Undaria pinnatifida, a biorefinery was set using low-cost industry-friendly methodologies, such as sequential solid–liquid extraction and liquid–liquid extraction. The obtained extracts were screened for their antioxidant and antimicrobial activity against five common wound pathogens and for their anti-inflammatory potential. The ethanolic wash fraction (wE100) had the highest antioxidant activity (114.61 ± 10.04 mmol·mg−1 extract by Diphenyl-1-picrylhydrazyl (DPPH) and 6.56 ± 1.13 mM eq. Fe II·mg−1 extract by and Ferric Reducing Antioxidant Power (FRAP)), acting efficiently against Gram-negative (Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria, and showing a nitric oxide production inhibition over 47% when used at 0.01 µg·mL−1. NMR and FTIR chemical characterization suggested that phlorotannins are present. Obtained fraction wE100 proved to be a promising candidate for further inclusion as wound healing agents, while the remaining fractions analyzed are potential sources for other biotechnological applications, giving emphasis to a biorefinery and circular economy framework to add value to this seaweed and the industry.Brown seaweeds are recognized sources of compounds with a wide range of properties and applications. Within these compounds, phlorotannins are known to possess several bioactivities  with potential to improve wound healing. To obtain phlorotannins enriched extracts from  The biodiversity that characterizes the marine environment has contributed to the bioprospection of unique compounds in the last years, culminating in an unprecedented development of blue biotechnology . A majorUndaria pinnatifida  is an invasive brown seaweed, originally from Northeast Asia , that was detected in the Atlantic Ocean, specifically in the north of Portugal, in 1999, and has progressed along the entire coast [re coast ,11,12,13re coast ,15. Thusre coast  state thStaphylococcus aureus, Pseudomonas aeruginosa, or Escherichia coli). Effective treatment against wounds’ bacterial infection remains a challenge, which has been further augmented by the development of antibiotic resistance by some pathogens, and new wound-healing products that possess antimicrobial effects are extremely required.The wound healing process is complex and requires a sequential cascade of events, where nitric oxide (NO) plays an important role in antimicrobial activities, vasodilation, inflammation response, cell proliferation, angiogenesis promotion, and matrix deposition, culminating in the reorganization of the injury . HoweverExtracting specific compounds from seaweed is a laborious task with several costs associated, particularly for phlorotannins, since these compounds are usually linked with proteins and polysaccharides and are easily coextracted with much more abundant sugars (mannitol) . The solDespite significant advances in novel extraction techniques for natural compounds, the majority have very high costs when used at the industrial scale. For this reason, the traditional methods are preferred, such as solid–liquid extraction (SLE) and liquid–liquid extraction (LLE). However, these processes usually generate leftovers and waste products that are disposed of, and in a world where global economies have been pressured to implement sustainable measures, new alternatives to avoid this scenario are crucial . Using aU. pinnatifida biomass using low-cost, sequential extraction and fractionation approaches, and posteriorly characterized for their further application in biomedicine, namely for wound healing.In the present study, several added-value extracts, among which a phlorotannins-enriched extract, were obtained from Undaria pinnatifida (Heterokontophyta: Phaeophyceae) was collected from Estai Cape  in Spain. After collection, the fresh algal biomass was sorted and sediments and epibionts removed, rinsed with freshwater, and dried in a wind tunnel at 25 °C, and after, ground to a fine flour-like powder (50 μm). This powder was then stored under vacuum, in the dark, at room temperature until usage.U. pinnatifida enriched in phlorotannins (less complex than a single-step crude extract), while promoting a circular economy approach to the fractions thereby obtained, a preliminary series of sequential solid–liquid extractions (SLE) and liquid–liquid extractions (LLE) was designed and performed (a comprehensive flowchart is presented in n-hexane (solid-to-liquid ratio 1:15), and agitated on a magnetic stirrer for 20 min, in the dark to avoid oxidative degradation of compounds, and on an ice bath to prevent temperature increase [g at 4 °C), 300mL of fresh solvent was added to the recovered leftover biomass and this was re-extracted for an additional 20 min. The procedure was then repeated a third time . The extracts recovered in each of the three washes of a given solvent were pooled and stored at 4 °C, resulting in the “hexane extract” (H). The extraction continued with water:acetic acid (99:1) (AQ), maintaining the previous extraction conditions: 300 mL per wash, 20 min, magnetic stirring, ice bath, darkness, and three washes with fresh solvent of the same type. Between different solvents, instead of centrifugation, biomass was recovered by filtration . After AQ, the extraction followed with ethanol:water:acetic acid (50:49.5:0.5) (E50), ethanol (E100), ethanol:acetone (1:1) (A50), acetone (A100), and ethyl acetate (AE).To recover a fraction of increase . To maxiThe extracts were semipurified to separate the different classes of compounds according to solvent affinity. The extract H (hexane) was washed with methanol. As methanol and hexane are partially miscible, a few drops of water were added to promote phase separation. Thus, the fraction hexane-methanol (HM), and the leftover fraction “washed hexane” (wH) were produced. The aqueous extract (AQ) was partitioned into three fractions. First, the liquid extract was fractionated with hexane, forming the aqueous-hexane fraction (AQH), and then it was extracted with ethyl acetate (AQAE), once again recovering the leftover fraction (wAQ). The aqueous ethanol extract (E50) was extracted with hexane, forming the aqueous ethanol-hexane fraction (E50H) and the leftover fraction (wE50). Following the same rationale, E100, A50, and A100 were each washed with hexane, forming E100H and wE100, A50H and wA50, and A100H and wA100. The last treatment was the wash with water:acetic acid (99:1) in the ethyl acetate (EA) extract, resulting in ethyl acetate-aqueous fraction (AEAQ), and the leftover, wEA.−1, depending on sample’s solubility. Extracts from this preliminary sequential extraction were tested for DPPH and FRAP antioxidant activities as well as antibacterial activity against Staphylococcus aureus by the disc diffusion method (methods in following sections).After LLE, all 15 fractions were evaporated to dryness in a rotary evaporator under vacuum and a water bath (maximum temperature of 45 °C), and yields were calculated based on the dry weight. Next, all fractions were resuspended in dimethyl sulfoxide , to obtain stock samples with concentrations between 10 and 100 mg·mLU. pinnatifida biomass were extracted under the following conditions: three washes per solvent, 20 min per wash, darkness, ice bath, and magnetic stirring. Separation of extract and biomass was maintained as before: centrifugation between washes of the same solvent and filtration between different solvents. The solvent pipeline was as follows: hexane > water:acetic acid (99:1) > ethanol:water (1:1) > and ethanol.Depending on the results of After, AQ and E100 crude extracts were semipurified through liquid–liquid extraction. The AQ extract was washed with ethyl acetate, originating the fractions AQAE and wAQ. Additionally, E100 extract was separated with hexane, forming E100H, and wE100 fractions . Before −1. Briefly, the extracts previously prepared in DMSO varying concentrations between 10 and 100 mg·mL−1 were used. In a 96-well microplate, 10 μL of each extract/standard concentration and DMSO as control, were pipetted per well (eight wells each). In four wells, 190 μL of working reagent was added, and in the other four, 190 μL of ethanol was added. The plate was incubated in the dark for 60 min at room temperature, and after this period, the absorbance was read at 515 nm . The amount of DPPH radical reduced was calculated using a standard curve previously obtained, and by subtracting the mean absorbance of the wells containing sample and ethanol to the mean absorbance of the wells containing sample and working reagent. Three independent assays were performed, and the results expressed as millimole per milligram of seaweed extract (mmol·mg−1 extract).This assay was performed according to Félix et al. , with mi3+ tripyridyltriazine) to a blue-colored ferrous complex (Fe2+ tripyridyltriazine) by the action of electron-donating antioxidants. The reduction is monitored by measuring the change of absorbance at 593 nm [2 (ferric chloride). The assay was conducted by pipetting 8 µL of each standard/sample’s concentration per well, or 8 µL of DMSO as control (eight wells/condition). In four of the eight wells, 272 µL of WR was added, and in the other four wells, 272 µL of water was added (blanks). Additionally, four wells containing 8 µL of FeSO4 (1 mM) and 272 µL of WR (positive control) and four wells containing 280 µL of water were added. The plate was incubated in the dark for 60 min at room temperature, after which its absorbance at 593 nm was read . The antioxidant activity of the samples was calculated by subtracting blanks to the samples, as well as the negative control, following which