet generation process. The experiment was stopped when the droplets extensively adhered to the channel surface, and the stability of droplet generation process was significantly disturbed. | 8 |
104266470 | Influence of the microstructure on the physico-chemical properties With the applied injection-moulding process, papilla-like structures could successfully be prepared on different polymers surfaces (Fig. 2a-c). The use of different polymers resulted in different morphologies of the papilla-like structures. The flattest papillae were observed on the surface of MST chips made from COC (Fig. 2c), whereas the PC2805 surfaces were characterised by rather sharp papillae (Fig. 2b). The different polymers also caused different heights of the papillae; i.e. heights of ~ 5 µm for PC2805 (Fig. 2b) and COC6017 (Fig. 2c) and heights of ~ 6 µm for PC2400 (Fig. 2a). These differences are caused by the different viscosities of the molten polymers. A lower viscosity of the melt improves its intrusion into the mould cavities and consequently increases the height of the papillae. The polymer PC2400 with the lowest viscosity (PC2400 < PC2805 < COC6017) resulted in the maximum papillae height. Compared to unstructured and uncoated polymer MST chip surfaces, the contact angles of uncoated microstructured MST chip surfaces increased from ~ 90° to ~ 120° (Fig. 2d). The highest water contact angle on uncoated but microstructured surfaces was measured with ~ 130° on the COC6017 MST surface. This also connotes that microstructured COC6017 FMS should be well suited for droplet-based applications even without plasma coating. The surface free energy of the unstructured and uncoated MST surfaces was ~ 40 mN/m (Fig. 2e). Caused by the microstructure, the surface free energy was increased by ~ 6 mN/m for the MST chips with smaller papillae height (PC2805 and COC6017) and by ~ 20 mN/m for the MST chips with maximum papillae height (PC2400, Fig. 2e). In the case of unstructured surfaces of MST chips made from the polymers PC2400 and PC2805, ~ 95% of the surface free energy was contributed by the dispersive component. For COC6017, the dispersive component was ~ 89%. For the microstructured surface of MST chips, the dispersive component was increased by ~ 3% for PC2400 and PC2805 and by ~ 10% for COC6017. In summary, the uncoated microstructure of the MST chip surfaces increases both the water wettability and the surface free energy. This phenomenon was described by Wenzel (Wenzel 1949): for a homogeneous wetting scenario where microstructures intensify the surface properties of the bulk material (hydrophobic solid materials become more hydrophobic and vice versa). | 9 |
104266470 | Influence of the plasma coating on the physico-chemical properties Plasma coating resulted in a significant increase of the water contact angle for both the unstructured and the microstructured MST surfaces (Fig. 2d, coated surfaces). The surface of an unstructured MST chips moulded from PC2805 revealed a water contact angle of 88.1° ± 3.4° and a surface free energy of 41.74 ± 1.0 mN/m (dispersive: 41.56 ± 1.1 mN/m, polar: 1.9 ± 0.3 mN/m). After plasma coating with C 4 F 8 , the wettability was significantly affected. The water contact angle increased to 119.4° ± 0.7°, and the surface free energy was reduced to 8.98 ± 0.2 mN/m (dispersive: 8.44 ± 0.3 mN/m, polar: 0.54 ± 0.2 mN/m). Interestingly, the wettability was not only reduced for aqueous test fluids but also for non-fluorinated oils like tetradecane. However, the coated surfaces were completely wettable with fluorinated oils like PFD (contact angle < 20°). From AFM measurements, the thickness of the C 4 F 8 -coating was estimated to be ~ 140 nm. For this, a glass slide was partially coated using the above-described coating procedure. For the microstructured surfaces, the water contact angle increased from ~ 110° to ~ 130° up to ~ 160° after plasma coating. Furthermore, the standard deviation of the water contact angles was reduced for all coated MST chips. After plasma coating the wettability of the three polymers were comparable even though there were significant differences in the wettability prior to the coating process (Fig. 2d). The polymer with the highest contact angle before coating (130° for microstructured COC6017) displayed the lowest contact angle after plasma coating (154°). Since the structure heights of PC2805 and COC6017 (Fig. 2d) are comparable, we assume that the rounded morphology of the COC6017 microstructure elements (Fig. 2c) causes an increase of the contact area to the polar phase resulting in a slightly increased wettability. In addition to the reduction of the water wettability, a significant reduction of the surface free energy was observed after coating both microstructured and not microstructured MST surfaces (Fig. 2e). For the unstructured surfaces, the surface free energy was reduced by more than 30 mN/m to values around 9 mN/m. An extremely low surface free energy of ~ 2 mN/m was observed for the microstructured surfaces after plasma coating. This reduction is predominantly due to a decrease of the dispersive part of the surface free energy. While the coating procedure does not significantly affect the polar component for unstructured surfaces (~ 1 mN/m), it is increased to ~ 4 mN/m for uncoated microstructured samples. When these microstructured surfaces are plasma coated, the polar part is reduced to values lower than 0.2 mN/m. In general, polar components account for less than 10% of the surface free energy irrespective of the coating. The high water contact angles additionally indicate a low contribution of the polar part of the surface free energy, especially since the coated microstructured polymer surfaces display an extremely low polar contribution. | 10 |
104266470 | Comparison of the coated microstructured polymer surfaces with the lotus leaf surface In order to compare the technically prepared biomimetic surfaces with the leaves of the lotus flower, AFM analyses were performed on microstructured and plasma-coated PC2400 MST chips and Nelumbo nucifera leaves. Comparative AFM analyses reveal that both surfaces are characterised by a hierarchical micro-and nanotopography (Fig. 3). The morphology of the individual microstructure elements of the injection-moulded polymer surface reveals a striking similarity to the papillae of the lotus leaf (Fig. 3b, e). They also display comparable heights, diameters and spacing. The arrangement of the microstructure elements is characterised by a lower degree of variability (in terms of height and spacing of individual microstructure elements, Fig. 3a, d). Furthermore, both surfaces display a similar nanotopography on top of the microstructure elements (Fig. 3c, f). Both surfaces reveal an extremely low water wettability (~ 160° for the lotus leaf (Barthlott and Neinhuis 1997) as well as for the microstructured, plasma-coated polymer surface). These superhydrophobic properties indicate a heterogeneous wetting according to the Cassie-Baxter theory (Cassie and Baxter 1944;Hüger et al. 2009;Wagner et al. 2003). | 11 |
