Listeria monocytogenes is a facultative Gram-positive bacterium that causes listeriosis, a severe foodborne disease. We previously discovered that ring-fused 2-pyridone compounds can decrease virulence factor expression in Listeria by binding and inactivating the PrfA virulence activator. In this study, we tested PS900, a highly substituted 2-pyridone that was recently discovered to be bactericidal to other Gram-positive pathogenic bacteria, such as Staphylococcus aureus and Enterococcus faecalis. We show that PS900 can interact with PrfA and reduce the expression of virulence factors. Unlike previous ring-fused 2-pyridones shown to inactivate PrfA, PS900 had an additional antibacterial activity and was found to potentiate sensitivity toward cholic acid. Two PS900-tolerant mutants able to grow in the presence of PS900 carried mutations in the brtA gene, encoding the BrtA repressor. In wild-type (WT) bacteria, cholic acid binds and inactivates BrtA, thereby alleviating the expression of the multidrug transporter MdrT. Interestingly, we found that PS900 also binds to BrtA and that this interaction causes BrtA to dissociate from its binding site in front of the mdrT gene. In addition, we observed that PS900 potentiated the effect of different osmolytes. We suggest that the increased potency of cholic acid and osmolytes to kill bacteria in the presence of PS900 is due to the ability of the latter to inhibit general efflux, through a yet-unknown mechanism. Our data indicate that thiazolino 2-pyridones constitute an attractive scaffold when designing new types of antibacterial agents. Listeria monocytogenes is a Gram-positive foodborne pathogen that can cause severe infections (listeriosis) in different animals and in humans. Listeriosis can appear as a sporadic infection or as a disease outbreak, with mortality rates of up to 30% , as well as to survive inside the gut.p to 30% . L. monoL. monocytogenes is a great concern for the food industry due to its ability to withstand and grow at a wide range of temperatures (−0.4°C to 45°C) and pH levels (4.3 to 9.6), as well as high salt concentrations (up to 20%)  and nitrate  are often added to food products, such as meat, to reduce L. monocytogenes growth L. monocy4.3 to 9., as wellL. monocytogenes enters the host through consumption of contaminated food. Once ingested, the bacterium enters the intestinal tract, where it able to reside for a couple of days before passing the intestinal barrier. In a human population study, L. monocytogenes was detected in the stool of 2.1% of asymptomatic individuals   and a bile exclusion protein (BilE)  . BrtA acactor A) . L. monon (BilE) –19. Bsh  (BilE) –.L. monocytogenes enters epithelial cells guided by the adhesin internalin A, which recognizes E-cadherin  in bacteria . CompareL. monocytogenes, PrfA, decreased the ability of the protein to interact with its DNA motif , 27L. mo10.1128/mbio.00449-23.1FIG S1FIG S1, EPS file, 2.0 MB.Fluorescence-quenching spectra of PrfA protein in the presence of DMSO (control). (A) Fluorescence quenching of various concentrations of MH44 (0.5 to 5.0 μM) in the absence of protein. (B) Fluorescence quenching of various concentrations of PS900 (0.5 to 5.0 μM) in the absence of protein. (C) A concentration of 5 μM PrfA in PBS solution was excited at 290 nm, and quenching of fluorescence was recorded in the presence of various concentrations of DMSO (0.2 to 2.0%).  Fluorescence-quenching spectra of BrtA protein in the presence of DMSO (control) (D) or MH44 (E). A concentration of 5 μM BrtA in PBS solution was excited at 290 nm, and quenching of fluorescence was recorded in the presence of various concentrations of DMSO (0.2 to 3.0%) or MH44 (0.5 to 5 μM). Download Copyright © 2023 Tükenmez et al.2023Tükenmez et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the L. monocytogenes, which is important for inlB expression  at a level comparable to that of MH44 without affecting the levels of PrfA . Interespression .L. monocytogenes, possibly by inactivating PrfA, as previously shown for other 2-pyridones   when theathogens  28). In. InL. moathogens .10.1128/mbio.00449-23.2FIG S2FIG S2, EPS file, 5.6 MB.Small growth inhibition of bacteria grown in BHI medium in the presence of PS900. Representative images showing the effects of MH44 and PS900 on bacterial growth and survival in BHI medium. Effects on bacterial growth (MICs) were scored by pellet formation and resazurin (blue)-to-resorufin (pink) conversion . Bacterial survival (MBC) was scored by growth on regular BHI agar plates. The WT strain was incubated overnight in BHI medium with various concentrations of DMSO (control), MH44, or PS900 at 37°C. Samples were then spotted onto regular BHI agar plates, followed by 24 h of incubation at 37°C. The images are representatives of three individual experiments. Download Copyright © 2023 Tükenmez et al.2023Tükenmez et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the L. monocytogenes is a soil bacterium that occasionally becomes ingested and can infect a eukaryotic host. Being exposed to a plethora of environments, it has developed a number of mechanisms, such as efflux pumps, to survive harsh conditions   . We thused in DM .10.1128/mbio.00449-23.3FIG S3L. monocytogenes BrtA protein. (A) AlphaFold model output of BrtA protein  color coded by model confidence score Copyright © 2023 Tükenmez et al.2023Tükenmez et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the L. monocytogenes growth and viability, we set up a plate experiment to select for suppressor mutants able to grow in DM in the presence of 40 μM PS900 , whereas the other suppressor mutant (HT015) had a +1 frameshift in the brtA gene, leading to a premature stop codon at position 126.To further elucidate the mechanism by which PS900 reduces μM PS900 . Two sucμM PS900  and B anμM PS900 . Unlike tor BrtA . One of 10.1128/mbio.00449-23.9TABLE S1Table S1, PDF file, 0.03 MB.(A) Details of Illumina sequence results. (B) SNP analysis of WT, HT014, and HT015 strains. Download Copyright © 2023 Tükenmez et al.2023Tükenmez et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the L. monocytogenes, BrtA has been shown to control the expression of the multidrug transporter MdrT by acting as a repressor . Md. MdL. momdrT in the WT strain, likely through CA interacting with and inactivating BrtA, as has been shown previously  resulted in increased mdrT expression in the WT strain  increases the expression of the MdrT efflux pump, thereby allowing bacterial growth in the presence of PS900 . To chaleviously ; Fig. S4eviously . The HT0T strain ; Fig. S410.1128/mbio.00449-23.4FIG S4mdrT efflux pump expression in the presence of MH44, PS900, and/or cholic acid (Northern blots). (A) Individual replicates of the Northern blots depicting the levels of mdrT and tmRNA. The total RNA was isolated from the WT, HT014, and HT015 strains, which were grown in the presence of 10 μM MH44, 10 μM PS900, or 0.1% DMSO (control), respectively, for 2.5 to 3 h at 37°C. Then, the cultures were divided into two subcultures. One set was incubated for another 30 minutes at 37°C with 10 mM cholic acid, whereas the other set was incubated with DMSO (control). The levels of mdrT, brtA, and tmRNA (control) were visualized by Northern blotting using specific probes. The mdrT and brtA levels from each sample were normalized to the tmRNA levels. The results for each sample were normalized to those of the WT strain grown in the presence of 0.1% DMSO without cholic acid.  Plots representing the normalized mdrT (B) and brtA (C) levels of each biological replicate normalized to the expression of tmRNA as shown in panel A. Error bars indicate the standard deviations among the replicates. Download FIG S4, EPS file, 7.7 MB.Copyright © 2023 Tükenmez et al.2023Tükenmez et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the mdrT expression, we asked whether the increased level of mdrT in bacteria exposed to PS900 was sufficient to allow bacteria to grow in the presence of CA. To examine this, WT bacteria were preexposed (or not) to 10 mM CA, 10 μM PS900, or 10 μM MH44 for 1 h before regrowing the bacteria in the presence of 10 mM CA for 15 h. Preincubation with dimethyl sulfoxide (DMSO) (control) or MH44 completely abrogated bacterial growth after the addition of CA  of other possible mutations. Plasmid constructs carrying the wild-type (WT) brtA (brtAWT) and the brtAF16S genes were created and introduced into the WT, the HT014 mutant (having the brtAF16S mutation on the chromosome), and the HT015 mutant (carrying a brtA frameshift mutation leading to a truncated BrtA protein), respectively. These constructs were inoculated into medium with or without CA and PS900, and their growth monitored  in the presence of CA and PS900 compared to the growth of the same strains carrying the vector control pMK4 (WT did not show a fully reduced growth rate (as in the WT strain) in the presence of either CA or PS900 could be that BrtAF16S acts as a dominant-negative mutant protein, partially reducing BrtAWT’s activity. Reintroducing BrtAF16S into the HT014 and HT015 strains resulted in slightly decreased growth rates compared to those of the vector controls. Our results thus suggest that an inactivated BrtA protein causes increased resistance of the HT014 and HT015 strains to CA and PS900, although we cannot exclude the possibility of other genetic differences contributing to the phenotype.Although both HT014 and HT015 had mutations in onitored . We hyporol pMK4 . The rea10.1128/mbio.00449-23.5FIG S5WT, or pMK4-BrtAF16S.  