this value was normalized to that of positive control subtracting the negative control. The FRAP test was performed in triplicate and the results expressed as millimolar of iron II equivalents per milligram of seaweed extract (mM eq. Fe (II) mg−1 extract).This method is based on the reduction, at low pH, of a colorless ferric complex (Fet 593 nm . The FRAStaphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 11296), and Proteus mirabilis (ATCC 29906).Relevant infectious agents of wounds were analyzed. The reference strains used in the antibacterial test were S. aureus following the directrices of the Clinical and Laboratory Standard Institute  reference documents M02 and M100-S24 [S. aureus was cultured in tryptone soya yeast extract agar , at 37 °C. Afterward, colonies of bacteria were resuspended in 0.85% saline solution, and turbidity was adjusted to 0.5 McFarland (approximately 1.5 × 108 CFU·mL−1). Then, the inoculum was spread with a swab on Muller-Hinton agar  plates. Four paper discs (90 mm in diameter) containing 15 μL of each extract having a concentration between 50 and 100 mg·mL−1 were placed in the above MHA plate. As positive controls, discs containing ciprofloxacin, , erythromycin  and gentamicin  were used. One disc containing only DMSO was used as a negative control. After incubating for 18 h at 35 °C, the diameter (mm) of the inhibition zone was measured. The experiment was carried out three times and the mean values and standard deviation were presented.For the initial screening of all 15 fractions, the disc diffusion method was performed using M100-S24 . Briefly7 CFU·mL−1 using a calibration curve of OD 625nm–CFU·mL−1 available for each bacterium. Of this diluted inoculum, 5 µL were used in each well so that a final concentration of 5 × 105 CFU·mL−1 was achieved. The fractions to be tested were diluted in Muller-Hinton Broth II  growth medium, in tubes, to a concentration of 1.5 mg·mL−1, originating the mix solutions. The mix solution, containing culture medium and extract, was filtered with a sterile syringe by sterile cellulose filters (0.2 μm) .To characterize the antimicrobial potential of the selected extracts, the methodology applied was the microdilution procedure adapted from Clinical and Laboratory Standards Institute, using the reference document M7   with min−1) was utilized, and as negative controls (full growth or 0% inhibition), culture medium with DMSO (1.5% v/v). For each extract tested a respective control with mixed solution and culture medium was performed. The assay was performed in sterile round bottom 96-multiwell microplates , using a final volume in each well plate of 200 μL. After incubating for 20 h at 35 °C, optical density (OD) of 96-well plates was measured at 625 nm in a microplate reader . The experiment was carried out in triplicate.In this procedure, three different controls were used. The sterility control was the culture media Muller-Hinton Broth II. For the positive control of inhibition, ciprofloxacin  and Kanamycin . Microplates containing 5 × 104 cells/well were incubated at 37 °C in 5% CO2 for 24 h. Cells were treated for 24 h with extracts . Each extract was used at 1.5 mg·mL−1 in phosphate-buffered saline . After the incubation period, the medium was removed by aspiration and washed with 100 µL of PBS. After that, 100 µL of DMEM with 5% FBS, without phenol red and supplemented with 3--2, 5 diphenyltetrazolium bromide  solution (0.5 mg·mL−1 in PBS) was added to each well to assess cell viability [2 for 4 h and then washed with PBS. After aspiration, 100 µL of DMSO was added and the microplates were agitated for a few minutes and kept in the absence of light until complete solubilization of formazan. The absorbance was read at 570 nm wavelength in a microplate spectrophotometer . PBS supplemented with the correspondent concentration of DMSO of each extract was used as a positive control and DMSO as a negative control. Each condition was tested using six technical replicates and three independent assays.A RAW 264.7 cell line  was grown and maintained according to manufacturer instructions, using Dulbecco’s modified Eagle medium , 10% Fetal Bovine Serum , and a mixture of Nystatin  of extracts which cell viability was above 90% for 6 h, following the addition of a lipopolysaccharide  solution at a final concentration of 1.5 µg·mL−1 for 22 h. Then, 150 µL of the supernatants of the cell culture was mixed with 50 µL of Griess reagent  and incubated for 15 min at room temperature. The absorbance was measured at 540 nm using a microplate spectrophotometer  and three independent assays were performed. The inhibition of NO production rates (%) was calculated as percentages of control.A Griess diazotization reaction was used to measure the production of NO in RAW 264.7 cells according to Bahiense et al.  with sliThe chemical characterization performed in this study was intended for evaluating the presence of phlorotannins in two specific fractions, AQAE and wE100, since their extraction procedure and bioactivities analysis suggest the presence of these compounds.2CO3 20% (w/v) was added to every well. Afterward, the plate was incubated for 45 min at room temperature, after which its absorbance at 765 nm was read . The TPC was expressed as milligrams of phloroglucinol equivalents (PGE) per milligram of seaweed extract (mg PGE·mg−1 extract). The assay was performed in triplicate.Total Phenolic Content was determined spectrophotometrically by using the Folin–Ciocalteu method, with some modifications . Briefly6  and NMR experiments (1H and HSQC) were performed in a Bruker Avance 400 spectrometer  with a frequency of 400 MHz for 1H and 100 MHz for 13C. Chemical shifts were expressed in ppm and reported to the residual solvent signals.Samples were dissolved in 500 µL of DMSO-d−1, at a 4 cm−1 resolution with 64 scans.Fourier transform infrared (FTIR) spectroscopy with attenuated total reflectance (ATR) was used to evaluate the chemical composition of the two crude extracts chosen. FTIR analyses were carried out using an Alpha-P Bruker FTIR-ATR spectrometer , in the range of 4000–400 cmp value under 0.05 was considered statistically significant.The data are presented as mean ± SD of three independent experiments. SigmaPlot (V11.0) was used to perform the statistical analysis. All variables were tested for normality using the Shapiro–Wilk test. Afterwards, one-way analysis of variance (ANOVA) followed by Tukey test was used to compare the antioxidant activity between extracts in DPPH and FRAP assays, as well as antimicrobial activity, NO production and in quantification of phenolic content. A The preliminary sequential extraction results  are presented in The yield of the six fractions obtained in the optimized sequential extraction protocol are presented in The yield of extraction was higher in water-containing solvents (AQ—22.2% and E50—6.1%) and lower in the hexane fraction (0.5%). Ethanol extract yielded 2.0%, which was further divided in two fractions (E100H—0.9% and wE100—1.1%). The aqueous extract partitioned with ethyl acetate yielded the lowest value—0.3%.−1 extract) > E100H (89.97 ± 14.62 mmol·mg−1 extract) > AQAE (56.54 ± 7.33 mmol·mg−1 extract) > H (47.24 ± 5.89 mmol·mg−1 extract) > wAQ (46.16 ± 48.34 mmol·mg−1 extract) > E50 (8.51 ± 6.80 mmol·mg−1 extract). In the case of FRAP, the highest values were obtained for wE100 and H. From highest to lowest, the extracts order was wE100 (6.56 ± 1.13 mM eq. Fe II·mg−1 extract) > H (5.44 ± 0.61 mM eq. Fe II·mg−1 extract) > AQAE (3.49 ± 1.15 mM eq. Fe II·mg−1 extract) > E100H (3.28 ± 1.07 mM eq. Fe II·mg−1 extract) > E50 (1.76 ± 0.45 mM eq. Fe II·mg−1 extract) > wAQ (0.49 ± 0.32 mM eq. Fe II·mg−1 extract).The antioxidant activity displayed in the optimized extraction, evaluated by DPPH and FRAP, is presented in U. pinnatifida extracts against S. aureus, P. aeruginosa, K. pneumoniae, E. coli, and P. mirabilis are presented in −1) and standard deviation. Ciprofloxacin control resulted in approximately 100% inhibition for all species. The extract AQAE was capable of totally inhibiting the growth of S. aureus. However, it did not inhibit the growth of the Gram-negative bacteria to a high degree (0–16.2% inhibition). Some extracts performed better overall than others: H and E100H had good intermediate inhibition results ; wE100 with medium values (19.49–44.79% inhibition); wAQ and E50 with lower values of inhibition .The results of antibacterial activity of the −1 (noncytotoxic concentration obtained from a dose–response curve with the cell line RAW 264.7) on the production of NO in lipopolysaccharide-stimulated RAW 264.7 is shown in The inhibitory effect of the six fractions obtained in this study when used at a concentration of 0.01 µg·mLBidimensional NMR spectra of proton and carbon-13 acquired by Heteronuclear Single Quantum Coherence (HSQC) experiments for wE100 and AQAE are presented in In The FTIR-ATR spectrum was used to identify the functional groups of the active components present in wE100 and AQAE extract based on the band’s values . A more −1 extract for AQAE, and 0.48 ± 0.047 mg PGE·mg−1 extract for wE100. These values were statistically different (p < 0.05) using Tukey’s test.To further corroborate the NMR and FTIR-ATR findings, AQAE and wE100 total phenolic content (TPC) was determined using the Folin–Ciocalteu assay and expressed in relation to phloroglucinol equivalents (PGE). The TPC values obtained were 0.24 ± 0.025 mg PGE·mgU. pinnatifida. Thus, an industry- and environmentally friendly approach was used to tentatively recover a fraction enriched in phlorotannins, while