104266470 | Microfluidic experiments The FMS microstructure was created on the surface of the main channel which is in direct contact with the test fluids. SEM and stereoSEM images of the main channel surface reveal that the microstructure that was created on the mould Table 2) could be successfully transferred onto the FMS chip elements (Fig. 4c, d). For the microfluidic experiments with DMEM and blood, the microstructured and plasma-coated FMS chips made from the above-mentioned polymers were investigated (Table 2,(1)(2)(3)(4)(5). Additionally, reference FMS chips ( Table 2, No. 6-7) were investigated to estimate the effect of a missing microstructure (unstructured PC2805) as well as of a missing C 4 F 8 coat (uncoated COC6017). Irrespective of the type of polymer, no significant adhesion of the cell culture medium to the channel surface was detected during the 30 min of droplet generation employing coated microstructured FMS (Table 2,(2)(3)(4)(5). However, severely reduced performance times of ~ 10 min for the unstructured but plasma-coated PC2805 reference FMS ( Table 2, No. 6) and ~ 15 min for the microstructured but uncoated COC6017 reference FMS ( Table 2, No. 7) were observed. This indicates that a combination of both the microstructure and the plasma coating is necessary to guarantee for a stable droplet generation process. In addition to these effects, the surface modification also affects both the droplet volume and the reproducibility of the droplet generation process (Fig. 4f). When the droplet generation was performed with microstructured and coated FMS, a consistent droplet volume of ~ 800 nL was obtained. For all microstructured and coated FMS investigated with DMEM ( Table 2, [3][4][5], there is a slight tendency of an increasing droplet volume for a decreasing water contact angle (and a decreasing surface free energy), compare (Table 2, No. 7). The droplet volumes increased to ~ 1100 nL (~+ 35%) when uncoated COC6017 FMS were employed (Fig. 4e, f). Each of these droplet volume increases correlates with a higher wettability of the surfaces (~ 30° lower water contact angles and higher surface free energy, Fig. 4e, f). The higher wettability of the FMS channel surface causes more pronounced interactions of DMEM with the channel surface, resulting in a delayed droplet break-up and thus in an increase of the droplet volume. For all microstructured and plasma-coated FMS, the CV values were smaller than 2.5%, indicating that the droplet generation process was highly reproducible. The lowest CV value of 1.45%, and thus, the highest reproducibility was observed for the microstructured and plasma-coated COC6017 FMS. For droplet generation from anticoagulated human whole blood, a coated microstructured PC2400 FMS was used ( Table 2, No. 1). Droplets could stably be generated during the whole process, lasting 30 min maximally. In contrast to the experiments with DMEM, droplets from blood could not be generated with microstructured but uncoated PC FMS. The blood droplet volumes and their CV values were higher than the respective data for droplets generated from DMEM. This may be due to the higher protein concentration of whole blood that supports interactions with the uncoated channel surface (Pham et al. 2016). Compared to the experiments without surfactants ( Finally, the experiments with DMEM and whole blood were continued with the four structured and coated FMS ( Table 2, No. 1-5) to study the long-term stability of the droplet generation. For this purpose, droplets were continuously generated on two successive days each for 6 h using the corresponding FMS as well as the test fluids DMEM and whole blood. Using three different, microstructured and coated FMS (Table 2, No. 2-5), droplets could be generated without disturbance over the complete test period of 12 h with DMEM. In the case of droplet generation from whole blood using the microstructured and coated PC2400 MST (Table 2, No. 1), punctiform adhesions of the blood on the channel surface were observed after approx. 10 h. However, these adhesions could be eliminated by increasing the flow rates and did not appear again. These experiments proved that injection-moulded, microstructured and plasma-coated FMS support a stable and long-term droplet generation process. In summary, both the introduced microstructure and the hydrophobic plasma coating lower the surface free energy of the FMS channel surfaces. Consequently, the tendency of biologically relevant fluids to adhere to the channel surface is lowered. According to Cassie's law (Cassie and Baxter 1945;Cassie 1948), the contact area between the droplets and the channel surface could be significantly reduced. Particularly (Absolom et al. 1987), this reduced contact area should also reduce the capability for protein adhesion (Fig. 1b, c). A combination of the microstructure and the hydrophobic plasma coating significantly reduces the adhesion of biologically relevant fluids to the channel surface. The resulting disposable FMS chips support a long-term stable droplet generation process with a highly reproducible droplet volume. | 12 |
233708770 | : In this study, cattle and pig slaughterhouse wastes (SHWs) were hydrothermally carbonized at 150–300 ◦ C, and the properties of SHW-derived hydrochar were evaluated for its use as a solid fuel. The results demonstrated that increasing the hydrothermal carbonization (HTC) treatment temperature improved the energy-related properties (i.e., fuel ratio, higher heating value, and coalification degree) of both the cattle and pig SHW-derived hydrochars. However, the improvements of cattle SHW-derived hydrochars were not as dramatic as that of pig SHW-derived hydrochars, due to the lipid-rich components that do not participate in the HTC reaction. In this regard, there was no merit of using HTC treatment on cattle SHW for the production of hydrochar or using the hydrochar as a solid fuel in terms of energy retention efficiency. On the other hand, a mild HTC treatment at approximately 200 ◦ C was deemed suitable for converting pig SHW to value-added solid fuel. The findings of this study suggest that the conversion of SHWs to hydrochar using HTC can provide an environmentally benign method for waste treatment and energy recovery from abandoned biomass. However, the efficiency of energy recovery varies depending on the chemical composition of the raw feedstock. | 1 |
233708770 | Introduction The increase in the consumption of meat and meat products has led to the expansion of the slaughtering industry and the consequent increase in biological waste production [1]. Slaughterhouse waste (SHW) is the animal product remaining after the manufacture of the principal commodity in slaughterhouses and formally consists of inedible offal and fats [2]. Most SHW is used as a raw material in the rendering industry for the production of pet and animal feed. However, outbreaks of livestock infectious diseases, such as foot-and-mouth disease, mad cow disease, and African swine fever, hinder the use of the SHW in pet and animal feed production, and even in application as fertilizers [3]. Thus, significant amounts of SHW are underutilized and are discarded via incineration or landfills. The disposal methods not only have a negative impact on the environment by generating secondary pollutants (e.g., odor and leachate), but also lead to an economic burden on the meat industry. Recent advances in the SHW utilization pathways include its application as a source of industrial proteins, enzymes, and lipids [4,5]. Despite being an organic-rich source for industrial raw materials, SHW has process-applicable limitations and is its use is considered challenging for functional applications due to its