Enhanced ORR activity of cobalt porphyrin co-deposited

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Synthesis,characterization,cytotoxic activity and DNA binding properties of the novel dinuclear cobalt(III)complex with the condensation product of 2-acetylpyridine and malonic acid dihydrazideRabia Eshkourfu a ,Bo židar Čobelji ća ,Miroslava Vuj či ćb ,Iztok Turel c ,Andrej Pevec c ,Kristina Sep či ćd ,Manja Zec e ,Sini ša Radulovi će ,Tatjana Srdi ć-Radi će ,Dragana Miti ća ,Katarina Andjelkovi ća ,Du šan Sladi ća ,⁎aFaculty of Chemistry,University of Belgrade,Studentski trg 12-16,11000Belgrade,SerbiabInstitute of Chemistry,Technology and Metallurgy,University of Belgrade,Njego ševa 12,P.O.Box 815,11000Belgrade,Serbia cFaculty of Chemistry and Chemical Technology,University of Ljubljana,A šker čeva 5,1000Ljubljana,Slovenia dBiotechnical Faculty,University of Ljubljana,Department of Biology,Ve čna pot 111,1000Ljubljana,Slovenia eInstitute for Oncology and Radiology of Serbia,Department of Experimental Oncology,Laboratory for Experimental Pharmacology,Pasterova 14,11000Belgrade,Serbiaa b s t r a c ta r t i c l e i n f o Article history:Received 23February 2011Received in revised form 25May 2011Accepted 26May 2011Available online 13June 2011Keywords:Dinuclear cobalt(III)complex Crystal structure Apoptosis DNA bindingA novel dinuclear cobalt(III)complex with the condensation product of 2-acetylpyridine and malonic acid dihydrazide,N ′,N ′2-bis[(1E )-1-(2-pyridyl)ethylidene]propanedihydrazide was synthesized and characterized by elemental analysis,spectroscopy (NMR and infrared),and X-ray crystal analysis.The complex showed a moderate activity towards Artemia salina .The highest cytotoxic potential of the complex was observed on the epithelial breast cancer (MDA-361)cell line.The investigated complex induced apoptosis,the early apoptotic cells comprising 28.18%,compared to 5.64%of control cells in the same phase.The interaction of the complex with calf thymus DNA (CT-DNA)was monitored by blue shift and hyperchromism in the UV –vis spectra.The observed intrinsic binding constant (K b =4.2×105M −1)together with structural analysis of the complex indicate the groove binding.©2011Elsevier Inc.All rights reserved.1.IntroductionAlthough metal complex shows an immense versatility of structures,only few of them have entered the clinical praxis as antitumor agents.Most of these agents target DNA molecules.The most important ones are platinum agents (cisplatin,carboplatin,oxaliplatin)[1,2].Unfortu-nately,because of intrinsic and acquired resistance a limited spectrum of cancers can be treated by platinum drugs.In order to overcome this problem,polynuclear metal complexes in which the metal centers are linked by ﬂexible alkyl chains [3,4]or positioned within a more rigid scaffold [5,6]have been synthesized.These agents can bind to DNA with larger af ﬁnity,impart unprecedented binding modes and recognize speci ﬁc DNA sequences.The complexes of polydentate dihydrazone ligands with adjacent coordination pockets,predominantly condensation derivatives of 2-acetylpyridine and malonic acid dihydrazide,are in the focus of interest of our research group [7–9].Dihydrazone type ligands have a large number of potential donor atoms and hence display versatilebehavior in metal coordination.The mode of coordination depends on the nature of the central metal atom,conformational ﬂexibility of the ligand as well as on the presence of other species capable to compete for coordination pockets.Malonic dihydrazide based ligands,like N ′,N ′2-bis [(1E )-1-(2-pyridyl)ethylidene]propanedihydrazide (H 2L,Scheme 1),are capable to bind to more than one metal center.In mononuclear octahedral Fe(III)complex,H 2L is coordinated through nitrogen atoms from both coordination pockets [7],while it binds two metal centers in bis-tridentate fashion in Cd(II)complex [8].Each Cd (II)atom has