also obtaining other bioactive fractions. Data of the preliminary sequential extraction is presented in In this study, the optimization of a sequential SLE followed by LLE, using the least toxic solvents possible, in low volumes, with low energy input, was developed for the brown seaweed n-hexane in order to avoid the downstream coextraction of lipidic components and/or pigments [Undaria pinnatifida possesses approximately 1% dry weight in total lipids [As common in several phlorotannins extractions, the current procedure started with a pretreatment of algal material, a defatting process using pigments . The yiel lipids . The nonl lipids , as welll lipids  are expel lipids . Presuma−1 extract, significantly higher than most fractions (p < 0.05). E100H and AQAE follow after wE100 with 89.97± 14.62 mmol·mg−1 extract and 59.54 ± 7.33 mmol·mg−1 extract, respectively. For FRAP activity , wE100 registered once more the highest value with 6.56 ± 1.13 mM eq. Fe II·mg−1 extract (significantly higher than most fractions (p < 0.05)), followed by H (5.44 ± 0.61 mM eq. Fe II·mg−1 extract), AQAE (3.49 ± 1.15 mM eq. Fe II·mg−1 extract), and E100H with 3.28 ± 1.07 mM eq. Fe II·mg−1 extract. These values are comparable to those of other ethanol extracts of brown seaweed, as demonstrated by Tenorio-Rodriguèz et al. [Eisenia arborea showed significantly higher mean FRAP (11.9 µM FeSO4 µg−1), followed by Padina concrecens (6.8 µM FeSO4 µg−1), and Cystoseira osmundacea (5.4 µM FeSO4 µg−1).In the wound healing process, oxygen is crucial, by eliminating invasive organisms, synthesizing collagen, acting on angiogenesis, and reepithelization . NeverthFucus vesiculosus by Wang and collaborators [In addition to the wE100 fraction, the AQAE fraction also stands out in both assays. Analogous results were obtained in the extraction of phlorotannins from borators , where aborators , and mosborators  and otheborators ,45,46,47U. pinnatifida was analyzed against five pathogenic bacteria, commonly found in the wound bed and hospital environment, by microdilution test and compared with the common antibiotic ciproxoflaxin (CP). The six extracts have shown antibacterial activity, especially against Gram-negatives, which are more difficult to combat with natural extracts (or even known antibiotics), due to their increased level of resistance mechanisms obtained from having an additional mutable outer membrane [S. aureus) AQAE fraction, hexane extract (H), and a fraction (E100H) are the most efficient, for Gram-negatives, the highest activity was found for E100H followed by wE100. wE100 registered the maximum activity against P. aeruginosa with a growth inhibition of 44.79%, which might be due to phlorotannins as reported by the work of Cox et al. [E. coli and P. mirabilis, with inhibitions of 37.62% and 31.56%, correspondingly, could also be partly due to these compounds. The results obtained for the inhibition of K. pneumoniae with wE100 are more distinct, only with 19.49%. Against S. aureus, the ethyl acetate fraction of the aqueous extract had the highest growth inhibition with 102.07%, having a spectrum of action as effective as that of the commercial antibiotic, at the tested concentration. On the other hand, leftovers derived from aqueous extract, wAQ, have registered inhibition rate < 30%, because of their content in non-bioactive coextractants such as sugars and polysaccharides. Overall, ethanol and hexane derived extracts revealed good antimicrobial activity for all pathogens with differences from the aqueous fractions (p < 0.05), which might be due to the presence of fucoxanthin or halogenated volatile organic compounds (HVOCs) [K. pneumoniae and P. mirabilis also prompts further investigation of this fraction, since K. pneumoniae is one of the most reported pathogens concerning antibiotic resistance [P. mirabilis is frequently found in the hospital environment and causes significant clinical infections.The antimicrobial activity of the six extracts of membrane . While fx et al. , where e (HVOCs) ,50,51. Tsistance ,53, and U. pinnatifida extracts at 0.01 µg·mL−1 and stimulated with LPS for 22 h revealed a significant reduction of NO production when compared with control treatment (p < 0.05) , and stearidonic acid (SA), already described to be present in this species, which possess anti-inflammatory effects [Myagropsis myagroides ethanolic extract had a significant anti-inflammatory activity in LPS-stimulated BV-2 microglia [Ecklonia cava ethanol extract, containing 10.6% w/w of dieckol, a phlorotannin type, decreased by more than half NO in LPS-stimulated RAW 264.7 cells, when treated with 100 μg·mL−1 [Fucus vesiculosus phlorotannin-enriched extracts. Contrarily, Dong et al. [U. pinnatifida sporophyll extracts had a higher anti-inflammatory effect. Nonetheless, in this work, extracts show an effective reduction of NO in very low concentrations, showing their potential to be used as an anti-inflammatory. Generally, other reports have NO reduction using seaweed extracts concentrations around 2.5–500 µg·mL−1 [In the early phases of wound healing, NO production is mediated by the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression , which a < 0.05) . The red effects . Similaricroglia . Eckloni μg·mL−1 . Notably μg·mL−1  with Fucg et al.  found th µg·mL−1 ,59,60,61Eisenia bicyclis [3 group of α-L-fucopyranose units [1H NMR, with signals at 1.32 and 1.26 ppm being assigned to C6 methyl protons of L-fucopyranose [3 protons of O-acetyl groups, which can possibly be from the monomers of fucoidan found in U. pinnatifida, e.g., fucose [13C NMR spectra at δ(C) = 95–105 ppm, which are also associated with phlorotannins  [In this study, both wE100 and AQAE did not go through extensive purification procedures since such methodologies impact negatively the scalability and cost effectiveness of the process. Thus, it is expected that both fractions still contain a mixture of compounds. As such, the techniques used  should not reveal a clear spectrum of a known phlorotannin, but if they were in appreciable amount, there should be indicative signals of their presence. In the case of wE100′s HSQC, intense signals can be observed in the region of δ(H) = 1–2 ppm and δ(C) = 20–40 ppm, which might be indicative of alkane derivates (carotenoids), such as fucoxanthin, as verified by Kim et al. on the seaweed bicyclis ,63 or itbicyclis . Additiose units , facts tpyranose . Narrow , fucose ,66. Some, fucose ,68,69, a, fucose ,72,73,74a [In the case of AQAE’s HSQC spectra, it did not show the signals characteristics of phlorotannins in the region previously mentioned. Rather, the presence of mannitol along with other sugars is likely considering the signals at δ(H) ~4 ppm . Similara . While N−1 region which can be assigned to hydroxyl O-H stretch in intermolecular bonded alcohols or even residual moisture. Such moiety can be found in carbohydrates and polyphenols, among other compounds [−1 corresponds to C-H stretch vibrations, which indicate the presence of alkanes [−1, corresponding to sulfate S=O stretching, while wE100 presents a band at 1168 cm−1, representative of sulfonic acid. These bands are most likely originated from sulfated residues of fucose from sulfated fucans of U. pinnatifida [−1, in wE100, can be attributed to C=O stretching vibration of O-acetyl groups, while, at the wavenumber of 1463 cm−1, a sharp band was found and attributed to the aromatic ring stretch (C=C). Normally, the wavenumber for this group is around 1450–1510 cm−1 [−1 [−1 to vibration of CH2 to galactose and xylose and CH3 to fucose [U. pinnatifida.ompounds . The ran alkanes , which cnatifida . The ban510 cm−1 ,33, and cm−1 [−1 . Howevero fucose , showing−1, the bands for wE100 and AQAE shown can indicate the presence of phlorethols/fuhalols with ether bridges. This type of linkage, as well as the number and arrangement of phenolic hydroxyl groups in their structure, has a deep influence on the antioxidant properties in Laminariales species, as U. pinnatifida [−1 for wE100 extract, which relates to methine bending vibration in benzene derivatives—a characteristic of phlorotannins [In the region of 1058 and 1020 cmnatifida . Howeverotannins . Togethep < 0.05). The values observed, 0.24 ± 0.025 mg PGE·mg−1 extract for AQAE and 0.48 ± 0.047 mg PGE·mg−1 extract for wE100, are in accordance with others found in literature [Fucus vesiculosus achieved the maximum phenolic content among other fractions, with 17.1 ± 1.5 mg PGE·g−1. Additionally, in the study of Wang and colleagues [F. vesiculosus also had the higher TPC value with 88.3 ± 2.2 g of PGEs/100 g of extract. In the present study, the ethanolic fraction was shown to be the phlorotannin-enriched fraction, as found in the Cho et al. [Sargassum siliquastrum had a significantly higher phenolic content (127.4 mg GAE−1 extract) than water or chloroform extracts. Additionally, when ethanolic and 50% EtOH extracts of Sargassum muticum, obtained at three different temperatures were compared, the highest content of phenolics was detected in EtOH extracts, while the lowest was found in water extracts [Eisenia bicyclis, Sargassum fusiforme, Saccharina japonica, and U. pinnatifida in comparison to the aqueous MeOH and Acetone extracts, neither the results from Leyton et al. [U. pinnatifida included [E. arborea, P. concrecens, and C. osmundacea can explain their antioxidant activity, since polyphenol compounds contain reducing properties, such as hydrogen or electron donating agents, which contribute to free radical scavenging potential. Lopèz and collaborators [F. vesiculosus obtained by Wang et al. possessed the highest TPC as well as the strongest DPPH scavenging activity and reducing power [In fact, the polyphenols quantification procedure used corroborated the NMR and FTIR results by showing the wE100 as the fraction with higher phenolic content  and metal ion reduction (FRAP), and was able to inhibit Gram-negative bacteria growth, including principal hospital infection and antibiotic-resistant bacteria such as U. pinnatifida for societal commodities and improved health.In summary, the wE100 has the potential to be a wound-healing agent. Future perspectives like the incorporation of this extract in smart wound dressings are routes of work that should be addressed and be pursued. Additionally, it provides a cost-effective reason for the removal of this invasive seaweed, contributing to marine ecosystem restoration, while maximizing the value of