heterogeneous composition (e.g., nonorganic compounds) and poor solubility [1,6]. More recently, SHW has received considerable attention as a feedstock for bioenergy production [7,8], particularly due to its high organic content. Increasing environmental concerns (e.g., fossil fuel depletion, carbon emissions, and waste management) necessitate efficient methods for energy recovery and waste management. Hydrothermal carbonization (HTC) is a thermochemical reaction-based method which occurs in a relatively low temperature range (150-300 • C) with moisture and autogenous pressure [9,10]. The relatively low energy input and mild treatment conditions of HTC have caused it to receive attention as an alternative biomass treatment technology rather than conventional thermal treatment technologies (e.g., pyrolysis, combustion) [11,12]. During the HTC reaction, various types of organic matter are converted into a valuable carbon-rich material (i.e., hydrochar). The operating conditions of the HTC (e.g., treatment temperature) affect the yield, physicochemical properties, and the functionality of hydrochar [13]. Hydrochar is a multifunctional carbonaceous material which can aid environmental remediation, soil amendment, and carbon sequestration, as well as serve as an alternative solid fuel [14]. Recently, the potential use of hydrochar as a solid fuel has received considerable attention owing to its enhanced heating value, thermal stability, and material structure, especially when compared to untreated biomass [15]. HTC boosts the energy-related properties of hydrochar, making it a sole and/or auxiliary fuel source for combustion facilities [16]. The production of hydrochar using waste biomass and its application as a solid fuel can double its profits, mitigate the environmental burden of SHW management, and help find the key to sustainable development. For the above reason, considerable studies on HTC reactions for hydrochar production and its use as a solid fuel have gained significant interest in this research area. Li et al. and Roy et al. reported on potential routes for solid fuel production using hydrochar derived from red jujube branch and peat moss, respectively [12,17]. Not only experimental research but also process efficiency modeling and cost analysis studies on HTC reactions have been published. Lucian and Fiori provide are a useful reference to evaluate the HTC process in terms of the environment and economics [18]. Herein, we focused on the conversion of cow and pig SHWs into hydrochar. The properties of the resultant hydrochars were evaluated for their use as solid fuels. We investigated the effects of the chemical feedstock composition on the hydrochar yield and properties. Finally, we determined optimal HTC treatment conditions for the tested SHWs to recover energy more efficiently. | 2 |
233708770 | Feedstock Cattle and pig SHWs were obtained from a domestic meat processing company and were used as feedstocks for hydrochar production by HTC. The SHWs were moved to the laboratory immediately after collection, and the impurities (e.g., horn, feather, toenail) were removed. The cattle SHW used in this study was mainly composed of fat, kidney, and genitals, whereas pig SHW consisted of fat, liver, lung, and brain tissues. The components of each SHW type were ground using a cryogenic grinder (SPEX 6875D Freezer/Mill, SPEX SamplePrep, Metuchen, NJ, USA) and homogenized by blending. The sample pulverizing was conducted following manufacturer's instruction, but grinding time and rate were modified referring to our previous work. The sample (~2 g) was continuously cooled until −196 • C, and the constant temperature was maintained during the entire grinding process. The grinding time was 60 s at a rate of 10 cycles per second. To avoid sample spoilage, the prepared SHWs were frozen (<−20 • C) until further use. | 3 |
233708770 | Hydrothermal Carbonization (HTC) SHWs were hydrothermally carbonized using a batch-type laboratory-scale reactor at four different temperatures (150, 200, 250, and 300 • C). The hydrothermal carbonization at each temperature was conducted in duplicate, and the products were mixed and analyzed together. The Teflon-lined stainless-steel reactor body with a total inner volume of 1 L was connected to a heating control system and a steam condenser. The HTC reaction was performed for 30 min at the preset temperature, and the pressure of the reactor was not regulated (autogenous pressure atmosphere was maintained during the HTC reaction). For each treatment, 300 mL of cattle or pig SHW was loaded with the same volume of deionized water into the reactor vessel and nitrogen gas was purged for 5 min to create anaerobic conditions, and it was then sealed. A mechanical agitator was operated at a speed of 200 rpm, and the contents in the reactor were continuously mixed during the reaction. After the HTC reaction, the residual steam was discharged, and the inner temperature and pressure of the reactor were lowered until they reached room temperature and atmospheric pressure, respectively. Both solid and liquid products of the HTC reaction were collected and oven-dried overnight at 105 • C. Finally, the hydrochar was obtained as dry matter and analyzed. The simple layout of the experimental setup is shown in Figure 1. Energy densification = HHV of hydrochar/HHV of feedstock where HHV is the higher heating value (2) Energy retention efficiency = Product yield × Energy densification (3) | 4 |
233708770 | Analytical Methods Proximate analyses of ash and volatile matter (VM) contents in the samples were conducted according to the ASTM D3174 and D3175 standard test methods, respectively. The fixed carbon (FC) content in the samples was analyzed based on the differences between the ash and VM contents. ASTM international standard test method E1758-01, AOAC international method 2001.11, and the procedure recommended by Bligh and Dyer (1959) [19] were followed to determine the total carbohydrate, protein, and lipid contents in the test samples, respectively. A high performance liquid chromatography (HPLC) system consisting of a Waters 2695 Separations Module (Waters, Milford, MA, USA) associated with a refractive index detector (Waters 2414, Waters, USA) and Sugar-Pak 1 column (Waters, USA) was employed for determination of total carbohydrate. Deionized waster was used as the mobile phase and the flow rate was 0.6 mL min −1 . The analytical grade for HPLC standard solution was used (Sigma-Aldrich). For the instrumental analyses, the dried raw SHW and hydrochars were ground into fine particles and sieved to particle sizes of less than 250 µm. An elemental analyzer (Flash1112, Thermo Fisher Scientific, Bremen, Germany) was employed to perform the final analyses. The HHVs of the test samples were evaluated using a bomb calorimeter (Parr6400, Parr Instrument, Moline, IL, USA), followed by a standard method for calorimetric analysis (US EPA 5050). The functional group changes during the HTC reaction were analyzed using Fourier transform infrared (FTIR) spectroscopy (Vertex70, Bruker, Karlsruhe, Germany). The absorbance values of the test samples were in the range of 4000-400 cm −1 . All analyses were replicated three times for precision, and the average values of the obtained variables were used. A one-way analysis of variance (ANOVA) test using Microsoft Office Excel 2013 was conducted to evaluate a significant difference between the analysis results, where the significance level was determined at p < 0.05. | 5 |