All strains were grown overnight in BHI medium with 10 μg/mL chloramphenicol at 37°C. Overnight cultures were diluted to an OD600 of 0.05 in BHI medium with 10 μg/mL chloramphenicol containing 0.1% DMSO without cholic acid (CA) (A) or 10 μM PS900 with 10 mM CA (B). The cultures were grown for 7.5 h at 37°C. All measurements were performed in triplicates, and the results were plotted using GraphPad Prism (version 9.3.1). Error bars represent standard deviations of replicates. Download FIG S5, EPS file, 1.7 MB.Growth of WT, HT014, and HT015 strains complemented with pMK4, pMK4-BrtACopyright © 2023 Tükenmez et al.2023Tükenmez et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the mdrT expression would be that PS900 is able to interact with and inactivate BrtA in a manner similar to that of CA. We therefore examined whether PS900 could interact directly with BrtA. Superimposing PS900 over CA at the ligand binding site of the BrtA homolog RamR from Salmonella enterica serovar Typhimurium and the QacR homolog inStaphylococcus aureus suggested that BrtA could accommodate PS900 (DK [equilibrium dissociation constant] of 4.6 μM), but not MH44 and BrtA. We next asked whether PS900 could displace BrtA from its DNA binding site located upstream from the mdrT gene. To test this, electrophoretic mobility shift assays (EMSAs) were conducted in the presence and absence of mdrT DNA and the BrtA protein, as well as CA, PS900, or MH44  bound the operator region more weakly than the BrtAWT protein did  within the ligand binding site of the QacR repressor from Staphylococcus aureus  and cholic acid (cyan) together with the X-ray crystal conformation of rhodamine 6G  binds DNA with a lower efficacy than BrtAWT. (A) EMSA using an 85-bp mdrT upstream fragment . Lanes 1 and 2, no protein added. Lanes 3 to 10 (left), binding reaction with various concentrations of wild-type BrtA protein (0.8 to 80.0 pmol). Lanes 3 to 10 (right), binding reaction with various concentrations of BrtAF16S protein (0.8 to 80 pmol). Lane 1, DNA ladder . Efficiency of protein-DNA interaction was determined by quantification of the unmigrated DNA. (B) Plots of decreases in levels of unmigrated DNA in the presence of increasing concentrations of wild-type BrtA (green) or BrtAF16S (blue). All measurements were performed in triplicates, and the results were plotted using GraphPad Prism (version 9.3.1). Black bars represent standard deviations of replicates. Download FIG S7, EPS file, 4.1 MB.BrtA protein carrying an F16S point mutation . Stern-Volmer plots of decreases in the fluorescence of BrtAF16S in the presence of various concentrations of PS900 were used to determine the dynamic quenching rate constant, Ksv . Logarithmic plots of relative fluorescence quenching of BrtAF16S against logarithmic concentrations of PS900 were used to determine DK  and the number of binding sites, n . Error bars indicate standard deviations calculated from three individual experiments. Download FIG S8, EPS file, 1.4 MB.Fluorescence-quenching spectra of the BrtACopyright © 2023 Tükenmez et al.2023Tükenmez et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Since PS900 has a bactericidal effect if bacteria are grown in defined medium and it is able to mimic some phenotypes observed after CA addition, we sought to examine whether the addition of PS900 together with CA could also affect bacterial viability in BHI medium (as we see for other Gram-positive pathogens exposed to PS900 ). TherefmdrT expression, i.e., not pretreated with CA, had a slow efflux of PS900  when it interacts with cytoplasmic DNA  (2+ channel inhibitor)  . In conthibitor) .mdrT expression. If PS900 only targets MdrT, the addition of PS900 to these mutants should reduce EtBr efflux. On the other hand, if PS900 has additional targets, EtBr efflux should remain similar. The addition of PS900 only marginally affected EtBr efflux, indicating that PS900 targets other efflux pumps as well   and potetor PrfA , 27, 29.S. aureus (MRSA) and vancomycin-resistant enterococcus (VRE), at low micromolar concentrations (L. monocytogenes in defined medium (DM), and not in BHI medium  uses MdrT for efflux . PretreamdrT ; Fig. S4l efflux . The prel efflux . Furtherr efflux .L. monocytogenes is dependent on osmolyte transporters like OpuC (a carnitine transporter), BetL (a betaine uptake system), and BilE (a bile exclusion system)  50-fold. Since KNO3 is important as a preservative in the food industry (E252), our findings identified a role for ring-fused 2-pyridones in combination with other food additives to prevent L. monocytogenes-mediated food spoilage. As for CA, we suggest that the bactericidal effect observed when PS900 was added together with the osmolyte was due to the ability of PS900 to inhibit efflux, thus increasing the intracellular concentration of the osmolyte. Alternatively, PS900 could interact with other targets, such as by affecting the level or activity of c-di-AMP, which is a secondary metabolite controlling the expression of many genes, including osmolyte transporters  . In thissporters . Also, csporters .L. monocytogenes virulence  in 25% glycerol or as colonies for up to 2 weeks at 4°C on brain heart infusion (BHI) agar (BD Biosciences). Growth experiments were performed at 37°C in BHI or in a chemically defined medium (DM) as previously described  and compounds (MH44 and PS900) was analyzed by the concentration-dependent effect of compounds on the tryptophan fluorescence emission of these proteins, as previously described by Tükenmez et al.  for docThe ligands were docked using Glide –47 imple–L. monocytogenes were diluted to an optical density at 600 nm (OD600) of 0.05 in BHI supplemented with 0.1% DMSO (vol/vol) or 10 μM 2-pyridones (MH44 or PS900) in 96-well plates. The cultures were grown at 37°C with shaking for 5 h to a final OD600 of 1.5 to 2.0. The cultures were then centrifuged at 18,000 × g for 5 min at room temperature. The supernatants were transferred to new tubes for trichloroacetic acid (TCA) protein precipitation, and the pellets were washed once with 1× PBS. The pellets were suspended with buffer A , and ~200 μL glass beads was added. The suspensions were disrupted using a bead-beater for 30 s at maximum speed 3 times and rested on ice for 1 min in between. The suspensions were subsequently centrifuged at 18,000 × g for 5 min at 4°C, and supernatants were TCA protein precipitated.Overnight cultures of g for 10 min at 4°C, and the pellets were washed with ice-cold 80% acetone. The dried protein pellets were suspended in 1× Laemmli buffer and boiled at 95°C for 10 min. The samples were then subjected to SDS-PAGE and Western blotting using anti-Bacillus subtilis CodY , anti-LLO , anti-ActA, and anti-PrfA R79IS4b  primary antibodies and a horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody . The signal levels were quantified with ImageJ (version 1.53t), and data analysis was done with GraphPad Prism (version 9.4.1).Both supernatant (secreted) and whole-cell lysate fractions were mixed with 1:10 volume of 2% sodium deoxycholate, followed by 10 min of incubation at room temperature. With the addition of a 1:4 volume of ice-cold 50% TCA, the samples were incubated on ice for an hour. Next, the samples were pelleted at 18,000 × L. monocytogenes were diluted to an OD600 of 0.05 in 20 mL BHI medium supplemented with 0.1% DMSO (vol/vol) or 10 μM 2-pyridone (MH44 or PS900) in 100-mL flasks. The cultures were grown at 37°C with shaking for 2.5 to 3 h to an OD600 of 0.5 to 0.7. The cultures were split into two subcultures and mixed at a 1:1 ratio with BHI medium supplemented with 2% DMSO (vol/vol) or 20 mM cholic acid, followed by incubation at 37°C with shaking for 30 min. The samples were mixed with 0.2× volume of a 5% phenol–95% ethanol solution and centrifuged at 4,000 × g for 15 min at 4°C. The pellets were then stored at −80°C.Overnight cultures of 32P]dATP-α-labeled DNA probes. These DNA probes were prepared using a Prime-a-Gene kit (Promega), and DNA fragments amplified by PCR with corresponding oligonucleotides for the mdrT (lmo2588) gene , the brtA (lmo2589) gene , and transfer mRNA (tmRNA) . Membranes were washed with 0.1% SDS, 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) at 65°C for 15 min, followed by washing with 0.1% SDS, 1 × SSC at 65°C for 15 min and 0.1% SDS, 0.5 × SSC. The membranes were exposed in a phosphoscreen cassette, which was then developed using the Typhoon FLA 9500 scanner . Signal levels were quantified with ImageQuant 8.2, and data analysis was done with GraphPad Prism (version 9.4.1).Isolation of RNA and Northern blotting were performed as previously described in Oliveira et al. , with miL. monocytogenes WT (EGD-e), HT014, and HT015 strains were cultivated in BHI medium at 37°C overnight. Genomic DNA from these cultures was prepared using the DNeasy blood & tissue kit (Qiagen) according to the manufacturer’s protocol. Illumina sequencing libraries were prepared by MicrobesNG  using the Nextera XT library preparation kit  according to the manufacturer’s protocol with the following modifications: input DNA was increased 2-fold, and the PCR elongation time was increased to 45 s. Libraries were sequenced with the Illumina NovaSeq 6000 platform using the 250-bp paired-end protocol. The reads were adapter trimmed using Trimmomatic 0.30 with a sliding window quality cutoff score of Q15 . Details of whole-genome-sequencing (WGS) data and single nucleotide polymorphism (SNP) data can be found in the supplemental material  upstream fragment. Complementary oligonucleotides bearing the mdrT upstream (−70…+15) sequence were annealed and amplified with oligonucleotides (5′-TGCTGTACTATTCAAAATTAATACCC-3′ and 5′-CTTGTTGGTCTTTTTTTATTATACTTC-3′). The EMSA reactions were performed in 10 mM Tris-HCl (pH 8.0), 150 mM KCl, 0.5 mM EDTA (pH 8.0), 0.1% Triton X-100, 12.5% glycerol, 0.2 mM DTT, 0.1 mg/mL bovine serum albumin (BSA), and 10 ng/μL poly(dI-dC) reaction buffer as previously described in Tükenmez et al. (Electrophoretic mobility shift assays (EMSAs) were performed with the 85-bp z et al. . The binL. monocytogenes WT, HT014, and HT015 cultures were grown overnight in BHI medium at 37°C with shaking. EtBr efflux was monitored in the presence of 10 μM MH44, PS900, or another known efflux inhibitor . All measurements were performed in triplicates, and the fluorescence signals from EtBr were normalized to the average EtBr signal of DMSO (vehicle control)-treated samples at zero minutes.The EtBr efflux assay was performed as previously described in Nye et al. , with miL. monocytogenes WT, HT014, and HT015 cultures were grown overnight in BHI at 37°C with shaking. Overnight cultures were diluted to an OD600 of 1.0 in 5 mL of BHI medium with or without 