In this study, S2− doped Bi2WO6-x coupled with twin crystal ZnIn2S4 nanosheets (Sov−BWO/T−ZIS) were prepared as an efficient photocatalyst by a simple hydrothermal method for the removal of tetracycline hydrochloride (TCH). Multiple methods  were employed to systematically investigate the morphology, structure, composition and photochemical properties of the as-prepared samples. The XRD spectrum indicated that the S2− ions were successfully doped into the Sov−BWO component. XPS spectra and photoelectrochemical analysis proved that S2− served as electronic bridge and promoted captured electrons of surface oxygen vacancies transfer to the valence band of T−ZIS. Through both experimental and in situ electron paramagnetic resonance (in situ EPR) characterizations, a defined direct Z-scheme heterojunction in S-BWO/T−ZIS was confirmed. The improved photocatalytic capability of S-BWO/T−ZIS results ascribed that broadened wavelength range of light absorption, rapid separation and interfacial transport of photoexcited charge, precisely regulated redox centers by optimizing the interfacial transport mode. Particularly, the Sov−50BWO/T−ZIS Z-scheme heterojunction exhibited the highest photodegradation rate was 95% under visible light irradiation. Moreover, this heterojunction exhibited a robust adsorption and degradation capacity, providing a promising photocatalyst for an organic pollutant synergistic removal strategy.The rational design of direct Z-scheme heterostructural photocatalysts using solar energy is promising for energy conversion and environmental remediation, which depends on the precise regulation of redox active sites, rapid spatial separation and transport of photoexcited charge and a broad visible light response. The Bi Organic pollutants in an aquatic ecosystem cause continuous environmental deterioration, which greatly threatens the ecological environment and human public health. Among various organic pollutants, the increasing presence of antibiotics and textile dyes in wastewater is one of the most serious issues. For instance, tetracycline hydrochloride (TCH), as one of the main components of pharmaceutical wastewater, is usually chemically stable and hardly removed by conventional biochemical and physical measures. It can bring about a series of environmental concerns such as causing the formation of resistant microorganisms and upsetting the natural balance ,2,3,4. T2−, h+, e−) produced by photocatalysts could immediately react with adsorbed contaminants, reviving the regenerate adsorption ability. Currently, bismuth tungstate (Bi2WO6), as the simplest Aurivillius-type member with alternating (Bi2O2)n(WO4)n construction and simultaneously containing the WO6 perovskite component, has attracted intense attention in the environmental remediation of refractory organic wastewater [2WO6 (Bi2WO6-x) is widely used in photocatalytic purify of organic contaminants due to its appropriate bandgap structure and positive valence band level, visible-light response and robust photochemical stability. In previous reports, introducing heteroatom has proven to be one of the most effective modification strategies, because the band structure and electronic structure can be effectively adjusted, ultimately optimizing the morphology, improving photoelectrochemical properties and enhancing the photocatalytic performance [Recently, photocatalysis as one kind of the most “green” AOPs, has emerged as a promising approach for the removal of TCH without bringing secondary pollution in the wastewater treatment process. Especially, various photocatalysts possess high specific surface area and ordered mesoporous layers, which facilitates the adsorption of contaminants within the mesoporous layer ,7,8. Morstewater ,10,11,12formance .To date, it is widely attracting more and more attention in Z-scheme heterojunction photocatalysts, due to their maximum redox capacity and effective separation and transfer of photoexcited carriers. However, the building of a non-intimate contact heterogeneous interface has been proven to be ineffective in the development of an extraordinary Z-scheme system ,15,16. I2− doped Bi2WO6-x coupling with twin crystal ZnIn2S4 (Sov−BWO/T−ZIS) composites were fabricated by self-assembly method via the tactics that synergistic adsorption with photocatalytic decontamination. Benefits from the S2− doping and the electron trap consisted of Sov, the as-prepared Sov−BWO/T−ZIS Z-scheme heterojunctions displayed the rapid separation and efficient utilization of carriers, thus exhibiting the enhancement of TCH removal efficiency.In this study, a series of S2), indium chloride (InCl3·4H2O), thioacetamide (TAA), glycerol, sodium tungstate (Na2WO4·2H2O), bismuth nitrate (Bi(NO3)3·5H2O) and N, N-dimethylformamide (DMF) were obtained from Shanghai Macklin Biochemical Co., Ltd. . All used chemicals were used as received without further purification.Zinc chloride 3·5H2O was added into 80 mL deionized water for 20 min with the aid of magnetic stirring. Then, 165 mg Na2WO4·2H2O was added into the aqueous solution and sustained another 10 min. Then, the appeared precipitate was centrifuged several times and transferred into 35 mL deionized water. Then, the mixed solution was transferred into a Teflon-lined autoclave and kept at 433 k for 18 h. Finally, the products were washed several times with deionized water and dried at 333 k for 12 h. The as-prepared products were denoted as Sov−BWO.The Bi2WO6-x/ZnIn2S4 composites were synthesized through a facile in situ solvothermal method, which was showed as 2WO6-x sample  was dispersed into the 15 mL DMF solution. Then, ZnCl2 (1 mmol) was dissolved into DMF and maintained ultrasonic for 10 min. InCl3·4H2O (2 mmol) was added into the mixture solution and maintained 10 min. TAA (4 mmol) was dispersed in the mixture solution, which was going on ultrasonic processing for 5 min. Subsequently, glycerol (3 mL) was added into the mixture and maintained for 5 min. The collected solution was then transferred into a Teflon-lined autoclave (50 mL) and heated at 453 K for 10 h. Finally, after cooling down to room temperature, the as-prepared product was obtained and labeled as Sov−25BWO/T−ZIS, Sov−50BWO/T−ZIS, Sov−75BWO/T−ZIS, Sov−100BWO/T−ZIS. For comparison, the synthetic procedure of the pure ZnIn2S4 sample was similar to the above steps except that the Bi2WO6-x was not added and labeled as T−ZIS.The Bi4 acted as the internal reflectance standard. Photoluminescence (PL) spectroscopy was measured using a PerkinElmer LS55 photoluminescence spectrometry testing system under the excitation of 325 nm light. Liquid chromatography mass spectrometry (LC-MS) was measured on the Agilent 1290 UPLC/6540 Q-TOF .X-ray Diffraction (XRD) was obtained by a Rigaku D/MAX 2500 V , a scanning scope from 10° to 80°. Fourier transform infrared spectroscopy (FT-IR) spectra of the samples were conducted using a Nicolet iS 50 . The microstructures were monitored by transmission electron microscopy . The morphology of the samples was monitored on a field-emission scanning electron microscopy  equipped with an energy-dispersive X-ray spectrometry (EDS). The positions of the XPS peaks were determined with reference to the carbon peak of C ls at 284.8 eV. The binding energies of Zn, In, S, Bi, W and O elements were acquired using an X-ray photoelectron spectroscopy . The electron paramagnetic resonance (EPR) analysis was performed to test surface oxygen vacancies on a Bruker A300 spectrometer at 298 K. In situ electron paramagnetic resonance (in situ EPR) spectra were obtained on a Bruker EMXplus spectrometer equipped with a 150 W mercury lamp as the illumination source. The details of the instrumental parameters were as follows: Scanning frequency: 9.82 GHz, central field: 3400 G, scanning power: 20 mW, and scanning temperature: 298 K . The optical absorption of samples was gotten by a UV-vis DRS spectrometer , in which BaSOUsing tetracycline hydrochloride (TCH) as a model pollutant, the decontamination activity of as-prepared materials was evaluated. The initial pH of the TCH solution was 6.3. The photocatalyst  was added into solution, increasing PH to 6.7. The adsorption capacity and photocatalytic activity of the as-prepared samples  was investigated in the glass vial with circulating cooling water system. A 300 W Xe lamp with a cut-off filter (λ > 400 nm) was used as the source of visible light. Typically, 30 mg as-synthesized sample was dispersed in 100 mL TCH aqueous solution (20 mg/L), and stirred magnetically in the dark for 30 min to achieve adsorption-desorption equilibrium. Then, 2 mL suspension was withdrawn each 15 min and then filter through 0.22 μm nylon membrane. The TCH concentrations were measured using a UV–vis spectrophotometer  at the absorption peak of 357 nm. The stability and reusability of the best-performing sample were tested for five runs under the same conditions.