233708770 | Properties of SHW and Hydrochar The raw SHWs were converted into hydrochar during the HTC reaction. The hydrochar properties at different treatment temperatures are shown in Table 1. Higher HTC temperatures led to lower VM and higher ash contents in the hydrochars due to the accelerated hydrolysis rate and dehydration of raw SHW [20]. The hydrochar ash content affects pollutant emissions and HHVs, thereby determining the suitability of the hydrochar for use as a fuel source [21]. Many countries regulate the maximum permissible level of ash content in bio-solid refuse fuel (SRF), and the domestic regulations in Korea allow a maximum ash dry weight of 15% in bio-SRF. The FC content in the hydrochar gradually increased with increasing HTC temperatures. The FC content in the cattle and pig SHWderived hydrochars increased from 0.10 to 0.55 wt.% (dry) and from 0.27 to 0.76 wt.% (dry), respectively. Higher FC content in fuels help maintain the stable state of the flame during the combustion process. The elemental composition of the obtained hydrochar is presented in Table 1. A gradual increase in carbon content was observed in both the cattle and pig SHW-derived hydrochars with an increase in the HTC temperature. The carbon content is known to be closely associated with energy capacity of combustible material [22,23]. The carbon contents in the cattle and pig SHW-derived hydrochars increased from 65.52 to 71.94 wt.% (dry) and from 50.91 to 71.24 wt.% (dry), respectively. Meanwhile, a drastic decline in nitrogen content in the cattle SHW-derived hydrochar was observed with increasing HTC temperatures. The observation was attributed to the devolatilization of volatile nitrogen in the raw cattle SHW and the elimination of devolatilized nitrogen into the gas or liquid phase [24,25]. The deterioration of nitrogen content in feedstock during HTC can lower NOx emissions during the combustion process if the hydrochar is used as a solid fuel. Furthermore, negligible sulfur levels in both the cattle and pig SHWs revealed the potential use of the hydrochar as a clean energy source without any risk of SOx emissions. Generally, the variable components in the feedstock undergo complex reactions during HTC and affect the hydrochar combustion characteristics [26]. The three main feedstock components (i.e., carbohydrates, proteins, and lipids) are associated with prominent changes in the physicochemical properties of hydrochar [27]. In the cattle SHW, ultrahigh lipid content was observed (74.75 wt.%, dry), whereas carbohydrates and proteins constituted only 1.45 and 1.00 wt.% (dry), respectively. However, these components were distributed more evenly in the pig SHW (the content of carbohydrates, proteins, and lipids equaled 13.86, 13.13, and 33.25 wt.% (dry), respectively). During HTC, carbohydrates tend to contribute to the formation of hydrochar, while proteins help develop N-heterocyclic functional groups; lipids do not participate in the formation of carbonization products [26]. Therefore, each component in the raw SHW can cause changes in the energy-related properties and functional groups of the SHW-derived hydrochar. This issue is addressed in more detail in the following sections. With an increase in the HTC temperature, the fuel ratios of both the cattle and pig SHWs gradually increased. However, there was no significant increase in the fuel ratio of the pig SHW above 200 • C. The higher the fuel ratio, the better the produced solid fuel. Because the FC and VM contents in a combustible material are correlated with the combustion atmosphere by flame violence and heat flow balance, the fuel ratio is essential for determining fuel source potential [16]. Furthermore, the HHVs of the SHW-derived hydrochars were investigated to evaluate the energy potential of the hydrochars. As the HTC treatment temperature increased, all SHW-derived hydrochars had enhanced HHVs. The HHV of the pig SHW-derived hydrochar markedly increased from 4674 to 8804 kcal kg −1 , while the equivalent value of the cattle SHW-derived hydrochar increased only by~1600 kcal kg −1 under the same HTC conditions. This is attributed to the aforementioned differences in the chemical compositions of the cattle and pig SHWs. Although higher lipid content in the cattle SHW led to the higher initial HHV of the raw feedstock, the increase was not dramatic because the lipids were nonreactive during the HTC reaction. Even so, the hydrolyzed lipids were probably adsorbed onto the hydrochar surface [26], and the highest HHV of the cattle SHW-derived hydrochar was comparable to that of the pig SHW-derived hydrochar. | 6 |
233708770 | Improvements in the SHW Properties The lower H/C and O/C ratios represent a greater coalification degree and advanced energy potential [13]. Both the cattle and pig SHWs underwent a combination of complex chemical reactions, especially dehydration and decarboxylation, during HTC. The coalification degree of the SHW-derived hydrochars gradually increased with an increase in the HTC temperature. Thus, it can be concluded that the economic value of raw SHWs for solid fuel use is improved by eliminating oxygen and hydrogen and proportionally increasing the carbon content in the product. In this study, the raw SHWs followed the trend of biomass coalification. However, due to the lipid-rich characteristics of the raw SHW, the SHW-derived hydrochars are positioned differently to the biomass-derived coals (i.e., in the upper left corner of Figure 3). The shift in the coalification degree during HTC was evaluated using the van Krevelen diagram, which compares the aromaticity (atomic H/C ratio) and the polarity (atomic O/C ratio) of the samples (Figure 3). Figure 4 shows the energy-related properties (i.e., energy retention efficiency, energy densification, and product yield) of the cattle and pig SHW-derived hydrochars. The ED of the SHW-derived hydrochars increased with an increase in the HTC temperature, and the ED value was improved to the raw SHW feedstocks (the ED of the raw SHW equaled 1.0). Owing to the aforementioned effects of chemical composition on hydrochar formation, a more substantial increase in ED was observed with the pig SHW-derived hydrochars. Meanwhile, the product yields of both the cattle and pig SHW-derived hydrochars was lowered by the continuous loss of VM and organic matter with the augmented severity in the HTC reaction. The product yield did not decline significantly, and ERE showed a similar trend to ED. When considering ERE and energy consumption at higher HTC temperatures, HTC for cattle SHW is not recommended, whereas a mild HTC temperature of~200 • C is optimal for converting pig SHW into value-added solid fuel. | 7 |