10 mM CA and incubated for an hour at 37°C. The cultures were then washed once with PBS and normalized to an OD600 of 1.0 in 5 mL of PBS. These PBS-washed cells were then incubated with either 0.1% DMSO (vehicle control) or 10 μM PS900 for 15 min at room temperature. The samples were washed once with PBS and then incubated again in PBS for 30 min at 37°C. At 0, 15, and 30 min, 1-mL amounts of these suspensions were harvested by centrifugation, resuspended with 1 mL of PBS, and aliquoted into flat-bottom black 96-well plates with clear bottoms . The fluorescence signals emitted from PS900 (excitation wavelength [λex] = 400 nm and emission wavelength [λem] = 440 nm), as well as OD600 values, were measured with a SpectraMax iD3 microplate reader (Molecular Devices). The fluorescence signals from PS900 were first normalized to the OD600 measurements, and then the dissociation results were plotted as the percentages of PS900 fluorescence relative to the initial fluorescence  over a 30-min time course. All measurements were performed in triplicates, and the results were plotted using GraphPad Prism (version 9.3.1).The PS900 dissociation assay was performed as previously described in Nye et al. , with miL. monocytogenes cultures were grown in BHI medium at 37°C with shaking for 5 to 6 h and then diluted to a final OD600 of 0.01 in BHI medium supplemented with 0.1% DMSO (vol/vol) or 10 μM 2-pyridones (MH44 or PS900), and various salts  were added at various concentrations (0.002 to 2.000 M) in round-bottom 96-well plates. After 18 h of incubation at 37°C, 20 μL (0.1× volume) of 400 μM resazurin (in PBS) was added to each sample for better visualization of the growth, as resazurin (blue dye) is oxidized to resorufin (pink) by living cells. The plates were also centrifuged at 3,500 × g for 10 min to score pellet formation as another indication of growth. Treated samples were also spotted onto BHI agar plates, followed by overnight incubation at 37°C to determine survival.SRR14598287 (EDGe WT), SRR14598286 (HT014), and SRR14598285 (HT015).The raw sequence reads from this study have been deposited in the SRA under accession numbers


H-tetrazoles was successfully achieved through one-pot multi-component condensation reactions of some aromatic aldehydes or indolin-2,3-dione with malononitrile and sodium azide using diverse reaction conditions to obtain considerable product yields. Furthermore, it has been achieved for the first time to construct desired products under neat condition. Molecular docking studies with CSNK2A1 receptor disclosed the lowest binding energy displayed by the dimethoxyphenyl derivative 4c with − 6.8687 kcal/mol. The synthesized tetrazoles were screened for their in-vitro cytotoxic activity against epidermoid cancer cell line (A431) and colon cancer line (HCT116) with respect to normal skin fibroblast cell line (BJ-1) using MTT assay, and antimicrobial activity against the bacteria: K. pneumonia, S. aureus, and the fungi: Candida albicans, as well as their antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl assay. In addition, the toxicity of tetrazole derivative was assessed by determination of their approximate lethal dose fifty (LD50), calculated via an oral administration to rats, through measurement of ALT and bilirubin levels in serum. The antitumor results can suggest that the potent tetrazole derivative namely, 3--2-(1H-tetrazol-5-yl)acrylonitrile (4c) could be a potential drug against epidermoid carcinoma. The antioxidant results indicated to tetrazoles exhibited great antioxidant properties even at very low doses. A molecular dynamics simulation was performed for the synthesized compounds (ligands) to investigate their tendency for binding with the active sites of protein.An efficient synthesis of 5-substituted 1 Recently, the synthesis of tetrazole (conjugated nitrogen-rich heterocycles) scaffolds has suited outstanding in the field of chemical and pharmaceutical research due to their utility as a pharmacophore in many interesting fields. In 2019, it was reported by drug bank that 23 approved medicines contained tetrazole as its core moiety13.Multi-component reactions (MCRs) combine three or more starting materials in a single chemical event to form a product containing most of the starting materials atoms. Mild conditions, efficiency, atom economy, straightforward reaction design, high affinity, and the progressive economy have been associated with modern multi-component reactions devices with their general compatibility with green chemistry15. Noteworthy, the tetrazole function is metabolically stable. Thus, this highlight and a close relationship between the acidic character of the tetrazole group and carboxylic acid group16 have encouraged medicinal chemists to prepare substituted tetrazoles as potential pharmacological agents acrylonitrile derivatives 4a–e using some catalysts and various techniques to achieve facile, eco-friendly, and one-pot process through a domino Knoevenagel condensation and 1,3-dipolar cyclo-addition reaction.Thus, design and synthesis of substituted tetrazoles have been approached in various methods like eco-friendly, non-toxic, water solvent, moderate conditions, easy setup, easy extractions, low cost, etc. with fair yields. A survey of literature revealed that 5-substituted 1H-tetrazol-5-yl)acrylonitriles 4a–e have been synthesized via preliminary investigation of one-pot multi-component reactions of different aromatic aldehydes like benzaldehyde, 4-methoxybenzaldehyde, 3,4-dimethoxybenzaldehyde, and 4-nitrobenzaldehyde 1a–d, or the ketone, indoline-2,3-dione (1e) with malononitrile 2 and sodium azide 3 under different conditions  and ultrasonic (US) irradiation conditions. All structures of the synthesized compounds were based on their spectral and elemental analyses. Thus, their infrared spectroscopic analysis displayed the appearance of the absorption bands at ν 3390–3300, 3080–3030, and 2229–2219 cm2), aluminum chloride (AlCl3), and ferric chloride (FeCl3) as a bulk materials and as a nanoparticles like ZnO, TiO2, NiFe2O4, CoFe2O4, and CuFe2O4. Also, eco-friendly techniques including microwave and ultrasonic irradiation were investigated. Last, the reaction was submitted to be investigated under fusion conditions. The yields, catalyst amounts, and reaction time were estimated. It was worthy that tetrazole derivatives 4a–e were acquired in spectacular yields under fusion condition   were evaluated. First, solvents like ethanol and acetic acid under reflux were employed. In next attempt, some catalysts have been used like sodium ethoxide (EtONa), ceric ammonium nitrate (CAN), sodium acetate, and some Lewis acids like zinc chloride . Otherwise, execution of the reaction under microwave irradiation conditions increased the reaction yield. Whereas, applying the ultrasonic irradiation conditions failed to give the desired products and afforded instead the arylidine malononitrile derivatives 5a–e (Scheme H-tetrazol-5-yl)acrylonitrile derivatives 4a–e have been successfully achieved upon carrying out the multi-component reaction under fusion condition, as a noticeable style toward environmentally clean synthesis of organic compounds.Therefore, using ZnO, TiO1a–d or indoline-2,3-dione 1e were treated with malononitrile 2 and sodium azide 3. Aldehydes with electron-withdrawing group (4-NO2) gave the desired product with excellent yield. Afterwards, aldehydes with electron donating groups 2) afforded the desired products with relatively lower yields.After reaching our optimal values, we subsequently studied the substrate scope for our synthetic protocol. Therefore, four substituted aromatic aldehydes 2 was introduced on the aldehydes, it raised the electron deficiency of the electrophilic center on aldehydic group which facilitated the nucleophilic attacking on it by the active methylene of malononitrile. In the meantime, the electron-donating groups such as –OCH3, and 3,4-(OCH3)2 moieties have counter effects.The formation of coveted products might be elucidated through the formation of arylidene malononitrile followed by aza-Michael addition of azide nitrogen ion on nitrile carbon atom of arylidene moiety forming the intermediate which underwent 1,5-endo-dig cyclization to form tetrazole as a final product (cf. Scheme H-tetrazol-5-yl)acrylonitrile derivatives was achieved by solvent-free one-pot multi-component reactions in the absence of catalyst with spectacular yields. Therefore, this fashion enables to construct a wide variety of 2-(1H-tetrazole-5-yl) acrylonitrile derivatives  compared to the ligand 2-(3-fluoro-4-hydroxyphenyl)-7-vinylbenzo[d]oxazol-5-ol (ERB-041)   higher than the reference ligand 041 with binding energy of − 6.4434 kcal/mol, this could be attributed to arene-H interaction between tetrazole ring of the ligand and ILE 174 of the enzyme and the ligand exposure. While compound 4d has analogous inhibitory activity compared to reference ligand which have a binding energies − 6.4305 kcal/mol. Hence, the high active participation of the tetrazole rings and various substituents of these compounds was found to be very important for locking their geometries in the active site of the CSNK2A1 receptor.While the compound 4a–e and the active sites of CSNK2A1 enzyme were showed in 2D and 3D diagrams  and colon cancer cell line (HCT116), as well as the normal skin fibroblast cells (BJ-1) at concentrations of 12.5, 25, 50, and 100 µg/mL. The well-known anticancer agent doxorubicin was used as a positive control. The results in Table 50 values were found against HCT116 (colon cancer) to reach 201.45 ppm. While 92.05 ppm for BJ-1 , and the lowest values were found against A431 at 44.77 ppm  and S. aureus (as Gram positive bacteria), and antifungal activity against Candida albicans. The results were displayed in Table K. pneumonia and S. aureus at concentrations as low as 6.25 and 1.56 mg/mL, respectively. Compounds 4e and 4c showed relatively higher MICs values against K. pneumonia  and S. aureus (50 mg/mL). All the tested compounds displayed weak antifungal activity (100 mg/mL), except compound 4c which disclosed good activity against C. albicans