−1 were acted as scavengers to capture hole (h+), hydroxyl radical (OH) and superoxide radical (O2−), respectively. The experiments were similar to the above best-performing photocatalytic degradation of TCH.Ethylenediaminetetraacetic acid disodium (EDTA-2Na), isopropanol, p-benzoquinone of 1 mmol·L0 was the absorbance of the original TCH solution. The Ct is the absorbance of the residual concentration of TCH under different degradation times.The adsorption and photocatalytic degradation rates were calculated by using the following equations:2SO4 aqueous solution. Electrochemical Impedance Spectroscopy (EIS) was carried out in 0.5 M Na2SO4 aqueous solution over the frequency ranging from 0.1 Hz to 105 Hz. Mott-Schottky plots were measured under the frequency of 1000 Hz and 2000 Hz in 0.5 M Na2SO4 aqueous solution. Photocurrent response (I-t) of the as-prepared samples was measured on IVIUM electrochemical workstation. A single wavelength (λ = 375 nm) was served as the light source.A CHI660E electrochemical workstation was performed to test. In a typical three-electrode system, Pt foil acted as the counter electrode and Ag/AgCl served as a reference electrode, in which the electrolyte was 0.5 M Na2WO6 , respectively [2S4 , respectively [2S4 (JCPDS 48–1778) [2− doping into Sov−BWO [2− doping, which promoted the rapid transfer of photoexcited charge on the unique interface. The FT-IR spectra of the as-obtained samples were shown in −1 and 726 cm−1 were considered as W–O bonds, while the characteristic peak at 1382 cm−1 was attributed to a W–O–W vibration bond, respectively [2− doped Sov−BWO/T−ZIS samples were around 1619 cm−1 and 3421 cm−1, respectively, which were caused by O–H bonds and water molecules adsorbed on the surface [ectively . Meanwhiectively . The pea Sov−BWO ,22,23. Fectively . Figure The morphology of prepared Sov−BWO, T−ZIS and Sov−50BWO/T−ZIS samples were investigated by FE-SEM. As shown in 2WO6 and the (102) plane of ZnIn2S4, respectively. The typical lattices of Sov−BWO and T−ZIS indicated the successful construction of the Sov−50BWO/T−ZIS hybrid. Furthermore, as shown in Furthermore, HRTEM images of Sov−50BWO/T−ZIS sample were presented to further analyze the morphology. HRTEM image in 1/2 and Zn 2p3/2, while the two peaks at 452.5 eV and 445.0 eV were distributed to In 3d3/2 and In 3d5/2, respectively. Similarly, as for Sov−50BWO/T−ZIS sample, the characteristic peaks at 1145.2 eV and 1122.1 eV are assigned to the Zn 2p1/2 and Zn 2p3/2 of Zn2+ species, while the peaks at 452.4 eV and 444.9 eV were consistent with the In 3d3/2 and In 3d5/2 of In3+, respectively [2+ and In3+ revealed the strong influence of T−ZIS, compared to the previous literature with heterojunction formation [1/2 and S 2p3/2 at 162.9 and 161.7 eV) of Sov−50BWO/T−ZIS hybrid exhibited the positive shift compared to T−ZIS (S 2p1/2 and S 2p3/2 at 162.8 eV and 161.6 eV). Whereas the Bi 4f characteristic peaks at 163.6 eV and 158.2 eV of Sov−50BWO/T−ZIS shifted toward lower binding energy compared to the peaks at 164.4 and 159.0 eV of Sov−BWO. Interestingly, the obvious new peaks approximately at 164.3 eV and 158.9 eV were attributed to the reason that S2− doped into Sov−BWO [2− acted as an efficient electronic bridge and facilitated the trapped electron carriers transfer from Sov−BWO to T−ZIS. In 2− served as an electronic bridge and facilitated priority trapped electron and transfer. In addition, the W 4f of pristine Sov−BWO exhibited two peaks at 37.5 eV and 35.3 eV, which could be assigned to W6+ species. Analogously, the lower shifted of Sov−50BWO/T−ZIS at 37.0 eV and 34.9 eV [2− doping and construction of electron bridge, which was conducive to the spatial separation and transport of charge in Sov−50BWO/T−ZIS Z-scheme heterojunction.The surface chemical state of surface elements was analyzed by XPS. ectively . These rormation ,30,31. M Sov−BWO . The str 34.9 eV . It was 2 adsorption-desorption isotherms and pore size distribution curves. As shown in 0 of about 0.8–1.0, revealing that the samples are all mesoporous structures [2·g−1 and 0.1455 cm3·g−1, which was higher than that of pristine Sov−BWO (13.98 m2·g−1 and 0.0385 cm3·g−1). The higher specific surface area and pore volume provided favorable conditions for the molecular diffusion of the reactants and the approachability of active sites. Hence, pure T−ZIS had better adsorption and decontamination capacity compared to pristine Sov−BWO. In addition, all composites  exhibited the enhanced specific surface area higher than single Sov−BWO and T−ZIS samples, which were 71.47 m2·g−1, 73.16 m2·g−1, 62.48 m2·g−1 and 50.61 m2·g−1, respectively. The self-assembly Sov−BWO and T−ZIS nanosheets probably possessed the tight heterogeneous interface contact and a puffier morphology, resulting in a larger specific surface area. With the addition of Sov−BWO, the adsorption capacity of TCH increased significantly, indicating that the construction of Sov−BWO/T−ZIS Z-scheme heterojunction was bifunctional in adsorbing and in situ decontamination to remove pollutants. Meanwhile, the Sov−50BWO/T−ZIS sample possessed the maximum specific surface area of 73.16 m2·g−1, which was responsible for the enhancement of adsorption capacity. However, the further addition of Sov−BWO resulted in a significant decrease in the specific surface area of Sov−75BWO/T−ZIS and Sov−100BWO/T−ZIS samples, which may be caused by excessive accumulation of Sov−BWO nanosheets during the self-assembly process. The specific surface area and pore structure of the samples were studied using Nructures . Table 12− served as electron bridge, accelerating the migration of photoexcited charge in Z-scheme heterojunction and enhancing photocatalytic kinetics. −1, 0.0108 min−1, 0.0226 min−1, 0.03 min−1, 0.0209 min−1 and 0.0195 min−1, respectively, which demonstrated that the construction of Z-scheme heterojunction retained the strong redox ability of T−ZIS and Sov−BWO. Meanwhile, due to the existence of electron bridge in the Z-scheme system, the separation of photoexcited carriers was accelerated, and more O2−, h+· and ·OH were produced in the reaction solutions, greatly improving the photocatalytic degradation rate of TCH.As an effective broad-spectrum antibiotic with outstanding antibacterial property, tetracycline hydrochloride (TCH) has excellent antibacterial properties and has been widely used in the medical field. Herein, performance test experiments using the synthesized samples as adsorbents and photocatalysts were performed to evaluate the removal efficiency of TCH. As displayed in +, OH and ·O2− scavengers, respectively. As shown in + and ·O2−, which were consistent with previous literature [In addition, the active species scavenging experiments were carried out to identify the active species involved in the TCH removal of Sov−50BWO/T−ZIS photocatalyst. EDTA-2Na, isopropanol and p-benzoquinone are chosen as hterature . For botUnder the same conditions, five successive photocatalytic experiments were carried out on decontamination and adsorbent regeneration. + attack. Then, the product (m/z = 346) was resulted from the deamination reaction [2 [To identify the potential decontamination pathway of TCH during the photocatalytic stage, the intermediate products were analyzed by LC-MS systems. The corresponding m/z (mass-to-charge ratio) were presented in reaction ,40. For ction [2 . Pathwayction [2 . Then, tction [2 . Finally2 versus the absorbed energy [To study the optical absorption and bandgap structure properties, the UV-vis DRS was performed to characterize. d energy . The Eg n-type characteristic. The flat band potential of T−ZIS and Sov−BWO were −0.71 and −0.41 V , respectively. The flat band potential of negative 0.1 V was considered to be the conduction band edge (ECB) of n-type semiconductors [VB) of T−ZIS and Sov−BWO were estimated to be 1.80 and 2.51 V versus NHE according to the formula: EVB = ECB + Eg.As displayed in nductors . Therefonductors . MeanwhiTo investigate the photoexcited carrier separation of as-obtained photocatalysts, PL spectra were performed at an excitation wavelength of 325 nm. As shown in Furthermore, I-t curves and EIS measurements were employed to examine the transport behavior of photoexcited carriers. As displayed in 2− of Sov−BWO, T−ZIS and Sov−50BWO/T−ZIS photocatalysts, respectively. − (1.99 V vs. NHE) and H2O/OH (2.34 V vs. NHE), resulting in the h+ in the CB of T−ZIS was unable to generate ·OH [2− signals were discovered in T−ZIS and the DMPO-·O2− signals could hardly be detected in Sov−BWO. The inexistence of·O2−  over Sov−BWO was due to the inadequate ECB potential [2− was significantly higher than that of T−ZIS. The coexistence of O2− and OH species confirmed that the charge separation pathway was a Z-type heterojunction, rather than the typical II-type heterojunction [To gain insight into the Z-scheme photocatalytic mechanism, we further performed in situ EPR studies to detect the responses of DMPO-·OH and DMPO-·Orate ·OH . As dispotential . Meanwhijunction . These r2− and h+) mineralized adsorbed pollutants into CO2 and H2O under visible light illumination. Herein, the electrons on the EVB of T−ZIS generated·O2− by reducing dissolved oxygen. The holes on the EVB of Sov−BWO produced OH by oxidized H2O and OH− [CB of Sov−BWO were injected into the EVB of T−ZIS through the Sov and rapidly transfer by S2− electronic bridge, and promptly recombined with the photoexcited holes, which was