233708770 | Changes of Functional Groups during the HTC Treatment The changes of functional groups on the SHW and hydrochar surfaces were analyzed using FTIR spectra analysis ( Figure 5). Some noticeable peaks were determined based on different peak intensities at various HTC treatment temperatures. The peak at 3000-2800 cm −1 is associated with the aliphatic C-H structure of hydrochars [28]. It represents the miscibility between lipids and water that increases when hydrogen bonding between water molecules weakens during HTC [29]. The lipid solubility in both the cattle and pig SHWs increased after HTC, and the lipid content in the raw feedstock probably became completely miscible under the supercritical conditions. The dissolved lipids were then adsorbed onto the hydrochar surface or ejected with steam after the HTC reaction. Thus, the relative peak intensity slightly decreased. The peak observed at 1650 cm −1 is related to the C=O bond of the carboxylic group [30], indicating that decarboxylation occurred in both SHW-derived hydrochars during the hydrothermal reaction. The reaction was more intense at higher HTC temperatures. The observation of the corresponding peak in hydrochars confirmed the results of the van Krevelen diagram and the H/C and O/C ratios obtained herein. The changes in peak intensity at 1130 cm −1 with respect to the C-O bond represent the carbohydrate component in the SHW [31]. The peak collapse is observed between HTC temperatures of 200 and 250 • C, revealing the thermal degradation of carbohydrates [32]. In summary, the FTIR spectra analysis demonstrated that the studied SHWs were well carbonized and converted into hydrochar after HTC. The differences in the hydrochar functional groups were attributed to the inherent properties of the cattle and pig SHWs. | 8 |
233622552 | : A new hybrid compound of chalcone-salicylate (title compound) has been successfully synthesized using a linker mode approach under reflux condition. The structure of the title compound has been established by spectroscopic analysis including UV-Vis, FT-IR, HRMS, 1D, and 2D NMR. Then, computational approach was also applied in this study through molecular docking and MD simulation to explore its potency against breast cancer. The results of the molecular docking study showed that the title compound exhibited more negative value of binding free energy ( − 8.15 kcal/mol) than tamoxifen ( − 7.00 kcal/mol). In addition, no striking change in the positioning of the interacting residues was recorded before and after the MD simulations. Based on the studies, it can be predicted that the title compound has a cytotoxic activity potency against breast cancer through ER α inhibition and it presumably can be developed as anticancer agent candidate. | 1 |
233622552 | Introduction Chalcones (1,3-diphenyl-prop-2-en-1-ones) are natural products that are found in several plant species and they have been reported for broad spectrum of biological activities. Their analogues and derivatives have been widely synthesized to explore their potential uses [1][2][3], especially in anticancer drug discovery researches [4]. Some researchers have reported that several hydroxylated and methoxylated chalcones exhibited potent cytotoxic activity against the MCF-7 cell line [4,5]. Besides chalcones, some salicylic acid derivatives have also been reported as potent cytotoxic agents against several cancer cell lines, including MCF-7 [6]. Based on the report, the IC 50 values of some tested compounds are close to the IC 50 of 5-fluorouracil. In addition, some hybrid compounds such as β-carbolinesalicylates have also exhibited potent cytotoxic activity against various cell lines [7]. A hybrid of 4-farnesyltiosalicylate-salicylate also possessed the higher cytotoxic activity against MCF-7 cell line than a withdrawn drug, sorafenib (Nexavar) [8]. Therefore, designing and synthesizing a new compound through the molecular hybridization approach is an interesting option in order to search for new anticancer agent candidates. Some workers have reported that this approach has been proven to be an effective way to develop new multifunctional compounds from hybridization or conjugation of two or more molecules that are expected to have a better biological activity than their parent compounds [9]. In this work, we report the synthesis of a new hybrid compound, (E)-4-(3-(3-(4methoxyphenyl)acryloyl)phenoxy)butyl 2-hydroxybenzoate (title compound) using a linker mode approach. The potency of cytotoxic activity of the title compound against breast cancer was studied in silico using computational approaches (molecular docking and MD simulation). | 2 |
233622552 | Results and Discussions 2.1. Synthesis of Title Compound (2) In this work, we have successfully synthesized a new hybrid compound of chalconesalicylate (2) by combining both structures of a substituted chalcone analog (1) and salicylic acid, using linker mode approach [9]. The linker was used because the steric hindrance from both molecules that will be linked usually became a problem in molecular hybridization [10,11]. Application of a linker is a way to minimize the steric hindrance [11,12]. In this work, we used the 1,4-dibromobutane as a linker. The reaction was performed under reflux condition in acetonitrile with the presence of potassium carbonate as catalyst, as described in Figure 1. more molecules that are expected to have a better biological activity than their parent compounds [9]. In this work, we report the synthesis of a new hybrid compound, (E)-4-(3-(3-(4-methoxyphenyl)acryloyl)phenoxy)butyl 2-hydroxybenzoate (title compound) using a linker mode approach. The potency of cytotoxic activity of the title compound against breast cancer was studied in silico using computational approaches (molecular docking and MD simulation). (2) In this work, we have successfully synthesized a new hybrid compound of chalconesalicylate (2) by combining both structures of a substituted chalcone analog (1) and salicylic acid, using linker mode approach [9]. The linker was used because the steric hindrance from both molecules that will be linked usually became a problem in molecular hybridization [10,11]. Application of a linker is a way to minimize the steric hindrance [11,12]. In this work, we used the 1,4-dibromobutane as a linker. The reaction was performed under reflux condition in acetonitrile with the presence of potassium carbonate as catalyst, as described in Figure 1. The title compound in pure form was obtained as clear yellow crystal in 33.13% yield, with melting point of 55-57 °C. The purity of the synthesized product was determined by TLC and HPLC analysis, as shown in Supplementary Material. The structure of the title compound has been confirmed by spectroscopic analysis including UV, FT-IR, HRMS, 1D, and 2D NMR. The FT-IR analysis was performed to ensure that the functional groups present in the synthesized compound match the functional groups present in target molecule. Based on the FT-IR spectra, the title compound showed the broad absorption band at 3112 cm −1 . This absorbance showed the presence of hydroxyl (O-H group) in ortho position bonded to carbonyl group of salicylate through intramolecular hydrogen bond formation. The absorption bands at 3076 cm −1 and 1594-1449 cm −1 showed the presence of aromatic C-H and C=C bonds in three aromatic rings of title compound, while the absorption band at 2953 and 2868 cm −1 showed the symmetrical and asymmetrical stretching of C-H in methoxy group of title compound. The presence of carbonyl group of ester and ketone is shown by the two vibration bands at 1672 and 1657 cm −1 , respectively. In addition, the absorption bands around 1298-1028 cm −1 showed the presence of C-O bonds in ether and ester groups of title compound. The other types of vibrations can be seen in Supplementary Material. In addition, the HRMS analysis was also performed to determine the molecular weight of the synthesized compound. Based on the mass spectral