at 12.5 mg/mL.The tetrazole derivatives 4c was tested for its antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay30. The radical scavenging activity of this tetrazole derivative was almost the same along the different concentrations against DPPH⋅. Ranging from 71.7% at 50 mg/mL to 72.5% at 300 mg/mL. DPPH⋅ scavenging activity of the compound against ascorbic acid is depicted in Table The tetrazole derivative 4c was tested for its lethal toxicity against rats, and the mortality was measured. At first concentration, there was no mortality. At the second concentration, two rats died, and at the third concentration, 50% mortality was observed. Only the final concentration (2500 mg/kg) showed 50% mortality, as well as severe toxic signs including malaise and severe weight loss. That the median lethal dose for the tested compound is 2500 mg/kg  higher in the treated group that in the control by 94.8%. Also, the direct bilirubin is significantly (P < 0.05) higher in the treated group by only 49%. Eventually, the indirect bilirubin is significantly (P < 0.01) higher in the treated group by 272%  characterize the electrophilic-attacking sites, while the LUMO imitates the nucleophilic-attacked sites. The low values of the energy gap (ΔE = ELUMO–EHOMO) will render good inhibition efficiencies because the energy required to remove an electron from the last occupied orbital will be low32. DFT based on quantum chemical computation outline the structure optimization of the intermediate that reacted to afford the final product.The optimization of molecular structures for the produced compounds was carried out with DFT method using the hybrid 4. The structures have been supported by spectral analysis and analytical data. Quantum chemical parameters calculation with DFT method can be used for the calculations of the produced compounds  are generally the most active centers in these molecules.So, DFT simulation helped to show the formation of the tetrazole derivative ds Table . The higThe calculated ΔE was compared with the theoretical reference data based on the corresponding experimental results in gas phase reaction. With ΔE being a criterion, three most typical and popular exchange–correlation functionals e.g., PW91 were systematically compared in terms of the typical synthesis in gas phase. In addition, the verification of geometrical and electronic properties of modeling the anticancer products, as well as the adsorption behaviors of typical probe reactants and solvents are also suggested for further screening proper functionalization. The present work provides general implications for how to choose a reliable exchange–correlation functional in the computational solvents and catalyst on reactant surface.EHOMO values but also, the number of heteroatoms, electron distributions, surface area, and lipophilicity must be considered. The dipole moment, and softness , for most potent synthesized compounds carrying hydrophobic groups were agreed to an excellent correlation between oxidation inhibition efficiencies. Also, the compounds of higher binding energy are of higher potency due to the strong interaction between these compounds and the active sites of receptors.Quantum chemical parameters calculations utilizing DFT method used for the calculations of produced compounds are in good agreement with the anticancer efficiency Table . The sca33. Their binding tendency can be expressed in terms of the calculated binding energies (Ebind) which are listed in Table Ebind calculated for these compounds reveal that their absolute magnitudes increase following the order: 4c > 4b > 4a > 4e > 4d, and this is the same trend for the values of ΔE confirming that the best binding capability of the synthesized compounds with the active sites of protein, and hence the best potency, is observed for compound 4c. This may be explained by such that the more polarizable the molecule, the better its interaction with protein. Figures 4a–e with the active sites of protein. It is evident that the three compounds get bonded mainly by means of the tetrazole moiety, which is the moiety over which HOMOs are distributed. This implies that HOMO regions are the most active sites for interacting with protein.Molecular dynamics (MD) simulation is an effective method for studying the binding capability between ligands and protein. MD simulation is performed for the synthesized compounds (ligands) to investigate their tendency for binding with the active sites of protein36. The synthesized compounds 4c–e, were found to meet Lipinski’s rule of five, having a total polar surface area  96.71, 124.07, and 107.35, respectively, in addition to good lipophilicity, expressed by the consensus Log Po/w which are in 1.20, 0.69, and 0.42, respectively. They exhibited a high GI absorption and were computed to possess a good bioavailability score (0.56), as displayed in Table P) parameter was − 6.94, − 6.93, and − 7.70, respectively, thus facilitating the accessibility of the bioactive molecules through the skin. The bioavailability of the synthesized compounds was also estimated based on their pink area on the radar chart  increased the inhibitory effect against the cell lines which may be attributable to the formation of hydrogen bonding with receptor acrylonitriles was obtained in good to excellent yields with great functional group compatibility. Our synthetic protocol included condensation of four benzaldehyde derivatives and/or indolin-2,3-dione with malononitrile and sodium azide to give five substituted tetrazoles. Molecular docking studies were carried out with the CSNK2A1 receptor to authenticate the epidermoid and colon cancer inhibition property of the synthesized molecules. The results concluded that compounds 4b, 4c, and 4d effectively inhibit the epidermoid cancer cell line among the other docked compounds. Also, the antimicrobial activity was evaluated against the bacteria: K. pneumonia, S. aureus, and the fungi: Candida albicans, as well as their antioxidant activity utilizing 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. In addition, the toxicity of the tetrazole derivative was assessed by determination of their approximate lethal dose fifty (LD50), calculated via an oral administration to rats. The antioxidant results indicated to tetrazoles exhibited great antioxidant properties even at very low doses. Molecular dynamics simulation has revealed that the synthesized derivatives tend to bind with protein following the same order shown by DFT study.A catalyst-free method was developed for the synthesis of structurally diverse substituted tetrazoles. A variety of 3-substituted 2- were run at Faculty of Science, Ain Shams University utilizing potassium bromide disks on FTIR Thermo Electron Nicolet iS10 infrared spectrometer . 1H and 13C NMR spectra  were run at 400 and 100 MHz on a BRUKER NMR spectrometer  utilizing tetramethyl silane (TMS) as an internal standard in deuterated dimethyl sulfoxide. Mass spectra were recorded on a Shimadzu GC-MS-QP-1000 EX mass spectrometer  operating at 70 eV utilizing Thermo X-CALIBUR software, at Faculty of Science, Ain Shams University. Elemental analyses were recorded at Faculty of Science, Ain Shams University utilizing Perkin-Elmer 2400 CHN elemental analyzer. The microwave reactions were done by a Microsynth instrument type MA143 (Microwave flux). Sonication was performed in a Toshcon typical SW4 char (40 kHz and 150 W).Solvents and chemicals obtained from Sigma Aldrich, Merck, Fluka, and El-Nasr pharmaceutical chemicals companies were purified and dried by standard techniques. The reactions and the purity of the produced compounds were followed and checked by TLC using TLC aluminum sheets silica gel F1a–d or indolin-2,3-dione 1e (0.01 mol) and malononitrile 2 (0.01 mol) in ethanol or glacial acetic acid (20 mL), sodium azide 3 (0.015 mol dissolved in 1 mL of water) was added. The reaction mixture was refluxed as depicted in Table To a solution of aromatic aldehydes 1a–d or indolin-2,3-dione 1e (0.01 mol), malononitrile 2 (0.01 mol), and the catalyst; sodium ethoxide (EtONa) (0.015 mol dissolved in 5 mL of absolute ethanol), ceric ammonium nitrate (CAN), aluminum chloride (AlCl3), zinc chloride (ZnCl2), or ferric chloride (FeCl3) (0.015 mol) in ethanol (20 mL), sodium azide 3 (0.015 mol) was added. The reaction mixture was refluxed as depicted in Table 5a–e.To a mixture of aromatic aldehydes 1a–d or indolin-2,3-dione 1e (0.01 mol), malononitrile 2 (0.01 mol), and catalyst; sodium acetate (0.015 mol) in ethanol (20 mL), sodium azide 3 (0.015 mol dissolved in 1 mL of water) was added, and the reaction mixture was refluxed as depicted in Table To a mixture of aromatic aldehydes 1a–d or indolin-2,3-dione 1e (0.01 mol), malononitrile 2 (0.01 mol), and the catalyst; ZnO, TiO2, NiFe3O4, CoFe2O4, or CuFe3O4 (0.015 mol) in ethanol (20 mL), sodium azide 3 (0.015 mol dissolved in 1 mL of water) was added, and the reaction mixture was refluxed as depicted in Table To a mixture of aromatic aldehydes 1a–d or indolin-2,3-dione 1e (0.01 mol), malononitrile 2 (0.01 mol), and sodium azide 3 (0.015 mol) in dimethyl formamide (2 mL) was sonicated as depicted in Table 5a–e.A mixture of aromatic aldehydes 1a–d or indolin-2,3-dione 1e (0.01 mol), malononitrile 2 (0.01 mol), and sodium azide 3 (0.015 mol) in dimethyl formamide (2 mL) was added to the reaction vessel of the monomodal Emrys™ Creator microwave synthesizer and allowed to react under microwave irradiation at 200–400 W power , malononitrile 2 (0.01 mol), and sodium azide 3 (0.015 mol) were fused in a sand bath at 140–200 °C : 3590 (NH), 2209 (C≡N). 1H NMR : 3.68 , 7.24–7.60 , 7.95 , 8.45 . Anal. Calcd. for C10H7N5 (197.20): C, 60.91; H, 3.58; N, 35.51; Found: C, 60.80; H, 3.52; N, 35.53%.Paly-yellow crystals, mp. 170–172 °C5. IR : 3320 (NH), 2235 (C≡N), 1605 (C=N). 1H NMR : 3.45 , 7.83 , 7.90 , 8.01 . Anal. Calcd. for C11H9N5O (227.23): C, 58.14; H, 3.99; N, 30.82; Found: C, 58.07; H, 3.90; N, 30.80%.Yellow crystals, mp. 153–155 °C5. IR : 3315 (NH), 2230 (C≡N), 1603 (C=N). 