consistent with the XPS analysis. Therefore, the Sov−BWO/T−ZIS Z-scheme heterojunction not only promoted the separation and transport of photoexcited carrier charges, but also retained the high redox capabilities of Sov−BWO and T−ZIS, thus exhibiting the high degradation efficiency.Based on the above analysis, a reasonable mechanism of adsorption and in situ decontamination in the sequential process was preliminarily confirmed between Sov−BWO and T−ZIS. As shown in  and OH− . Meanwhi2− and h+ were mainly species, which were responsible for photodegradation. Therefore, the best-performing Sov−50BWO/T−ZIS sample exhibited an excellent TCH removal rate of 95%, higher than Sov−BWO (47%) and T−ZIS (67%). This study uncovers the correlation between adsorption and degradation, and provides a new insight in the design and application of direct Z-scheme heterojunction photocatalysts, which exhibits remarkable potential for the removal of pollutants.In summary, we proposed the Z-scheme heterojunction via coupling Sov−BWO with T−ZIS nanosheets, which acted as an efficient functional photocatalyst for removal of pollutants. The addition of T−ZIS nanosheets significantly optimized the cohesion of nanosheets and improved the adsorption ability of pristine Sov−BWO for removal of tetracycline. The enhanced adsorption capacity of self-assembly Sov−BWO/T−ZIS nanosheets was attributed to the increase in specific surface area, which was favorable for the subsequent photocatalytic process. The radicals ·O


P1, carbazole P2, and 3,4-alkoxythiophene P3) or electron-deficient moieties  were synthesized via direct C–H (hetero)arylation polymerization (DHAP) in moderate to excellent yields (60–98%) with medium to high molecular weights . Owing to their various electronic and structural properties, different absorption spectra  as well as different specific capacitances of 70, 68, 75, 51, and 25 F/g with 19, 10, 21, 26, and 69% of capacity retention after 1,000 cycles were observed for P1–P5, respectively. After careful study through multiple experimental measurements and theoretical calculation, appropriate electronic characteristics, small molecular conformation differences between different oxidative states, and well-ordered molecular stacking could improve the electrochemical performance of CPs.Over the past decades, π-conjugated polymers (CPs) have drawn more and more attention and been essential materials for applications in various organic electronic devices. Thereinto, conjugated polymers based on the 3,4-ethylenedioxythiophene (EDOT) backbone are among the high-performance materials. In order to investigate the structure–property relationships of EDOT-based polymers and further improve their electrochemical properties, a series of organic solvent–soluble EDOT-based alternative copolymers consisting of electron-rich fragments (fluorene  Accordingly, the molecular structure types of polymers synthesized by these two methods are relatively limited, which is not conducive to the study of the structure–property relationship. For example, the A–B alternative copolymer cannot be readily synthesized by classical electropolymerization or oxidative chemical polymerization. Moreover, the products of electropolymerization generally have problems in further processing, large-scale preparation, and limited molecular structure diversity, due to their infusibility and poor solubility in common solvents. And the products of oxidative chemical polymerization also have problems in the metal ion residue and low reactivity for electron-deficient monomers. In other words, to design more appropriate synthetic methods is of crucial importance to the development of high-performance CPs.With the rapid development in the field of wearable and flexible electronics during the past decades, π-conjugated polymers (CPs) have drawn more and more attention and been essential materials for applications in organic solar cells (OSCs) , organicvia DHAP have been widely applied in the field of high-performance organic semiconductors and have shown a variety of merits, such as a well-defined structure, a diversified molecular structure, high reproducibility, large-scale throughput, and good solubility  were synthesized effectively via DHAP in moderate to excellent yields (60–98%) with medium to high molecular weights . Although several similar examples of these polymers have been synthesized before, the relationships between the photophysical and electrochemical properties and their structures have not been systematically investigated arylation polymerization (DHAP) of non-preactivated (hetero)arenes with (hetero)aryl halides is one of the most ideal and effective methods to construct conjugated polymers, avoiding tedious reaction steps and formation of stoichiometric toxic organometallic byproducts in the traditional organometallic couplings . Furtherlubility . Based ovia DHAP , we herestigated . The rel2 (5 mol%), K2CO3 (2.5 equiv.), and 1-adamantanecarboxylic acid  in DMAc at 100 C for 24 h was used to synthesize our EDOT-based CPs. As shown in P1, carbazole P2, and 3,4-alkoxythiophene P3) and electron-deficient units  were successfully engaged in the reactions, offering structure-diversified EDOT-based CPs in moderate to excellent yields (60–98%) with medium to high molecular weights . The yield and molecular weight of P2 can be further improved up to 81% and 7,900 Da after 48 h, respectively, indicating its relatively low reactivity of 3,6-dibromocarbazole. The good yields of P1–P5 with comparable or even higher Mn compared to those obtained in previous works indicated the high reactivity of EDOT under this catalytic system and good applicability of DHAP , films for characterization of UV-Vis absorption, electrochemical properties, and microscopic packing properties were fabricated by solution processing, such as drip coating and spray coating.Owing to the wide application of EDOT-based CPs and high reactivity of C–H bonds, the DHAP of EDOT was early and extensively studied by several research groups . Based o of DHAP . It is oterature . Br termP1–P5 were optimized through density functional theory (DFT), as shown in P1–P5, covering the near-UV and blue, green, and red color regions, suggest their distinct electronic and structural properties. The obviously red-shifted absorption of P1–P5 in the thin film in comparison with that in the solution could be generated from the strong intermolecular interaction between the polymer backbones in film states. On comparison with P1 and P2 containing electron-rich units , P4 and P5 with electron-deficient units displayed significantly red-shifted absorption , which should be assigned to the intramolecular charge transfer (ICT) from the EDOT donor to corresponding electron-deficient acceptor units. When connecting 3,4-alkoxythiophene with EDOT, P3 also displayed a red-shifted absorption (λsolution = 547 nm and λfilm = 558 nm), owing to its almost planar backbone  induced by an intramolecular O∙∙∙S interaction (o), P5 exhibited the most red-shifted maximum absorption wavelength among these five polymers, implying its efficient electron delocalization, transport property, and close molecular stacking.The UV-Vis spectra and data of these five EDOT-based CPs in diluted solution and in films are shown in eraction . Thus, c+ wire (silver wire in 0.01 M AgNO3 in acetonitrile) as the reference electrode, and a Pt disk or foam-nickel electrode drop-coated with the polymer as the working electrode and using 0.1 M tetrabutylammonium hexafluorophosphate in dry acetonitrile as the electrolyte solution. Firstly, cyclic voltammetry (CV) measurements of P1–P5 films on a Pt disk electrode were performed to analyze their p-doping/dedoping processes at a scan rate of 100 mV s−1  eV, where Eox,onset is the onset potential of the oxidation peak with respect to the ferrocene/ferrocenium (Fc/Fc+) redox couple. The HOMO energy levels of P1 (−5.17 eV), P2 (−5.06 eV), and P3 (−4.79 eV) are in good agreement with those reported by other groups  (P5 (−5.35 eV) among P1–P5, the coplanar and electron-rich P3 showed the highest HOMO energy level (−4.79 eV), indicating its easiness of losing electrons and the potential development of new polymeric anodes. The arrangement trends of HOMO energy levels of P1–P5 estimated by CV were in accordance with those of HOMO values calculated by DFT, besides slightly overestimated HOMO and LUMO levels by DFT  curves, and electrochemical impedance spectroscopy (EIS) and are summarized in −1 demonstrated their fast redox behaviors and high rate charge/discharge performances (P1–P5 exhibited obvious pseudocapacitive energy storage properties. The measured specific capacitances of the polymers (P1–P5)/foam-nickel electrode were calculated to be 70, 68, 75, 51, and 25 F/g (at 100 mV s−1) with 19, 10, 21, 26, and 69% of capacity retention after 1,000 cycles, respectively. These specific capacitances were higher than those of some other conducting polymer electrodes deposited by solution processing methods  . Compares by DFT . Subsequormances . For theormances , suggestormances . From thormances , it can  methods , probablcy range , indicatcy range .