analysis, the synthesized compound has a molecular weight corresponding to the molecular weight of the title compound. The molecular ion peak [M + Na + ] of the title compound is found at m/z 469.1616 with intensity of 100%, while the calculated mass is 469.1627, as shown in Supplementary Material. The title compound in pure form was obtained as clear yellow crystal in 33.13% yield, with melting point of 55-57 • C. The purity of the synthesized product was determined by TLC and HPLC analysis, as shown in Supplementary Material. The structure of the title compound has been confirmed by spectroscopic analysis including UV, FT-IR, HRMS, 1D, and 2D NMR. The FT-IR analysis was performed to ensure that the functional groups present in the synthesized compound match the functional groups present in target molecule. Based on the FT-IR spectra, the title compound showed the broad absorption band at 3112 cm −1 . This absorbance showed the presence of hydroxyl (O-H group) in ortho position bonded to carbonyl group of salicylate through intramolecular hydrogen bond formation. The absorption bands at 3076 cm −1 and 1594-1449 cm −1 showed the presence of aromatic C-H and C=C bonds in three aromatic rings of title compound, while the absorption band at 2953 and 2868 cm −1 showed the symmetrical and asymmetrical stretching of C-H in methoxy group of title compound. The presence of carbonyl group of ester and ketone is shown by the two vibration bands at 1672 and 1657 cm −1 , respectively. In addition, the absorption bands around 1298-1028 cm −1 showed the presence of C-O bonds in ether and ester groups of title compound. The other types of vibrations can be seen in Supplementary Material. In addition, the HRMS analysis was also performed to determine the molecular weight of the synthesized compound. Based on the mass spectral analysis, the synthesized compound has a molecular weight corresponding to the molecular weight of the title compound. The molecular ion peak [M + Na + ] of the title compound is found at m/z 469.1616 with intensity of 100%, while the calculated mass is 469.1627, as shown in Supplementary Material. | 3 |
233622552 | Synthesis of Title Compound The 1D NMR spectra was measured to confirm the structure of the title compound based on the number and chemical environment of protons and carbons. The 1 H NMR spectra of the title compound in CDCl 3 showed a singlet peak (1H) at δ 10.82 ppm due to the presence of hydroxyl group in the aromatic ring of the salicylate moiety. In addition, the aromatic proton signals around δ 7.84-6.87 ppm (12 H) were assigned as aromatic protons in three substituted aromatic rings of the title compound, whereas the signals around δ 4.46-2.01 ppm (11 H) were also assigned as aliphatic protons. Then, the two doublet signals at δ 7.80 ppm (1H) and 7.40 ppm (1H) were assigned as Hβ and Hα protons, respectively. Based on the calculation, the coupling constants (J) of both doublet signals are 15.5 Hz. The coupling constants showed that both protons are in trans (E) position. Based on the interpretation of 1 H NMR spectral data, the synthesized compound has 26 protons, corresponds to the number of protons present in the expected structure. The presence of a methoxy group was observed by the appearance of a singlet signal at δ 3.87 ppm. Then, the appearance of two triplet signals and a multiplet signal in the aliphatic proton region were assigned as four methylene groups (-CH 2 -) 4 in the title compound, as described clearly in Supplementary Material and Table 1. The 13 C NMR spectra of the title compound in CDCl 3 showed that two specific signals at δ 190.19 and 170.15 ppm were assigned as carbonyl signals of ketone and ester, respectively. In addition, the carbon signals around δ 161.70-112.46 ppm were assigned as aromatic carbon signals on three aromatic rings of the title compound including three oxyaryl carbons C2 (161.67), C1 (159.13), and C4 (161.70). Then, five carbon signals around δ 67.42-25.44 ppm were assigned as aliphatic carbon signals of a methoxy group and four methylene carbons in the title compound, as described in Supplementary Material and Table 1. Based on the interpretation of 13 C NMR spectral data, the synthesized compound has 27 carbons, and it is matched with the number of carbon in the expected structure. The assignments of 1 H and 13 C NMR spectra of the title compound were supported by 1 D-TOCSY, 13 C-DEPT, COSY, HSQC, and HMBC spectra, as described clearly in Supplementary Material. The 1 H-1 H correlations in HMBC spectra ensured that the linker is bound to carboxyl group, as depicted in Figure 2. The interpretation result of COSY, HSQC, and HMBC spectra showed that the 1 H-1 H and 1 H-13 C correlations in the title compound are matched with the expected structure. δ 67.42-25.44 ppm were assigned as aliphatic carbon signals of a methoxy group and four methylene carbons in the title compound, as described in Supplementary Material and Table 1. Based on the interpretation of 13 C NMR spectral data, the synthesized compound has 27 carbons, and it is matched with the number of carbon in the expected structure. The assignments of 1 H and 13 C NMR spectra of the title compound were supported by 1 D-TOCSY, 13 C-DEPT, COSY, HSQC, and HMBC spectra, as described clearly in Supplementary Material. The 1 H-1 H correlations in HMBC spectra ensured that the linker is bound to carboxyl group, as depicted in Figure 2. The interpretation result of COSY, HSQC, and HMBC spectra showed that the 1 H-1 H and 1 H-13 C correlations in the title compound are matched with the expected structure. | 4 |
233622552 | Molecular Docking Study and MD Simulation The molecular docking study was performed to predict the ability of the title compound to bind with the estrogen receptor. This receptor is classified into two subtypes, ERα and ERβ. Both receptors are present in the mammary gland [13]. ERα plays an important role in cell proliferation [14] and pathogenesis of breast cancers [15]. Approximately 75% of breast cancers have positive expression of this specific type of hormonal receptor [16]. Therefore, targeting this receptor is an attractive option for finding a new anticancer agent for breast cancer. In this work, the molecular docking study was performed for the starting material (chalcone analogue), title compound (hybrid of chalcone-salicylate), and tamoxifen as a reference drug to treat breast cancer. The crystal structure of ERα was downloaded from rcsb.org with PDB ID of 3ERT. This crystal structure is bound to a co-crystalized ligand, 4-OHT, one of the major active metabolites of tamoxifen [17]. The docking study was performed in several steps, as described in the procedure section. After the protein and ligands were prepared, a validation of docking protocol is required to ensure the accuracy of the docking result [18][19][20]. The validation of docking protocol can be performed by redocking the co-crystalized ligand (4-OHT) to the prepared receptor. The validation result of docking protocol is presented in Supplementary Material. The result showed that the similarity of binding poses between co-crystalized ligand and re-docking ligand is 81.48% with RMSD value less than 2. These results showed that the docking protocol was valid. The 3D and 2D binding poses of re-docked ligand (4-OHT) is depicted in Supplementary | 5 |