1H NMR : 3.46 , 3.77 , 3.82 , 7.61–7.72 , 7.87 , 8.20 . EIMS : 260.57 , 255.26 , 254.16 (48.63), 238.17 (100), 226.27 (67.78), 211.16 (55.50), 180.27 (54.37), 177.16 (53.80), 148.77 (53.02), 127.99 (61.40), 120.66 (86.42), 105.06 (33.28), 69.50 (87.96). Anal. Calcd. for C12H11N5O2 (257.25): C, 56.03; H, 4.31; N, 27.22; Found: C, 55.97; H, 4.26; N, 27.25%.Yellow crystals, mp. 190–192 °C5. IR : 3354 (NH), 2253 (C≡N), 1608 (C=N), 1573, 1436 (NO2). 1H NMR : 3.52 , 8.18 , 8.36 , 8.48 . Anal. Calcd. for C10H6N6O2 (242.20): C, 49.59; H, 2.50; N, 34.70; Found: C, 49.51; H, 2.44; N, 34.67%.Beige crystals, mp. 167–169 °C−1): 3375, 3240 (NH), 2219 (C≡N), 1714 (C=O). 1H NMR : 7.36–6.84 , 9.09 , 10.84 . 13C NMR : 166.93 156.77, 143.50, 134.94, 133.27, 128.67, 121.80, 121.21, 116.71, 110.18, 107.36. EIMS : 240.96 , 236.82 , 210.27 (12.56), 202.37 (15.20), 178.96 (26.93), 166.27 (27.01), 156.03 (53.49), 154.24 (48.82), 137.99 (46.83), 121.50 (40.19), 107.23 (20.14), 82.85 (35.52), 75.06 (42.31), 70.27 (41.32), 41.65 (100). Anal. Calcd. for C11H6N6O (238.21): C, 55.46; H, 2.54; N, 35.28; Found: C, 55.37; H, 2.50; N, 35.30%.Red crystals, mp. 220–222 °C. IR  were selected from a pool of many ferro-spinels from which the copper ferrite was selected after some comparative trials for being the most active toward the target reaction. Where a simple co-precipitation method was used to prepare all three spinels for this simple comparison in the activity toward the desired reaction, resulting in copper ferrite being the most active of the three selected spinels due to the presence of copper ions in its formula and crystal lattice leading to an improved catalytic activity toward nitro phenols reduction. The preparation process was further improved by the addition of a polymer to further reduce the copper ferrite size and catalytic activity. The polymer was also selected from a pool of four polymers for comparison and optimization for the selected model reaction, being polyethylene glycol (PEG), where the most effective polymer in stabilizing the formed particulates was PEG which did not only improve stability and size but also introduced further surface defects improving the catalytic activity and was optimized by using different amounts from the polymer to stabilize the formed particles. Where the 2% weight ratio was selected for the comparison between different polymers.The simple preparation method done using a mixture containing (1:2) ratio of  and ferric ions which was stirred together first, and the appropriate amount of the polymer was then added to the mixture and stirred for 1 h. Sodium hydroxide solution (1 M) was then added dropwise to begin the precipitation of the copper-iron double hydroxide until the mixture pH reached a basic pH ~ 10. The mixture was then filtered off and washed with copious amounts of distilled water to ensure the removal of chloride ions (using silver nitrate) and the excess sodium hydroxide. The obtained slurry was then dried in and oven at 100 °C overnight to obtain the powder, which was then crushed into a mortar to ensure size uniformity. The obtained fine powder was then calcinated at 600 °C for 3 h to ensure polymer removal and the complete transformation of the formed copper-iron double hydroxide to a spinel. The calcinated powder was then crushed again to obtain the final ferrite spinel in powder form.37. X-ray diffraction (XRD) analysis was performed to assess the nature and size of titania crystalline phases. The diffraction pattern of titania can be illustrated in Fig. Titanium tetraisopropoxide (20 mL) was dissolved in isopropanol (80 mL), then hydrolyzed by the slow addition of distilled water. The mixture obtained was kept for stirring for 2 h and then left for two days for complete hydrolysis. The precipitate formed was filtered off, washed, and then dried at 100 °C overnight and calcined at 600 °C for 2 h38. The ZnO-NPs were assessed by XRD for their crystalline nature. The diffraction pattern showed four main peaks at 2θ values of 31.8, 34.5, 36.27, 47.5, 56.7, 62.8, and 67.5 , which are mostly relevant for the ZnO structure. Observable line broadening in the XRD peaks indicates that the material which is synthesized is in the nm range. TEM analysis was performed to better recognize crystalline properties and size of the synthesized nanoparticles. The TEM images , cyclohexane (50 mL) and ethanol (50 mL) were added. The mixture was stirred for 15 min. Then, an aqueous solution of ammonia (40%) was added to the solution dropwise with stirring for 10 min. The precipitate formed was washed with water several times and then dried in an oven at 100 °C for 1 h, followed by calcinations at 500 °C for 2 hcf. Fig. . The meacf. Fig.  of ZnO cd]oxazol-5-ol], the molecular docking study has been executed on the synthesized tetrazole derivatives as unprecedented ligand in inhibition of CSNK2A1 which was restored from protein data bank (PDB: 6QY7) (http://www.rcsb.org/pdb) as a reference ligand for docking score. Al enzymes were fitted for docking study by reconnecting the bond broken, fixing the potential, and adding the lost hydrogen. Moreover, the known ligand ERB-041 was exchanged by our synthesized tetrazole derivatives at the identified active sites.As known, CSNK2A1 enzyme was inhibited by the ligand ERB-041, [2-(3-fluoro-4-hydroxyphenyl)-7-vinylbenzo[2 and 95% humidity. Cells were sub-cultured using trypsin 0.15%29.The cancer cell lines: A431 (Epidermoid carcinoma), HCT116 (colon cancer), and the normal cell line (BJ-1) were obtained from Karolinska Institutes, Sweden. As a gift from Dr. Stig Linder. Each one of them was maintained in a suitable medium with 10% FBS. Cell lines were incubated at 37 °C in 5% CO3 cells/well (96 well plate), while for BJ-1, cell seeding was conducted at count 50 * 103. After 24 h, the medium was discarded, and a new serum free medium was added with the tested compound at different concentrations of 12.5, 25, 50, and 100 µg/mL. After 24 h of incubation, cell viability was determined using MTT -2,5-diphenyltetrazolium bromide) assay29. Doxorubicin was used as a positive control and DMSO was used as a negative one. The exact IC50 was calculated with calculated using SPSS.For A431 and HCT116 cell lines, cell seeding was conducted at count 10 * 10Klebsiella pneumonia ATCC 700603, Staphylococcus aureus ATCC 29213, and Candida albicans . Each well is inoculated with a microbial inoculum prepared in the same medium and adjusted to 0.5 McFarland scale (1–5 × 105 CFU/mL). After well-mixing, the inoculated microplates are incubated for 24 h at 37 °C and the lowest concentration of the tested compound that prevented microbial growth (showed no turbidity) was measured visually and recorded as its MIC. The study was approved by the Research Ethics Committee at Ain Shams University and was performed in accordance with the guidelines of the National Institute of Health (NIH).The minimal inhibitory concentrations (MIC) of the tested compound were determined by a modified version of Clinical and Laboratory Standards Institute (CLSI) protocol. The procedure involves preparing twofold dilutions of the tested compounds (from 100 to 0.05 mg/mL) in a liquid growth medium dispensed in a 96-well microplate. The tested organisms were 30. DPPH⋅ has a characteristic absorbance at 515 nm which disappears after its reduction by an antiradical compound. The reduction in DPPH thus can be monitored by measuring the decrease in its absorbance at 515 nm during the reaction.Prior to any animal testing, the compound antioxidant capacity was evaluated against ascorbic acid using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assayAnimals: A total of 24 male albino rats (Rattus norvegicus) were randomly selected for the present study, each weighing 160 ± 20 g. These rats were housed in animal houses with a natural 12 h. Dark/light cycle at 27 °C. They had free access to food and tap water. All animals were healthy and were allowed to adapt to the laboratory conditions for at least a week prior to the start of the experiment. The rats were allotted into two groups, (1) the control group (n = 6), received only tap water through the gavage. (2) Treated group (n = 18), which received the tetrazole derivative dose for LD50 calculation. The LD50 was determined following the OECD 423 guidelines.The tetrazole derivative was administered orally by gavage. First, at concentration of 500 mg/kg, then 2000 mg/kg and eventually at 2500 mg/kg .Animals were observed individually after dosing at least once every 30 min. For the following 5 h after intubation, and twice a day along the following 14 days. Afterwards, the following parameters were measured.Body weight change calculation: The body weights of rats were recorded at the beginning and the end of the experiment, then the change in body weight was calculated by subtracting the rats body weights at the beginning of the experiment from that at the end of it. The body weights of rats were recorded at the beginning and the end of the experiment, then the change in body weight was calculated by subtracting the rats body weights at the beginning of the experiment from that at the end of it.Biochemical methods in serum: The LD50 calculation was confirmed through measuring ALT activity and bilirubin content in serum.Testing for alanine-amino transferase (ALT): ALT was measured through the kinetic method according to the International Federation of Clinical Chemistry (1986). The assay principle depends on assaying the rate of NADH oxidation, which is proportional to the reduction in Absorbance at 340 nm over time.Bilirubin toxicity: was measured through the colorimetric method provided by the BioMed-Diagnostics bilirubin kit. The principle depends on the fact that total bilirubin reacts in acidic medium with diazotized sulfanilic acid to form a red colored azobilirubin and the intensity of the color produced is directly proportional to the amount of bilirubin present.This study was approved by the Research Ethics Committee at Ain Shams University  and was performed in accordance with the guidelines of the National Institute of Health (NIH). All methods are reported in accordance with ARRIVE guidelines.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.