a pentagonal bipyramidal e of Zn(BF 4)2·6H 2O afforded formation of a dinuclear Zn(II)complex,with one molecule of the ligand bridging two Zn atoms,the remaining coordination sites being occupied by solvent molecules [9].In the reaction with Ni(ClO 4)2·6H 2O two binuclear complexes were obtained [3].Each complex cation is built of two ligands,each acting as a bis tridentate (NNO)system,bridging two metal centers.Such coordination results in octahedral environment around each Ni(II)center.Relative rigidity of the ligand might afford complxes with appropriate supramolecular architecture for ef ﬁcient DNA binding.Malonic acid based dihydrazones and their complexes showed versatile biological activities [8,10].In comparison to other acyl hydrazones of 2-acetylpyridine [10],H 2L ligand shows better cytotoxic activity on HeLa (human cervix carcinoma cells)and B16(murine melanoma)tumor cell lines.The heptacoordinated binuclear Cd(II)Journal of Inorganic Biochemistry 105(2011)1196–1203⁎Corresponding author at:Faculty of Chemistry,University of Belgrade,Studentski trg 12-16,P.O.Box 158,11000Belgrade,Serbia.Tel.:+381113336679;fax:+381112636061.E-mail address:dsladic@chem.bg.ac.rs (D.Sladi ć).0162-0134/$–see front matter ©2011Elsevier Inc.All rights reserved.doi:10.1016/j.jinorgbio.2011.05.024Contents lists available at ScienceDirectJournal of Inorganic Biochemistryj o u r n a l ho m e p a g e :w w w.e l s ev i e r.c o m /l o c a t e /j i n or g b i ocomplex with H 2L showed very good cytotoxic activity already in the low micromolar range [8].The activity was,at least in part,a consequence of strong binding to DNA [11].These promising results prompted us to evaluate if other analogous metal complexes exhibit similar behavior.In this paper we report synthesis,characterization,cytotoxic activity and DNA binding properties of a novel dinuclear cobalt(III)complex with the condensation product of 2-acetylpyridine and malonic acid dihydrazide.Cobalt(III)was selected for its high positive charge and redox properties.2.Experimental2.1.Materials and methods2-Acetylpyridine (99%),and malonic acid dihydrazide (97%)were obtained from Aldrich.Starting salt Co(NO 3)2·6H 2O (purum p.a.≥99%)was obtained from Merck.All solvents (ethanol,DMSO)and chemicals used for DNA studies were reagent grade and used without further puri ﬁcation.All buffer solutions for investigations of in-teractions of compounds with DNA were prepared in deionized sterile water and ﬁltered through 0.2μm ﬁlters (Nalgene,USA).Calf thymus DNA (CT-DNA)(lyophilized,highly polymerized),ethidium bromide and plasmid pUC18were purchased from Serva,Heidelberg.Stock solutions (10mM and 50mM)of salt Co(NO 3)2·6H 2O (S),ligand H 2L and complex [Co 2L 2](NO 3)2·2H 2O·0.5C 2H 5OH were prepared in water and DMSO.Elemental analyses (C,H,and N)were performed by standard micro-methods using the ELEMENTARVario ELIII C.H.N.S O analyzer.IR spectra were recorded on the Perkin –Elmer FT-IR 1725X spectrometer using the KBr-pellet technique in the region 4000–400cm −1.The NMR spectra were recorded on Bruker Avance 500equipped with broad-band direct probe.All spectra were measured at 26°C.The complex was characterized on the basis of NMR spectroscopy results:1D (1H,13C,DEPT —distortionless enhancement by polarization transfer),2D (COSY —correlation spectroscopy)as well as 2D 1H –13C heteronuclear correlation spectra.Chemical shifts are given on δscale relative to tetramethylsilane (TMS)as internal standard for 1H and 13C.UV –visible (UV –vis)spectra were recorded on a UV Cintra 40UV –visible spectrometer operating from 200to 800nm in 1.0cm quartz cells.A Submarine Mini-gel Electrophoresis Unit (Hoeffer HE 33)with an EPS 300power supply was used for electrophoresis experiments.The stained gels were illuminated under a UV transilluminator Vilber-Lourmat (France)at 312nm and photographed with a Panasonic DMC-LZ5Lumix Digital Camera