233622552 | Molecular Docking Study and MD Simulation The molecular docking study was performed to predict the ability of the title compound to bind with the estrogen receptor. This receptor is classified into two subtypes, ERα and ERβ. Both receptors are present in the mammary gland [13]. ERα plays an important role in cell proliferation [14] and pathogenesis of breast cancers [15]. Approximately 75% of breast cancers have positive expression of this specific type of hormonal receptor [16]. Therefore, targeting this receptor is an attractive option for finding a new anticancer agent for breast cancer. In this work, the molecular docking study was performed for the starting material (chalcone analogue), title compound (hybrid of chalcone-salicylate), and tamoxifen as a reference drug to treat breast cancer. The crystal structure of ERα was downloaded from rcsb.org with PDB ID of 3ERT. This crystal structure is bound to a co-crystalized ligand, 4-OHT, one of the major active metabolites of tamoxifen [17]. The docking study was performed in several steps, as described in the procedure section. After the protein and ligands were prepared, a validation of docking protocol is required to ensure the accuracy of the docking result [18][19][20]. The validation of docking protocol can be performed by re-docking the co-crystalized ligand (4-OHT) to the prepared receptor. The validation result of docking protocol is presented in Supplementary Material. The result showed that the similarity of binding poses between co-crystalized ligand and re-docking ligand is 81.48% with RMSD value less than 2. These results showed that the docking protocol was valid. The 3D and 2D binding poses of re-docked ligand (4-OHT) is depicted in Supplementary Material and the comparison of binding poses of co-crystalized (yellow color) and re-docked ligand (atomic coloring) are depicted in Figure 3. Based on the figure, we can observe that they are superimposed. Based on the docking result, the chalcone analogue, title compound, show similar interactions to 4-OHT. Based on the Table 2, we observed tha pound had more negative binding free energy (−8.15 kcal/mol) when co Based on the docking result, the chalcone analogue, title compound, and tamoxifen show similar interactions to 4-OHT. Based on the Table 2, we observed that the title compound had more negative binding free energy (−8.15 kcal/mol) when compared to the chalcone analogue (−6.32 kcal/mol) and tamoxifen (−7.00 kcal/mol). More negative binding free energy indicates that the title compound is more easily bound to the ERα [18]. In addition, we also observed that the binding free energy of the title compound was very close with the binding free energy of 4-OHT as native ligand (−9.02 kcal/mol). The chalcone analogue is able to form one hydrogen bond to Glu353 residue in the active site of ERα. This interaction was also observed between 4-OHT and ERα. However, 4-OHT has more van der Walls and other hydrophobic interactions that might cause 4-OHT to have more negative value of binding free energy than chalcone analogue. In addition, the title compound is also able to form hydrogen bond, but with different amino acid residue (Cys530). This interaction is not observed for other docked compounds. However, there are 18 similar interactions between the title compound and tamoxifen, and there are 19 similar interactions between the title compound and 4-OHT, as depicted in Table 2 MD simulation was also performed to observe the stability of the protein-ligand complex. This simulation treats both protein and ligand as flexible entities, involves the binding and breaking of the hydrogen bonds and other interactions in the protein-ligand complex caused by the continuous motion of the molecules, and also computing movements as a function of time [21]. This simulation can aid us to ensure weather the interactions between the compound and protein were still maintained or not during the simulation [22][23][24]. The MD simulation result showed that the hydrogen bond between hydroxyl group of title compound and Cys530 (2.79 Å) was broken, caused by conformational changing. However, most of interactions between the title compound and ERα are still maintained, and amazingly, after MD simulation, two new hydrogen bonds are formed, between the hydroxyl group of the title compound with Asp351 (1.66 Å) and between MD simulation was also performed to observe the stability of the protein-ligand complex. This simulation treats both protein and ligand as flexible entities, involves the binding and breaking of the hydrogen bonds and other interactions in the protein-ligand complex caused by the continuous motion of the molecules, and also computing movements as a function of time [21]. This simulation can aid us to ensure weather the interactions between the compound and protein were still maintained or not during the simulation [22][23][24]. The MD simulation result showed that the hydrogen bond between hydroxyl group of title compound and Cys530 (2.79 Å) was broken, caused by conformational changing. However, most of interactions between the title compound and ERα are still maintained, and amazingly, after MD simulation, two new hydrogen bonds are formed, between the hydroxyl group of the title compound with Asp351 (1.66 Å) and between MD simulation was also performed to observe the stability of the protein-ligand complex. This simulation treats both protein and ligand as flexible entities, involves the binding and breaking of the hydrogen bonds and other interactions in the protein-ligand complex caused by the continuous motion of the molecules, and also computing movements as a function of time [21]. This simulation can aid us to ensure weather the interactions between the compound and protein were still maintained or not during the simulation [22][23][24]. The MD simulation result showed that the hydrogen bond between hydroxyl group of title compound and Cys530 (2.79 Å) was broken, caused by conformational changing. However, most of interactions between the title compound and ERα are still maintained, and amazingly, after MD simulation, two new hydrogen bonds are formed, between the hydroxyl group of the title compound with Asp351 (1.66 Å) and between ketone carbonyl group with Thr347 (2.02 Å). Based on the literature [20], the hydrophilic interactions with both residues are playing important role in antagonist activity of 4-OHT to ERα. The MD simulation results are presented in Table 3 and depicted in Supplementary Material. A comparison of binding poses of the title compound before and after MD simulation is depicted in Figure 6. Notably, no striking change in the positioning of the interacting residues was recorded before and after the MD simulations. In addition, they are still superimposed. ketone carbonyl group with Thr347 (2.02 Å). Based on the literature [20], the hydrophilic interactions with both residues are playing important role in antagonist activity of 4-OHT to ERα. The MD simulation results are presented in Table 3 and depicted in Supplementary Material. A comparison of binding poses of the title compound before and after MD simulation is depicted in Figure 6. Notably, no striking change in the positioning of the interacting residues was recorded before and after the MD simulations. In addition, they are still superimposed. | 6 |