θ = 3.43, 6.79, 9.94, and 25.54°, corresponding to d-spacings of 25.73, 13.01, 8.89, and 3.48 Å, respectively, representing the existence of lamellar stacking and π–π stacking of polymer main chains. With the lowest roughness and best XRD crystallinity of P5 among all polymers, the relatively good cyclic stability could be attributed to the homogeneous surface and well-ordered stacking of molecules in films. All the thermal degradation temperatures (Td) over 300°C of P1–P5 at 5% weight loss manifested their good thermal stability  through direct (hetero)arylation polymerization. Owing to the structural diversity of obtained EDOT-based copolymers, the varied absorption spectra, specific capacitance, and capacity retention were observed, which should be attributed to the electronic characteristics of various units introduced, molecular torsions in the polymer backbone, and thus resultant different surface morphologies and molecular packings. Clear redshifts in absorption of P3, P4, and P5 were observed, due to their good coplanarity and/or ICT property. When using a foam-nickel electrode drop-coated with the polymer as the working electrode in a three-electrode cell, good specific capacitances of 70, 68, 75, 51, and 25 F/g (at 100 mV s−1) with 19, 10, 21, 26, and 69% of capacity retention after 1,000 cycles for P1–P5 were obtained, respectively, owing to the good redox activity of the EDOT unit. The results of our present study confirm that appropriate electronic characteristics, fast charge transfer, good coplanarity, low RMSD between different oxidative states, homogeneous surface, and well-ordered molecular stacking could improve the cycling stability of polymeric capacitive performance which is still one of the main obstacles in further applications of supercapacitors based on π-conjugated polymers. It is anticipated that such study will provide a novel strategy for the molecular design of π-conjugated polymers regarding electrochemical properties.In this work, a series of novel soluble EDOT-based conjugated copolymers consisting of electron-rich or electron-deficient units were synthesized steadily in moderate to excellent yields (60–98%) with medium to high molecular weights (


In high grade serous ovarian cancer (HGSOC), there is a spectrum of sensitivity to first line platinum‐based chemotherapy. This study molecularly characterizes HGSOC patients from two distinct groups of chemotherapy responders (good vs. poor).Following primary debulking surgery and intravenous carboplatin/paclitaxel, women with stage III–IV HGSOC were grouped by response. Patients in the good response (GR) and poor response (PR) groups respectively had a progression‐free intervals (PFI) of ≥12 and ≤6 months. Analysis of surgical specimens interrogated genomic and immunologic features using whole exome sequencing. RNA‐sequencing detected gene expression outliers and inference of immune infiltrate, with validation by targeted NanoString arrays. PD‐L1 expression was scored by immunohistochemistry (IHC).BRCA2 mutations and had a significantly higher nonsynonymous mutation rate compared to PR tumors (p = 0.001). Samples from the PR cohort were characterized by mutations in MGA and RAD51B and trended towards a greater rate of amplification of PIK3CA, MECOM, and ATR in comparison to GR tumors. Gene expression analysis by NanoString correlated increased PARP4 with PR and increased PD‐L1 and EMSY with GR. There was greater tumor immune cell infiltration and higher immune cell PD‐L1 protein expression in the GR group.A total of 39 patient samples were analyzed . Median PFI for GR and PR patient cohorts was 32 and 3 months, respectively. GR tumors were enriched for loss‐of‐function Our research demonstrates that tumors from HGSOC patients responding poorly to first line chemotherapy have a distinct molecular profile characterized by actionable drug targets including PARP4. In patients with suboptimally‐debulked, advanced high grade serous ovarian cancer (HGSOC), the spectrum of responses to first line platinum‐based chemotherapy (PtC) vary from durable to non‐existent, highlighting an unmet medical need for those with poor responses. Our study molecularly characterizes a cohort of HGSOC patients with distinct responses, either good or poor, to PtC. The good and poor responders have median progression‐free intervals of 32 and 3 months, respectively, and molecular analysis of tumors identifies differences in DNA damage response pathways and immunity, namely, tumors from poor responders to first line PtC have a distinct molecular profile characterized by actionable drug targets including PARP4. Patient2.2Formalin‐fixed paraffin embedded (FFPE) tumor blocks were utilized for molecular analysis following quality review of a hematoxylin and eosin (H&E) slide. A reference pathologist (H.S.) identified specific tumor‐rich areas which were then macrodissected from 10‐µm tissue sections using a sterile razor blade followed by deparaffinization in 1 ml of xylene. Genomic DNA and total RNA were isolated using the Qiagen AllPrep FFPE Tissue Kit (QIAGEN) followed by fluorometric quantification using a Qubit 3.0 instrument (Life Technologies). RNA integrity was assessed using the Agilent TapeStation on a High Sensitivity RNA ScreenTape.2.3Transcriptome sequencing libraries were constructed from 200 ng of total RNA using the Illumina TruSeq Stranded Total RNA Library Prep Gold kit from which >69 million paired‐end sequencing reads were generated on the Illumina NextSeq550 platform using V2 chemistry and reagents. Exome libraries were made from 100 ng of genomic DNA using the Kapa HyperPrep Kit which were enriched using Agilent SureSelect XT Human All Exon V6+ COSMIC reagents. Exome libraries were sequenced to a mean exon coverage of 100X on the Illumina HiSeq2500 platform using V4 chemistry and reagents. All reads were processed following the GATK Best Practices framework including read alignment against the hg19 human reference using BwaMem v 0.7.12,2.4Total RNA data quality was assessed using FastQC and ReSeOC v 2.6.42.5An H&E stain was performed on one tumor section per patient, and a blinded pathologist reviewed the sections for tumor content (M.R.). The tumor component was maccrodissected from FFPE, and RNA was extracted using RNeasy FFPE Extraction kit (Qiagen) following manufacturer's instructions. RNA was quantified using Qubit 2.0 Flourometer (Life Technologies) and hybridized 150 ng of each sample with the codeset at 65°C for 21 h and kept at 4°C < 1 h before preparing cartridges. Patient samples were randomly distributed in four cartridges and included tumor reference and background controls (water). NanoString assays were performed by following the standard protocol “Setting up 12 nCounter Assays ”. AZ‐designed DDR‐max codeset was used in this project. Cartridges were read immediately after being prepared, on the AZ GEN2 Digital Analyzer station with high resolution selected . In addition to 27 housekeeping genes, the AZ‐custom designed DDR‐max codeset includes 753 genes from the core DDR pathways and  and previously published gene signatures predicting for response to DNA damaging chemotherapy.2.6FFPE tissues were cut into 4‐µm serial sections and mounted on charged slides. The H&E was assessed by a blinded pathologist (M.R.) for the percentage of tumor surface containing immune cells (ICs) . PD‐L1 staining was conducted using Ventana PD‐L1 (SP263) IHC assay, investigational use only, according to the manufacturer's instructions on the Benchmark Ultra stainer (Ventana). PD‐L1 IHC stained slides were blindly assessed for tumor cell (TCs) PD‐L1 expression and for percentage of PD‐L1 positive ICs . Ataxia telangiectasia mutated (ATM) IHC staining was performed using the ab32420 antibody as previously described,2.7t test or a Wilcoxon rank‐sum test for continuous outcomes, significance cut‐off p < 0.05, were used. Associations of protein expression with clinical, pathological and molecular features, and chemotherapy response were evaluated with Fisher's exact and Wilcoxon–Mann Whitney tests, as required. To find genes associated with PFI, PFI was treated as the outcome variable in a survival model and gene expression values, as measured by the NanoString technology, were used as predictor variables in univariate analyses. PFI was modeled, adjusting for censoring, as a lognormal distribution and therefore patients with PFI values equal to zero had 0.5 added to the PFI value .For analyses where responder status was the predictor or independent variable, Fisher's exact test for categorical outcomes and an independent samples 33.1BRCA1 and BRCA2 carrier status, residual disease, vascular invasion, or number of lines of therapy between the two groups. There was a higher median CA125 in the PR group compared to the GR group . At data cut‐off, the median PFI for GR and PR cohorts was significantly different at 32 and 3 months (p < 0.001), respectively. The median OS was 65.5 months in the GR group and 23 months in the PR group (p < 0.001), in support of the two groups having extremes in overall outcomes. Patients with pathogenic germline BRCA1 and BRCA2 mutations trended towards both a higher PFI and OS than the germline BRCA1 and BRCA2 negative patients.Chart review identified 39 patients that met study inclusion/exclusion criteria Figure . Baselin3.2BRCA1, BRCA2, and TP53, as well as additional oncogenic mutations in genes associated with PI3K/AKT/mTOR signaling, DDR and epigenetic regulation . The most frequently mutated genes with known relevance to cancer were TP53 , BRCA1 and BRCA2 , DDR genes MLH3, MSH2, MSH6, and RAD51B , PIK3CA , MGA , CSMD3 , NF1 , AURKA , and CNTRL . Of the eight somatic BRCA2 mutations, seven were found in GR tumors (p = 0.044) and included an in‐frame deletion, two nonsense mutations, two frameshift insertions, and two missense mutations . The single BRCA2 mutation in a PR tumor was a p. Arg2108Lys missense mutation outside of a functional domain. Whereas BRCA2 nonsense, insertion/deletion, and splice‐site loss‐of‐function (LOF) mutations were found almost exclusively in the GR group , LOF BRCA1 mutations were found in both GR and PR tumors . LOF mutations in MGA (2/39) and RAD51B (2/39) mutations were only found in PR tumors.Exome evaluation of somatic mutations in all 39 tumors revealed previously identified genes of relevance in HGSOC including n Figure . Comparip = 0.77) , MECOM (4/20 vs. 9/19), and ATR (3/20 vs. 7/19). CCNE1 and MYC were each amplified in 18% of all cases (7/39), and this number was nearly equally distributed among PR and GR groups. In the GR group, one deep deletion was observed in CCNE1.Tumors were characterized by considerable aneuploidy consistent with HGSOC in both GR and PR patients, yet the percentage of genome altered by a copy number amplification or deletion was not significantly different between the two groups  Figure . An anal3.3TFIIH, POLR2B, CCNC, LIG3, POLD3, RFC2, POLE3 genes were amongst the most highly expressed in GR samples. In contrast, PR tumors had elevated levels of PARP4, POLK, CDK7, RAD17 and TP53BP1  showed that GR tumors were enriched for expression of genes involved in DNA replication/cell cycle, BER, NER, and HRD pathways  . The PR tumors also showed an enrichment of genes involved in NER  showed that 127 genes were expressed at significantly higher levels in GR patients, whereas 13 genes were significantly more expressed in PR patients. 