The morphology of the prepared Cu@Pani/MoS2 nanocomposite was characterized using FE-SEM and Cs-STEM, and electrochemical analysis was performed using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and chronoamperometry techniques. Electrostatic interaction between Cu@Pani and MoS2 greatly enhanced the charge dispersion, electrical conductivity, and stability, resulting in excellent electrochemical performance. The Cu@Pani/MoS2 was used as an electrocatalyst to detect glucose in an alkaline medium. The proposed glucose sensor exhibited a sensitivity, detection limit, and wide linear range of 69.82 μAmM−1cm−2, 1.78 μM, and 0.1–11 mM, respectively. The stability and selectivity of the Cu@Pani/MoS2 composite for glucose compared to that of the potential interfering species, as well as its ability to determine the glucose concentration in diluted human serum samples at a high recovery percentage, demonstrated its viability as a nonenzymatic glucose sensor.A Cu@Pani/MoS  •2 nanocomposite was prepared via in-situ oxidative polymerization followed by hydrothermal method.Cu@Pani/MoS•2 nanocomposite exhibits flower like nanosheet morphology.Cu@Pani/MoS•2 and Cu@Pani greatly enhance the sensitivity and selectivity of glucose sensor.The π-π interaction between MoS•2 composite reveal high sensitivity of 69.82 μAmM−1cm−2 and detection limit of 1.78 μM.The Cu@Pani/MoS Copper  glucose ,24. To t glucose ,26. Inor glucose . Conduct glucose .2), has been used in nanoelectronics, lithium-ion batteries 2 FTIR sp aniline . The incani/MoS2 ,62. The 65-1951) . Similar aniline ,64. The 2 composite FESEM and CS-TEM images were recorded to study the surface morphology, and they are presented in 2 revealed that MoS2 nanosheet were grew on the surface of Cu@Pani, as represented in 2 in 2 nanosheet structure, which confirmed the formation of MoS2 nanosheets [2, as shown in The Cu@Pani and Cu@Pani/MoSnosheets ,68. Cs-S3/2 and Cu 2P1/2 respectively, and the small peaks at 944 eV and 940 eV represent satellite peaks of copper, which indicate the paramagnetic behavior, of the Cu2+ oxidation state. The binding energy difference of 19.6 eV between the 2P3/2 and 2P1/2 states of Cu resembles the reported 20 eV binding energy difference, as shown in 1/2, S 2P3/2 and oxidized sulfur, respectively [3/2 respectively [4+ 3d5/2, Mo6+ 3d5/2, Mo4+ 3d3/2, and Mo6+ 3d3/2, respectively, as represented in The chemical composition of the nanocomposite material was studied by using XPS, and the results are shown in  species . Bindingectively ,76,77. Tectively . Binding3.12 electrodes were analyzed using 0.1 M NaOH solution as working electrolyte before fabricating a modified electrode for glucose sensing. As shown in 2 and Cu@Pani/MoS2 in the presence of 2 mM glucose were measured using a three-electrode system [2 electrode was higher than that of the Cu@Pani electrode. In the presence of 2 mM glucose, the Cu@Pani/MoS2 electrode demonstrated a significantly increased current response, which can be attributed to glucose oxidation [2O2 for polymerization instead of ammonium persulfate (APS), and simultaneous use of oxidizing agent (H2O2) and reducing agent (NaCt), which leads to the incomplete oxidation of aniline to polyaniline, is also represented in 2 CV spectrum exhibits a current response increment compared to that of Cu@Pani, which can be attributed to the two - dimensional layered structure of MoS2 providing a large surface area for electron dispersion at the electrode surface. This greatly increases the electrocatalytic performance of the material [The electrochemical properties of the Cu@Pani, and Cu@Pani/MoSe system . The Cu@xidation . The commaterial .Fig. 5, Rct, double-layer capacitance (Q1), and Warburg impedance (Q2), where the electrolyte resistance Rs, is fitted in series combination, and double layer capacitance (Q1), Rct, and impedance of the faradaic reaction are arranged in parallel combination [An electrolyte containing a mixture of 5 mM [Fe (CN)lectrode . The insbination .2 when exposed to 0, 1, 2, 3, 4, and 5 mM glucose solutions. 2 = 0.9804), reveals that a diffusion-controlled reaction occurred on the Cu@Pani/MoS2 electrode surface [2 nano structure improves the electrocatalytic activity because of its high surface area, electron transfer abilities and rapid diffusion of the electrons and ions on the electrode surface, providing active sites for the electrocatalytic reactions during electrochemical glucose oxidation [2 electrode involves the following reaction mechanisms [3+, it yields gluconolactone, which produces gluconic acid via hydrolysis [2+ to Cu3+, which greatly enhances the electrocatalytic activity and electron transfer process at the surface of the electrode material [ surface ,91. MoS2xidation . The elechanisms .(1)Cu + drolysis ,94. The material ,96.3.22 electrode with glucose are presented in 2 electrode led to a gradual increase in the current response, which demonstrate the applicability of the Cu@Pani/MoS2 electrode for measuring different glucose concentration. The plot of the observed oxidation current against the glucose concentration, follows a linear regression line with a co-relation coefficient of R2 = 0.9950, as illustrated in −1cm−2. The calculated limit of detection (LOD) was found to be 1.78 μM, which was obtained using the formula LOD = 3.3 σ/m, (where m = slope and σ = standard deviation) [2-based electrode materials available in the literatures, as detailed in The results of the chronoamperometry analysis of the Cu@Pani/MoS3.32 electrode by adding 1 mM same volume of glucose solution and different interfering biomolecules such as, maelic acid (MA), potassium chloride (KCl), ascorbic acid (AA), urea (UR), uric acid (UA), and sodium chloride (NaCl) (0.1 mM) [2-based electrochemical non-enzymatic glucose sensor. Our results indicate that there is no noticeable current for interfering biomolecules; but increased current response for glucose, both before and after the addition of interfering biomolecules [We further analyzed the selectivity of the Cu@Pani/MoS(0.1 mM) ,104, [10(0.1 mM) . Fig. 6 sample was measured using the Cu@Pani/MoS2-based electrode [2 electrode for electrochemical non-enzymatic glucose sensing of biological samples in the presence of interfering species [Successive chronoamperometric measurements are performed over ten days using 2 mM glucose solution to confirm the stability of the electrode material; the corresponding results are compared in lectrode . For thelectrode . Subsequlectrode . Our res species .Table 2R42 nanocomposite. The structural morphology of the nanocomposite was studied using different analytical tools, and the fabricated nanocomposite was used as a non-enzymatic electrochemical glucose sensor. Cu@Pani/MoS2 composite-based sensor revealed a sensitivity of 69.82 μAmM−1cm−2, detection limit of 1.78 μM, and linear range of 100 μM to 11 mM with 92.75 % stability. The Cu@Pani/MoS2 nanocomposite exhibited effective sensitivity, good selectivity, stability, and a short electrochemical response time, which indicates that the Cu@Pani/MoS2 composite electrode is viable for electrochemical non-enzymatic glucose sensor. Our work provides a new method for the design of composite electrode materials for electrochemical applications such as supercapacitor, electrochemical sensor, batteries etc.A conclusion, combination of in-situ oxidative polymerization and a hydrothermal process was employed to synthesize Cu@Pani/MoSData will be made available on request.Krishna Prasad Sharma: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing - original draft, Writing - review & editing. Miyeon Shin: Data curation, Formal analysis, Project administration, Resources, Software, Writing – review & editing. Kyong Kim: Data curation, Project administration, Resources, Software. Kyungmin Woo: Data curation, Formal analysis, Investigation, Resources, Software. Ganesh Prasad Awasthi: Data curation, Formal analysis, Methodology, Validation. Changho Yu: Formal analysis, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing – review & editing.There is no conflict of interest to disclose for this research paper. The human serum samples were not taken form human patients. We have used commercially available human serum  for the real sample analysis.


Phenolic compounds are dominant antioxidant factors in whole grains and are essential quality traits in future breeding programs. We proposed a robust set of methods for extraction, screening, and quantitative analysis of soluble and wall-bound (WB) phenolic compounds from fine powder and fine powder products using a 96 Wells UV Flat Bottom and subsequent UHPLC-DAD validation of candidate samples. The plate-UHPLC strategy significantly simplifies the screening of phenolic-enriched grains, reduces the screening cost, saves harmful organic chemicals, and contributes to developing novel health-promoting varieties.  Specifications tableDescription of protocol:Methods detailsThe antioxidant characteristic of wholegrain phenolics benefits human health by lowering the risks of cardiovascular disease (CVD), gastrointestinal (GI) cancer, and diabetes We screened wheat mutants enriched with ferulic acid by UHPLC-DAD to increase the antioxidant potential of foods. Screening mutant grains led to identifying mutants accumulating ferulates in the grains We purchased the following chemicals: hydrochloric acid , sodium hydroxide , ferulic acid , methanol (UPLC grade), acetic acid , and ethyl acetate  from Sigma-Aldrich .Step 1: Wheat sample preparation1.Cover two grains with foil and crush them with the pliers.2.Ground the samples into a fine powder (60 meshes) with two stainless steel beads in high-throughput grinding equipment .3.Set the grinding program as 20 HZ for 4 min, 25 HZ for 4 min, 30 HZ for 4 min, 40 HZ for 1 min twice, 50 HZ for 1 min twice, 60 HZ for 1 min twice, 65 HZ for 30 s 20 times.4.Store the samples at -80°C until subsequent chemical analyses.We recommend the following method for the rapid preparation of large quantities of trace samples (less than six grains):Step 2: Extraction of cell wall-bound (WB) phenolic compounds1.Wash the ground samples (100 mg ±0.5) with 1 mL methanol (80% in water).2.Centrifuge at 13,000g for 10 min; carefully drain out the supernatant3.Repeat twice to eradicate soluble flavonoids.4.Resuspend the debris in 4N NaOH solution (3mL).5.Shake the samples at 200 rpm overnight at 37 °C.6.Centrifuge the samples at 10,000g for 20 min.7.Extract 100 µL supernatant for subsequent chemical analyses.Step 3: Ultraviolet spectrophotometry analysis of phenolic acid1.Mixed 100 µL supernate with 300 µL 4 N NaOH.2.Remove 200 µL into a UV Flat bottom 96-well plate .3.Read the absorbances at 320 nm in a MULTISKAN GO .4.Calculate phenolic acid content according to the standard curve with 4N NaOH as the negative control.Step 4: Wheat sample preparation and pasta-making1.Grind the grains into whole wheat flour (60 meshes) in a stone mill.2.Mix the whole wheat flour and 25% water (w/w) to prepare raw pasta.3.Prepare threaded pasta with ATLAS 150 .4.Cook the raw pasta for ten minutes in boiled water.5.2O to remove potential residue soup contamination.Wash the pasta twice with ddH6.Dry the raw and cooked pasta in a 65°C oven to achieve constant weight.7.Grind the samples into a fine powder in the high-throughput grinding equipment for 60 s at 60 HZ.8.Store the samples at -80°C until further analyses.Step 5: Extraction of wall-bound (WB) phenolic compounds1.Add methanol (80% in water) at ten microliters per mg into dried powders.2.Sonicate the samples in a water bath for 30 min.3.Centrifugate at 15,000 g for 15 min.4.Wash the samples with methanol (80% in water) three times to remove the soluble part.5.Treat the debris with 4N NaOH (3mL) containing p-toluic acid .6.Shake the samples overnight at 37 °C.7.Adjust the samples with 2 mL hydrochloric acid (6N) to pH 4.0.8.Add 600 µL ethyl acetate (water-saturated) for extraction and mix them well by vigorous shaking.9.Centrifugated at 10000 g for 15 min,10.Collect the supernatant and repeat the wash three more times.11.Pass the samples through 1 mL anhydrous sodium sulfate in a 150 mm Glass Pasteur straw Jiangsu Shitai, Jiangsu, China) to remove any polar contamination.Dry the samples under a stream of nitrogen gas.12.Resolve the samples with HPLC-grade methanol at one microliter per mg dry weight grain powder ratio and analyze by UHPLC Step 6: Quantitative phenolic acid analysis2O with 0.1% acetic acid (v/v), while mobile phase C was acetonitrile with 0.1% acetic acid (v/v). The gradient details are in The analysis was performed using an Agilent 1290 Infinity system with a CNW Athena C18-WP HPLC Column  coupled with a DAD detector  ,8. 