233622552 | Instrumentations The instruments used in synthesis, purification, and structure characterization of the title compound are a set of reflux apparatus, vacuum rotary evaporator (Buchi ® , Flawil, Figure 6. The 3D visualization of superimposition of binding poses of the title compound before and after MD simulation. Binding pose before MD simulation is presented in blue and after MD is presented in yellow. The amino acid residue with hydrogen bond is presented in green and the other maintained interactions during MD simulation are presented in red. | 7 |
233622552 | Synthesis of (E)-4-(3-(3-(4-Methoxyphenyl)acryloyl)phenoxy)butyl 2-Hydroxybenzoate As much as 9 mmol (1.9431 g) of 1,4-dibromobutane was diluted in 50 mL of acetonitrile in a round bottom flask (mixture 1), 3 mmol (0.762 g) of chalcone analogue was dissolved in 15 mL acetonitrile (mixture 2), and 6 mmol (0.8292 g) of potassium carbonate was added into mixture 2. Then, the mixture 2 was poured into mixture 1. The reaction mixture was refluxed and stirred on the oil bath at 80-85 • C until the reaction was completed (27 h). After the reaction was completed (observed by TLC analysis), the intermediate solution was used immediately without further purification. A mixture of 6 mmol (0.828 g) of salicylic acid and 4 mmol (0.5528 g) of potassium carbonate in 7.5 mL acetonitrile was added to the intermediate solution and then refluxed and stirred at 80 • C until the reaction was completed (48 h). After the reaction was completed (observed by TLC analysis), the mixture of the product was poured in a separatory funnel and washed by distilled water (3 × 15 mL). Then, the solvent was evaporated using vacuum rotary evaporator to afford the crude product. This crude product was purified through a SiO 2 column chromatography with a mixture of n-hexane and ethyl acetate (8:2) as mobile phase to get pure product of chalcone-salicylate. The purity of hybrid compound was confirmed by TLC and HPLC analysis. Then, the structure of hybrid compound was confirmed by spectroscopic analysis including FT-IR, HRMS, 1D NMR ( 1 H NMR, 13 13 | 8 |
233622552 | Molecular Docking Study The structure of ligands were drawn using ChemDraw Professional 15.0 and a database of ligand was created using MOE 2019.0101 software package (Chemical Computing Group, Tokyo, Japan). The crystal structure of ERα with PDB ID 3ERT was downloaded from http://www.rcsb.org [18][19][20] and was prepared in Discovery Studio 2020 Client to remove the water, co-crystalized ligand, and then, it was further prepared using MOE to minimize the energy. Before running the docking, the placement and refinement method were set as alpha triangle and rigid receptor, respectively. The placement and refinement scores were set as Afinity dG. Then, the placement and refinement poses were set as 100 and 30, respectively. The other parameters were set as default. The 4-OHT as native ligand was re-docked to the prepared protein to validate this docking protocol. After the protocol was validated, the other ligands were docked with same method. The best poses of docking results were selected based on some parameters such as binding free energy, RMSD, and the similarity of interactions compared to the 4-OHT as native ligand and to tamoxifen as breast cancer drug. The best poses of the docked ligands were presented in 2D and 3D visualization using Discovery Studio 2020 Client. | 9 |
233622552 | MD Simulation The MD simulation was also performed using MOE. Before run the simulation, the algorithm was set as NPA. The start time, checkpoint, and sample time were set as 0, 300, and 0.01, respectively. Then, the forcefield was set as CHARMM27, and the other parameters were set as default. The result of MD simulation was presented in 2D and 3D visualization using Discovery Studio 2020 Client. Then, the protein-ligand interactions before and after MD simulation were compared. | 10 |
233622552 | Supplementary Materials: The following are available online, Figure S1: the clear yellow crystal of the title compound (hybrid compound), Figure S2: TLC chromatogram of hybrid compound (HC) compared to chalcone analog (CA) and salicylic acid (SA) as starting materials under UV lamp, 254 nm. H = n-hexane and E = ethyl acetate, Figure S3: the result of TLC analysis of hybrid compound using various mobile phases: (a) n-hexane: ethyl acetate (9:1), (b) DCM: n-hexane (8:2), (c) D100% = DCM 100%, Figure S4: HPLC chromatogram of hybrid compound, analysis was performed using reverse phase column Shim-Pack VP-ODS (150 × 4.6 mm) with gradient elusion method using water and acetonitrile (HPLC grade) as mobile phase for 20 minutes with flow rate 1 ml/minute, Figure S5: the FT-IR spectra of hybrid compound, Figure S6: The HRMS spectra of hybrid compound, Figure S7: the 1 H NMR spectra of hybrid compound in CDCl 3 (500 MHz), Figure S8: the 1 H NMR spectra of hybrid compound in CDCl 3 (500 MHz), expansion in aromatic region, Figure S9: the 1 H NMR spectra of hybrid compound in CDCl 3 (500 MHz), expansion in aliphatic region, Figure S10: the 1D TOCSY spectra of aromatic region of hybrid compound in CDCl 3 (500 MHz), Figure S11: the COSY spectra of hybrid compound in CDCl 3 (500 MHz), Figure S12: the COSY spectra of hybrid compound in CDCl 3 (500 MHz), expansion in R aromatic region, Figure S13: the COSY spectra of hybrid compound in CDCl 3 (500 MHz), expansion in R" aromatic region, Figure S14: the COSY spectra of hybrid compound in CDCl 3 (500 MHz), expansion in R"' aromatic region, Figure S15: the COSY spectra of hybrid compound in CDCl 3 (500 MHz), expansion in aliphatic region, Figure S16: the 13 C NMR spectra of hybrid compound in CDCl 3 (125 MHz), Figure S17: the overlay of 13 C NMR and DEPT-135 spectra of hybrid compound in CDCl 3 (125 MHz), Figure S18: the overlay of 13 C NMR and DEPT-135 spectra of hybrid compound in CDCl 3 (125 MHz), expansion in aromatic region, Figure S19: the HSQC spectra of hybrid compound in CDCl 3 (500 MHz), expansion in aromatic region, Figure S20: the 1 H-13 C correlations of R aromatic region in HSQC spectra of hybrid compound in CDCl 3 , Figure S21: the 1 H-13 C correlations of R" aromatic region in HSQC spectra of hybrid compound (title compound) in CDCl 3 , Figure S22: the 1 H-13 C correlations of R"' aromatic region in HSQC spectra of hybrid compound in CDCl 3 , Figure S23: the 1 H-13 C correlations of aliphatic region in HSQC spectra of hybrid compound in CDCl 3 (500 MHz), Figure S24: the HMBC spectra of hybrid compound in CDCl 3 , Figure S25: the important 1 H-13 C correlations of aliphatic protons in HMBC spectra of hybrid compound in CDCl 3 , Figure S26: the important 1 H-13 C correlations of hydroxyl group in HMBC spectra of hybrid compound in CDCl 3 , Figure S27: the important 1 H-13 C correlations of R" and R"' aromatic protons and α, β protons in HMBC spectra of hybrid compound in CDCl 3 (a) correlation with C190.19-C159.13, (b) correlation with C144.47-C127.60, Figure S28: 3D and 2D binding poses of re-docked native ligand (4-OHT) to ERα, Figure S29: the 3D and 2D visualization of binding modes of docked compounds to ERα, (a) chalcone analogue, (b) title compound, (c) Tamoxifen, Figure S30: the visualization of MD simulation result of the title compound using Discovery Studio 2020 Client (a) 3D visualization (b) 2D visualization, Table S1: mobile phase composition for HPLC analysis of hybrid compound, Table S2: the result of docking protocol validation. | 12 |
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