1 Figure . Analysi1 Figure , or top ) Figure . In contR Figure .EMSY (p = 0.008), a repressor of BRCA2 transactivation; and programmed death ligand 1 (PD‐L1) (CD274) (p = 0.014), an immune inhibitory receptor ligand frequently used as a biomarker for immune checkpoint inhibition  from RNA‐seq trended towards correlation with gene copy number, NanoString analysis showed no statistically significant difference in mRNA log2 gene expression when amplified cases were examined compared to all others, independently of platinum‐response . Furthermore, no significant differences in mRNA expression were found between the GR and PR groups for the DDR genes: BRCA1, BRCA2, ERCC1, SLFN11, TP53, and XRCC1  but not with BRCA1 mutations   Figure . EMSY exs Figure .3.4p < 0.001 Mann–Whitney)  than in the PR group at 8% (±7% SD) Figure . In addi) Figure ,F, based) Figure . Immune ) Figure , observe) Figure  or diffu) Figure .To assess whether these patterns may be associated with specific immune infiltrates, RNA‐seq of 29 tumors (Table S5) was performed to assess total infiltration  and infiltration of specific immune cell types (CIBERSORT). Overall, we did not find a significant difference in aggregate immune or stromal scores between the GR and PR groups Figure , nor wer44.1Platinum resistance poses a great clinical challenge in the treatment of HGSOC as up to 30% of patients develop recurrent disease within 6 months of first line treatmentTP53, which is 100% in our tumor cohort.The GR and PR cohorts in the present study are noteworthy for their extreme differences in median PFI and OS . In suboptimally debulked stage III, HGSOC patients treated with platinum doublet chemotherapy, the previously reported PFI and OS were 14.1–16.9 and 35–45.1 months, respectively,4.2BRCA1and BRCA2 mutation rate of 38% (15/39) in the present study is higher than the The Cancer Genome Atlas (TCGA) reported rate of 22%,BRCA mutant cohort. Our finding that BRCA2 mutations were significantly more frequent in the GR group is in accordance with evidence that mutations in BRCA1 and especially BRCA2, confer chemo‐sensitivity in ovarian cancer.BRCA1 and BRCA2 mutations, higher tumor mutation burden and infiltration by T cells with improved survival in HGSOC,BRCA2 mutations were found to be enriched among long‐term responders to PARP inhibition.The BRCA1 and BRCA2 and TP53, mutated tumor suppressor genes are uncommon in HGSOC.EMSY, a repressor of BRCA2 transactivation that localizes to sites of DNA repair and the amplification of which has been associated with an adverse prognosis.EMSY, and consequent suppression of BRCA2 transcriptional activity, has been proposed as an additional mechanism of defective DNA double‐strand break (DSB) repair in HGSOC.BRCAness on the chemo‐responsiveness of a tumor is dependent on the mechanism by which BRCA is silenced .EMSY at the DNA level, but EMSY mRNA levels were significantly increased within the GR group, supporting a role for EMSY upregulation as a marker of chemosensitivity.Aside from expected mutations in ATR was not mutated in our cohort and its expression was not significantly different in GR and PR samples, we report ATR amplification, as determined by exome sequencing, at a higher frequency  than in the TCGA datasets (8%).ATR amplification noted in 15% of GR and in 37% of PR, but this was not statistically significant in this relatively small cohort.Ataxia telangiectasia mutated (ATM) and Rad3‐related protein (ATR) are core players in the DDR pathway, with central roles coordinating response to DSBs and replication stress (RS).4.3ATR, MECOM, and PIK3CA. That PIK3CA, which is commonly amplified in HGSOC (18%),MECOM and CCNE1 in our study cohort was consistent with TCGA data.MGA that were concentrated in the PR group. MGA, a transcription factor which interacts with c‐MYC, is a putative tumor suppressor gene. Inactivating mutations in MGA may contribute to solid tumor development and have been detected in colorectal cancer, adenocarcinomas of the lung and small‐cell lung cancers.Amplification of Chromosome 3q (Chr3q) has been frequently described in up to 50% of HGSOCs.4.4The broad screen of mRNA expression data using the NanoString DDRmax codeset showed association between longer survival and high expression of genes involved in DNA replication, NER, BER, and HR repair pathways.TFIIH, POLR2B, POLR2C, CCNC, RFC2, LIG3, and POLD3, involved in NER and transcription, correlated with PFI  in patients with long PFI links enhanced replication potential, on which platinum relies to exert DNA damage, with GR  exhibited the same trend but lacked significance , and tumor infiltration of PD‐L1 expressing immune cells. Platinum resistant tumors were characterized by increased expression of PARP4, NER pathway genes, and lower expression of DNA replication genes as compared to platinum sensitive tumors. These findings may be applied to larger studies to advance therapeutic options for patients experiencing inherent platinum resistance.Despite these limitations, our gene expression data supports a clear role for DNA repair genes in both the favorable and poor response groups. Our work suggests interplay between DDR pathways and the tumor immune microenvironment in shaping platinum response. Durable remissions were associated with a high tumor mutation burden, evidence of DNA repair disruption (via GG has acted as principal investigator on an AstraZeneca‐sponsored clinical trial, outside the submitted work. GG and JW have participated in AstraZeneca advisory boards, outside the submitted work. GG has received travel grants from AstraZeneca, outside the submitted work. GG and JW have received honoraria from AstraZeneca, outside the submitted work. MR, GNJ, SEL, PMC, DH, and NL are/were employees of AstraZeneca, and MR, GNJ, PMC, DH, and NL own AstraZeneca stock. The other authors declare no potential conflicts of interest relevant to this publication.Johanne I Weberpals: study conception, study design, data acquisition, data analysis, data interpretation, manuscript writing, manuscript review, and approval; Trevor J. Pugh: study design, data acquisition, data analysis, data interpretation, manuscript writing, manuscript review, and approval; Paola Marco‐Casanova: study design, data acquisition, data analysis, data interpretation, manuscript writing, manuscript review, and approval; Glenwood D. Goss: study conception, study design, data analysis, data interpretation, manuscript review, and approval; Natalie Andrews Wright: data acquisition, data analysis, data interpretation, manuscript writing, manuscript review, and approval; Prisni Rath: data acquisition, data analysis, data interpretation, manuscript review, and approval; Jonathon Torchia: data acquisition, data analysis, data interpretation, manuscript review, and approval; Alexander Fortuna: data acquisition, data analysis, data interpretation, manuscript review, and approval; Gemma N Jones: data acquisition, data analysis, data interpretation, manuscript review, and approval; Martine P Roudier: data acquisition, data analysis, data interpretation, manuscript review, and approval; Laurence Bernard: data analysis, data interpretation, manuscript review, and approval; Bryan Lo: data analysis, data interpretation, manuscript review, and approval; Dax Torti: data acquisition, data analysis, data interpretation, manuscript review, and approval; Alberto Leon: data acquisition, data analysis, data interpretation, manuscript review, and approval; Kayla Marsh: data acquisition, manuscript review, and approval; Darren Hodgson: study conception, study design, data interpretation, manuscript review, and approval; Marc Duciaume: data acquisition, data analysis, manuscript review, and approval; William J Howat: data acquisition, manuscript review, and approval; Natalia Lukashchuk: data analysis, data interpretation, manuscript review, and approval; Stanley E. Lazic: data analysis, data interpretation, manuscript review, and approval; Doreen Whelan: data acquisition, data analysis, manuscript review, and approval; Harmanjatinder S. Sekhon: study design, data acquisition, data analysis, data interpretation, manuscript review, and approval.Fig S1‐S7Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.