5 µL Our data demonstrated that the UV-spectrophotometer screening method effectively screened phenolic-enriched grains, although its precision and sensitivity were weaker than UHPLC. Given the cost and time reductions, the UV spectrophotometer/UHPLC strategy improves efficiency. Analyzing 96 samples take less than 15 min compared with 1920 min by the UHPLC method National Natural Science Foundation of China (31972350).This work was supported by the Yi-Lin Gong: Methodology, Data curation, Writing – original draft. Jin-Ying Gou: Conceptualization, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The modification of the surface of the photocatalyst was performed by wet impregnation, and the obtained solids were characterized using X-ray diffraction, XPS, SEM, EDS, TEM, N2 physisorption, Raman and UV-Vis DRS, which revealed the structural and morphological stability of the modified material. BET isotherms are type IV, with slit-shaped pores formed by nonrigid aggregate particles and no pore networks and a small H3 loop near the maximum relative pressure. The doped samples show increased crystallite sizes and a lower band gap, extending visible light harvesting. All prepared catalysts showed band gaps in the interval 2.3–2.5 eV. The photocatalytic degradation of aqueous phenol over TiO2-P25 and Co(X%)/TiO2 was monitored using UV-Vis spectrophotometry: Co(0.1%)/TiO2 being the most effective with NUV-Vis irradiation. TOC analysis showed ca. 96% TOC removal with NUV-Vis radiation, while only 23% removal under UV radiation.One of the main challenges of photocatalysis is to find a stable and effective photocatalyst, that is active and effective under sunlight. Here, we discuss the photocatalytic degradation of phenol as a model pollutant in aqueous solution using NUV-Vis (>366 nm) and UV (254 nm) in the presence of TiO Many activities, such as agriculture, medicine, or industry, release pollutants into the environment, creating one of the most serious problems we currently face: environmental pollution. Water pollution, in particular, poses important risks to human health and the environment. Therefore, the control of pollutants released into aqueous media has led to increasingly restrictive environmental regulations. Phenol is considered a major pollutant because of its toxicity, bioaccumulation, low biodegradability, and carcinogenicity . It caus2O3, ZnO, Fe2O3, and TiO2) [2, of which the photoactive forms are anatase and rutile. One of the most effective forms, and by far the most widely used, is a mixture of anatase/rutile at a ratio of ca. 80%/20%, with a small percentage of amorphous form, commercialized as Evonik-P25 (referred to here as P25) [−/h+ recombination [2-P25 is known for its lack of toxicity when presented as aggregates or macroparticles, chemical stability in aqueous solution, and low cost [Considerable efforts have been devoted to developing approaches aiming to a complete removal of organic pollutants. However, the application of conventional methods, such as membrane filtration, adsorption on activated carbon, ion exchange on synthetic resins, purification by chemical coagulation systems, etc., are not cost effective and may generate wastes that require further treatment stages and additional costs ,6. Convend TiO2) , the mos as P25) . The impbination . TiO2-P2low cost . Despite2-P25 under Vis irradiation, including doping/loading with metal ions or nonmetals [−/h+ recombination and decreases the band gap, shifting the photocatalyst light absorption towards the Vis region, which facilitates the use of sunlight as the irradiation source [2 as a photocatalyst [2 was found to have high photocatalytic activity for the degradation of acetaldehyde [Numerous strategies have been used to increase the photocatalytic efficiency of TiOonmetals ,19. Dopin source . Recentln source ; many ofcatalyst ,23. Co loaded with Co ions at different concentrations (Co(X%)/TiO2, where X = 0.1%, 0.3%, 0.5%, and 1%). The samples were prepared by the wet impregnation method. Extensive characterization of the samples was carried out using X-ray diffraction (XRD), SEM, EDS, TEM, X-ray photoelectron spectroscopy (XPS), Raman and UV-Vis diffuse reflectance spectroscopy (UV-Vis DRS), and N2 physisorption. Its photocatalytic activity was evaluated by monitoring phenol transformation, taken as a model organic pollutant, and total organic carbon (TOC) evolution. The photoreaction was conducted in an aqueous solution under near-UV light at wavelengths of 366 nm and longer and also under UV light (254 nm).Here, we present an unprecedented extensive study of phenol photodegradation under UV and near-visible light irradiation, using TiO2-P25 was from Evonik : surface area of 55 ± 15 m2·g−1 [4·7H2O, ≥97%) was from Aldrich, and phenol  from Sigma. Acetonitrile (HPLC grade) was from J.T. Baker. O2 gas (purity ≥ 99.995%) was used in some experiments. All chemicals were used without further purification. Distilled water was obtained from a Millipore apparatus (Milli-Q water), resistivity of 18.2 MΩ at 298.0 K and total organic carbon (TOC) less than 5 μg·L−1.TiO5 m2·g−1  and part5 m2·g−1 . Cobalt 2 nanoparticles to prepare Co/TiO2 photocatalysts [4·7H2O was dissolved in distilled water and impregnated on Degussa TiO2-P25 nanoparticles under vigorous stirring.Wetness impregnation was used to immobilize Co(II) on the surface of TiOatalysts  with enh−1. Finally, the so-prepared catalysts were ground thoroughly and labeled as Co(X%)/TiO2, where X denotes the Co(II) mass percent relative TiO2, X = 0.1%, 0.3%, 0.5%, and 1%. TiO2 particles turned from white to yellowish as the cobalt percentage increased.This suspension was stirred for 24 h, and then dried in an oven at 50 °C to dry it completely. Then, the photocatalysts were calcined in a furnace  at 600 °C for 4 h with a ramp rate of 10 °C·minn+ leaching; after filtration, the solution was analyzed using ICP/MS and the dry solid by XRD. No evidence of Con+ leaching was found  measurements were conducted with a Bruker Siemens D5000 diffractometer following Bragg–Brentano geometry and θ/2θ configuration and equipped with a graphite monochromator. The optics consist of primary and secondary 2° Soller slots, a variable output slot, a 1mm receiver slot, a 0.2 mm monochromator slot, and a 0.6 mm detector slot. The detector was a scintillation counter. The acquisition conditions were a sweep range (2θ): 2–80°, a jump size (step): 0.050°, and the acquisition time at each jump was about 2.5 s (time per jump). The DiffracPlus software v. 8.0.0.2 (Socabim) was used for data processing. 2-P25 and Co(X%)/TiO2 were studied using a Jeol JSM-IT100 scanning electron microscope (SEM) equipped with an energy dispersive X-ray (EDX) system to obtain information on the surface elemental composition. The samples were deposited on thin films of amorphous carbon and coated with gold (2 nm). Transmission electron microscopy (TEM), using a Jeol JEM 1100 instrument, operating at an acceleration voltage of 80 kV, was also employed to examine the photocatalysts’ surface morphology. Samples were prepared by depositing a few drops of nanoparticle suspension on carbon-coated copper grids  and air drying.The morphology of TiO2, as adsorption gas, at 77.4 K . Helium was used for the measurement of the dead volume of the sample holders. Isotherms were measured in the range P/P0 = 0.1–1.0. The range of the BET area used for the calculation of the specific surface area was P/P0 = 0.05–0.3. “Microactive for Tristar II Plus”, v.2.03 (Micromeritics), was the software for control, acquisition, and data processing.Textural characterization was carried out using adsorption/desorption isotherms of N−1. Raman spectra were acquired at room temperature using a 20× objective over the spectra range of 90–2500 and 90–1800 cm−1 for 633 and 785 nm, respectively, laser powers at the samples of 21 mW (633 nm), 28 and 35 mW (785 nm), and acquisition times in the range of 0.5–5 s with 10 accumulation. The Raman spectra were processed with the Project5.2 Witec software for peak assignment including position, height, and FWHM. SpectraGryph 1.2 software was used for spectral processing involving baseline, background removal , and cosmic ray removal correction. Raman shift calibration was performed using a silicon wafer (peak at 520 cm−1). The spectra were normalized to the Eg peak (146 cm−1) for a better comparison between samples.Raman measurements of the dried samples on glass were performed using a Witec Alpha 300 R confocal Raman system equipped with 633 and 785 nm excitation laser lines and a grating of 600 gr·cm2 photocatalyst was analyzed by X-ray photoelectron spectroscopy (XPS) using a Thermo Scientific K-Alpha ESCA instrument equipped with an Al Kα monochromatized radiation (hν = 1486.7 eV) X-ray source. Measurements were carried out in a Constant Analyzer Energy mode with a 100 eV pass energy for survey spectra, and 20 eV pass energy for high-resolution spectra. The spot size selected was 400 μm and K-Alpha’s charge compensation system was employed during analysis. In data analysis, C 1s = 285.0 eV was taken as the binding energy reference. Surface elemental composition was determined using the standard Scofield photoemission cross sections.Co(0.1%)/TiOThe UV-Vis diffuse reflectance spectra (DRS) (200–800 nm) of the solid photocatalyst was measured on a JASCO V-560 UV-Vis spectrophotometer with a double monochromator and double beam optical system, equipped with an integrating sphere attachment . Reflectance spectra were converted to equivalent absorption Kubelka–Munk units.6H5OH) concentration in aqueous solution. Two types of photocatalytic experiments were carried out. The first was performed under NUV-Vis light, in which