through ﬁlter DEEP YELLOW 15(TIFFEN,USA).2.2.Synthesis of [Co 2L 2](NO 3)2·2H 2O·0.5C 2H 5OHTo a solution containing N ′,N ′2-bis[(1-E )-1-(2-pyridyl)ethylidene]propanedihydrazide (H 2L)(0.0338g,0.1mmol)in ethanol (15mL)were added solutions of 2,2′-dipyridylamine (0.0171g,0.1mmol)and Co(NO 3)2·6H 2O (0.0291g,0.1mmol)each dissolved in 5mL of ethanol.The mixture was re ﬂuxed for 2h forming a brick-red solution.After ﬁve days dark brown –red crystals suitable for X-ray diffraction were formed.Yield 52%;(Found:C 42.80H 3.99N 19.79%.C 70H 78Co 4N 28O 25calcd.C 43.18H 4.04N 20.14%).IR spectrum (KBr;ν,cm −m =medium,w =weak,s =strong,vs =very strong):3416(m),1602(w),1501(s),1457(s),1382(vs),1358(vs),1329(s),1257(m),1147(m),1074(m),1034(w),944(w),831(w),776(m),735(m),692(w),654(m),578(w).UV –vis (DMSO):λmax 370nm (ε,550M −1cm −1)1A 1g →1T 2g (0.275),λmax 320nm (ε,950M −1cm −1)1A 1g →1T 1g (0.475).2.3.Crystallographic structure determinationCrystal data:C 35H 39Co 2N 14O 12.5,M =973.66,triclinic,space group P 1,a =15.0282(4),b =16.3112(4),c =17.4548(5)Å,α=69.609(2),β=86.147(2),γ=88.014(2)°,V =4001.19(18)Å3,Z =4,D c =1.616g cm −3,μ=0.912mm −1.A red prism of compound [Co 2L 2](NO 3)2·2H 2O·0.5C 2H 5OH with dimensions of 0.28×0.10×0.10mm was greased on a glass thread.Diffraction data were collected on a Nonius Kappa CCD diffractometer with an area detector at −123°C.A Cryo-stream Cooler (Oxford Cryosystems)was used for cooling the sample.A graphite monochromated MoK αradiation (λ=0.71073Å)was employed.The data were processed by using DENZO [12].A total of 25,530re ﬂections were measured,14,384were independent,and 9066[I N 2σ(I )]were considered observed.The structure was solved by direct methods using SIR-92[13]and re ﬁned by a full-matrix least-squares procedure based on F 2using SHELXL-97[14].All the non-hydrogen atoms were re ﬁned anisotropically.All hydrogen atoms were included in the model at geometrically calculated positions and re ﬁned using a riding model.The residual density peaks,observed in the difference Fourier map,were unre ﬁnable and probably can be attributed to disordered solvent molecules.In our ﬁnal model,the scattering contributions from all of these diffuse moieties have been removed using the SQUEEZE routine in PLATON [15].A potential solvent-accessible volume of 236Å3was found.This volume corresponds to six additional molecules of water present in the unit cell,three per asymmetric unit.The empirical formula above is thus also in accordance with the elemental analysis and expresses two molecules of H 2O and 0.5molecules of EtOH per complex cation.All the calculations were performed by using the WinGX System,Ver.1.80.05[16].Final R indices [I N 2σ(I )]R1=0.0614,wR2=0.1317,and (all data)R1=0.1111,wR2=0.1504were found.2.4.Biological activity evaluationA teaspoon of lyophilized eggs of the brine shrimp Artemia salina was added to 1L of the arti ﬁcial sea water containing several drops of yeast suspension (3mg of dry yeast in 5mL distilled water),and air was passed through the suspension thermostated at 301K,under illumination for 24h.The tested substances were dissolved in DMSO.In a glass vial,into 1mL of arti ﬁcial sea water 1–2drops of yeast extract solution (3mg in 5mL of distilled water)and 10–20hatched nauplii were added,and ﬁnally solutions of all derivatives to the appropriate concentrations.For each concentration,two determina-tions were performed.The vials were left at 28°C under illumination for 24h,and afterwards live and dead nauplii were counted.LC50was de ﬁned as the concentration of a drug that causes death of 50%nauplii.DMSO was inactive under applied conditions.2.5.Cytotoxicity assayHuman epithelial breast cancer (MDA-361),human cervical carci-noma (HeLa),murine melanoma (B16),human leukemia cells (K562)and human lung ﬁbroblasts (MRC 5)were maintained in RPMI 1640medium (Sigma-Aldrich,Cat.No.R7755),in a humidi ﬁed atmosphere