the photoreactor was equipped with a Heraeus TQ 150 medium-pressure Hg-vapor lamp, with intense emission lines at 254, 313, 366, 405, 436, 546, and 578 nm. A Duran 50® glass jacket, filled with water, was used to cut off the radiation below 366 nm. For the second experiment, the photoreactor was equipped with a Heraeus TNN 15/32 low-pressure Hg-vapor lamp, with a single intense emission line at 254 nm, located inside a quartz tube. The photon flux at 366 nm, as determined by potassium ferrioxalate actinometry, [−6 Einstein·s−1, and at 254 nm, it was of 8.33 × 10−8 Einstein·s−1.The photocatalytic activity of the synthetized samples was tested in an axial photoreactor, under UV and near-UV-Vis light (NUV-Vis), monitoring changes in phenol . Replicate experiments showed differences in rate constants within 5% error.Phenol concentration was monitored using a Biochrom Libra S70 spectrophotometer to measure the UV-Vis absorbance at 270 nm and using HPLC UV-Vis at 210 and 270 nm, in a Thermo Fisher instrument equipped with a 6000 LP UV detector, an AS 3000 autosampler, and a P4000 solvent pump. A KROMAPHASE C18 column (4.6 mm × 150 mm × 5 μm) was used, with a volume of 50 μL injected, a flow rate of 1.0 mL·min−1. The gradient followed was 0–1 min, 95–95% of A and 5–5% of C; 1–8 min, 95–5% A and 5–95% C; 8–10 min, 5–5% A and 95–95% C; 10–11 min, 5–95% of A and 95–5% C; and 11–15 min, 95–95% A and 5–5% C. The amount injected was 5–25 μL. The analyses were carried out using full-scan data-dependent MS scanning from m/z 50 to 500. The obtained photoproducts were the same found in the photocatalyzed degradation of phenol using surface-impregnated TiO2  as photocatalysts [Total organic carbon (TOC) removal was measured using a ShimadzuTOC-5000A analyzer. Photoproducts were identified using HPLC/MS (Thermo Scientific LTQ Orbitrap Discovery), equipped with an electrospray interface operating in negative ion mode (ESI-), with a Phenomenex Kinetex XB-C18 column (100 mm × 2.6 μm) operating at 30 °C with elution solvents A (0.1% formic acid) and C (0.1% methanol) at a flow rate of 200 μL·minatalysts .2-P25 shows eight primary peaks at 25.20°, 36.52°, 37.78°, 48.00°, 53.96°, 56.86°, 62.74°, and 75.08°, which are assigned to anatase diffraction planes: (1 0 1), (1 0 3), (0 0 4), (2 0 0), (1 0 5), (2 1 1), (2 0 4), and (2 1 5). The four peaks located at 27.34°, 36.02°, 41.14°, and 55.00° can be attributed to rutile diffraction planes: (1 1 0), (1 0 1), (1 1 1), and (2 1 1) [2, Co(0.3%)/TiO2, Co(0.5%)/TiO2, and Co(1%)/TiO2 photocatalysts were very similar to that of nonimpregnated TiO2-P25. No other metal and/or metal oxides were observed, which could be related to the low Co content  and its uniform distribution onto the TiO2 surface [2 due to the dopant Co2+ promoting anatase to rutile phase transformation [The crystal structure of the synthesized samples was evaluated using XRD analysis, and the results are shown in  (2 1 1) . The dif surface . Table 2 surface  to Co [Crystallite size was calculated using the Scherrer equation . The obsr rutile . Doping,ination) . The fac2 sample in 1 L of distilled water was stirred for 2 h. The solid recovered after filtration and drying was XRD analyzed. The same diffraction peaks of Co(0.1%)/TiO2 were found before and after the test /TiOthe test ; therefo2, which are compared with the Raman spectrum of undoped TiO2, acquired using two different lasers, 633 and 785 nm, and two laser powers. Anatase polymorph, a tetragonal structure, is present. The Raman active “lattice vibrations” shown in 4h point group.−1, Eg mode of the rutile phase, does not allow a rough estimation of the weight ratios of anatase phase to rutile phase from the Raman spectrum. The table also includes Ti-O and Ti-Ti bond lengths calculated using covalence/length/frequency relations [2 are similar to those reported for P25, which agrees with the tiny structural effect of 0.1% Co doping observed using XRD.The very low intensity band at ca. 445 cmelations . Values 2 synthetized using sol-gel [2) sample  upon the excitation of 633 nm with a laser power of 21 mW. However, these shifts were not observed at lower powers of the 633 nm laser (−1). This laser-induced phase transition  in Co-doped anatase TiO2 nanoparticles was previously reported [The method of preparation of the doped photocatalysts determines its particle size, which has to do with oxygen vacancies and electron–phonon coupling. Both factors may influence shifts and broadening of Raman peaks. Contrary to Co-doped TiO sol-gel , the as-) sample , the correported . This la2 samples were studied using scanning electron microscopy/energy dispersion spectroscopy (SEM/EDS). SEM images, 2 [2 doping percentages, the presence of Co is lower on the surface /TiOmages, 2 , with an surface , which cadiation .2 samples were analyzed using XPS, in an attempt to understand the bonding states of the surface elements on the doped photocatalyst. The survey XPS spectrum is shown in 2 photocatalyst was 27.8%, 20.1%, 50.3%, and 1.8%, respectively. The characteristic satellite (shake up) of Co at around 786 eV is evidence of the presence of Co(II). The high-resolution Co 2p spectrum showed spin-orbit splitting into 2p1/2 and 2p3/2 components  [2 [Co(X%)/TiOmponents . The Co compound . The domo Ti(IV) , Figure i(IV) [2 .2 adsorption–desorption isotherm of Co(0.1%)/TiO2 is displayed in 0), suggesting the mesoporous nature of the photocatalyst [The Ncatalyst . The adscatalyst .2) as prepared by the hydrothermal method, where the isotherm, also type IV, shows a large H3 hysteresis loop (0.4–0.8 P/P0), and the reported values of SBET (22.8 m2·g−1) and pore volume (0.033 cm3·g−1) are smaller than those of the as-synthesized photocatalyst [The BET isotherm was used to calculate the surface area (SBET—multipoint), and the textural properties were analyzed based on the Barrett, Joyner, and Halenda (BJH) model  Table 5Table 5. catalyst . Pore si2-P25 and the prepared photocatalysts are shown in 2-P25, occurring from 200 to 380 nm, is associated with the O2− (2p) → Ti(IV) (3d) transitions and tetrahedral symmetry [2 samples, band tailing appears at ca. 400 nm; such light absorption increase in the Vis region, as compared to pure TiO2-P25, relates to the charge transfer from O2− to Co(II) [g) was obtained using the Tauc plot, 2-P25, the band gap was found equal to 3.3 eV in agreement with the literature [2 photocatalysts, Eg narrows and depends on Co concentration  by Co(II), which implies new d-states coming from Co- in the forbidden band of TiO2, and to the addition of energy levels in the same zone from oxygen vacancies [The UV-Vis DRS spectra of TiOsymmetry . In the o Co(II)  and to to Co(II) . Visibleterature . For cobntration , with vaacancies .−/h+ pair recombination, thus enhancing the photocatalytic efficiency, but an excess of dopant facilitates hole–electron pair recombination, reducing the photocatalytic activity [The presence of cobalt energy levels over the Fermi level enables visible light absorption and prevents eactivity ,37.2 and Co-TiO2-doped samples was tested by monitoring phenol photodegradation upon UV and NUV-Vis radiation. After irradiation, e−/h+ pairs are formed according to the usual scheme of photocatalysis with semiconductors, [−, thus reducing the probability of recombination. O2 and H2O/HO− adsorbed onto the photocatalyst surface scavenge the trapped e− and h+ to form reactive oxygen species , which are involved in the oxidative photodegradation of phenol, same as h+.The photocatalytic activity of TiOductors, ,58 and t2, the degradation of phenol was found to be effective under UV light; which was not the case under NUV-Vis light /TiO2 photocatalysts. The as-prepared photocatalysts slightly degrade phenol under UV light, whereas complete phenol photodegradation was observed under Vis light. 2, there is only a very small phenol disappearance; a similar behavior was observed with Cu-doped titanium [In the case of Co(X%)/TiOP25-TiO2 . Figure titanium . On the 2. As stated above, the fastest phenol photodegradation happen with the Co(0.1%)/TiO2 photocatalyst, probably due to a better dispersion of metal ions on the TiO2-P25 surface /TiO−1 with a measured TOC value of 48 mg·L−1 was used.To determine the degree of mineralization during photocatalysis, TOC analyses were carried out after 60 min of photoirradiation under UV light and 240 min under NUV-Vis light . The ini−1 under NUV-Vis light in the presence of Co(X%)/TiO2 photocatalysts, i.e., a 96% TOC removal, whereas the observed removal was much lower (23%) under UV light. For both irradiation conditions, the maximum TOC removal was observed for the lowest dopant content (Co(0.1%)/TiO2), and in accordance with kinetics results, it decreased as the Co percent was increased.TOC values decreased drastically upon photocatalysis, from 48 to 1.97 mg·LTOC results are in agreement with kinetic results; the observed absorbance decay follows the same pattern found for TOC, irrespective of the irradiation wavelength: small decay and TOC reduction with UV and almost complete elimination with NUV-Vis.λ/Einstein·L–1·s–1) at the irradiation wavelength (λ), the molar absorptivity at λ (ελ/mol·dm–3·cm–1), the pathlength of the photoreactor (l/cm), and the apparent pseudo first order rate constant (k/s–1), the photocatalysis quantum yield (Φphotodegradation) can be calculated (–1), the electric power consumed by the lamp (P/kW), and the solution volume (V/L), the energy efficiency parameter (EEO/kW·L –1·s –1), i.e., the energy required to reduce the pollutant concentration per volume and time unit, can be obtained [Using the light intensity , the catalyst can be washed with no lixiviation or structural consequences after use, and when reutilized, it showed similar kinetic and TOC reduction results.2 samples with different concentration, Co(X%)/TiO2, with X = 0.1%, 0.3%, 0.5%, and 1.0%, under NUV-Vis (>366 nm) and UV (254 nm) radiation. The TiO2-P25 surface was impregnated, and the obtained solids were characterized by XRD, Raman, XPS, SEM, EDS, and TEM. Crystallite size increased in the doped samples, where Ti(IV) was substituted by Co(II) and oxygen vacancies were created as well as interband states, which reduced the band gap as found with UV-Vis DRS measurements. The N2 adsorption isotherm corresponds to type IV with a small H3 loop near the maximum relative pressure. The photocatalytic activity over phenol photodegradation was found to be the best for Co(0.1%)/TiO2, at the lowest found band gap (Eg ca. 2.3–2.4 eV), when using NUV-Vis radiation. The TOC test showed an approximately complete removal (96%) for phenol photodegradation under NUV-Vis radiation and Co(0.1%)/TiO2, while it only removed 23% under UV radiation.The degradation of phenol was investigated using cobalt-impregnated TiO2 (P25) photocatalyst showing extended light harvesting into the Vis region and were able to completely photodegrade phenol in an aqueous solution (50 ppm) after 3 h under NUV-Vis irradiation. The so-prepared catalyst was found to be chemically and mechanically stable and showed similar kinetic and TOC results upon reutilization.In summary, we easily prepared, by impregnation, and characterized a Co- doped TiO