containing 5%(v/v)CO 2.Human endothelial cells EA.hy 926were maintained in high glucose DMEM —Dulbecco's modi ﬁed eagles'medium (PAA,Cat.No.E15-883).Both media were supplemented with 10%heat-inactivated fetal bovine serum (Sigma-Aldrich,Cat.No.F4135),penicillin (100units/mL),and streptomycin (100μg/mL).Except for the K562and MRC 5,all cell lines were seeded at a density of 2000cells/well in 96-well plates,whereas K562and MRC5Scheme 1.N ′,N ′2-bis[(1E )-1-(2-pyridyl)ethylidene]propanedihydrazide.1197R.Eshkourfu et al./Journal of Inorganic Biochemistry 105(2011)1196–1203were seeded at a density of5000cells/well.The cells were left for24h to rest,and afterwards investigated agents were added.Preparation of tested solution was performed immediately before experiment,by dissolution in DMSO to the stock concentration of10mM,whereas further dilutions were performed with the RPMI—Roswell park memorial institute(or DMEM)medium.Final concentrations achieved in treated wells for all tested agents were3,9,30,90and 300μM,whereas1,3,10,30,and100μM for MDA-361,HeLa and K562cells.Each concentration was tested inﬁvefold on particular cell line,and left for48h of incubation.Except for the K562cells,the cytotoxic activity of each agent was evaluated by means of sulphorhodamine B(SRB,Sigma-Aldrich,Cat.No.S1402-5G) colorimetric assay[17].Since the K562is the cell line that grows in suspension,the cytotoxicity potential was determined by means of MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide,Sigma)assay[18].The%of control was further calculated as the following:%of control=[ODt−ODb]/[ODc−ODb],ODt—mean optical density of treated wells,ODb—mean optical density of blank wells,ODc—mean optical density of control wells.Results areﬁnally expressed as IC50 values(concentration of investigated agent that declines the number of viable cells by50%in treated cell population compared to non-treated control),determined using sigmoidal dose–response curve ﬁtting on Graph Pad Prism software[19].Survival curves were determined using sigmoidal dose–response curveﬁtting on Graph Pad Prism software.2.6.Flow-cytometric analysis of cell cycle phase distributionHuman epithelial breast cancer(MDA-361)cells were seeded in six well plates(200,000cells/well)in duplicate,and left overnight to rest,afterward the investigated substance was added.For the cell cycle analysis the investigated compound was applied in two concentrations,theﬁrst one equal to IC50value,and the second one corresponding to2×IC50.For cell cycle analysis,cells were harvested after24h of drug treatment,washed once in phosphate buffered saline (PBS),andﬁxed in ice-cold ethanol(70%).Fixed specimens were kept at 4°C for24h,when ethanol was removed by two washing cycles in PBS. Following the last spinning cycle the cells were treated with0.2mL ribonuclease(1mg/mL)for30min at37°C.Then,cells were put on ice with propidium iodide(PI)(0.2mL,50μg/mL)added15min before analysis[20].2.7.Apoptotic assayApoptosis of MDA-361cells treated with investigated complex [Co2L2](NO3)2·2H2O·0.5C2H5OH was evaluated by Annexin V–FITC—ﬂuorescein isothiocyanate apoptosis detection kit(BD Biosciences Cat. No.65874x,Pharmingen San Diego,CA,USA).Brieﬂy,1×106cells/mL treated with the concentration of1×IC50of investigated compound for 24h,were washed twice with cold PBS and then resuspended in200μL binding buffer(10mM HEPES/NaOH(HEPES—(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)pH7.4,140mM NaCl,2.5mM CaCl2). 100μL of the solution(1×105cells)were transferred to a5mL culture tube and5μL of Annexin V–FITC and5μL of PI was added.Cells were gently vortexed and incubated for15min at25°C in the dark.After that, 400μL of binding buffer was added to each tube and then analyzed using a FACS Calibur Becton Dickinsonﬂow cytometer and Cell Quest computer software[21].2.8.DNA binding experimentsCT-DNA was dissolved in Tris buffer(10mM Tris–HCl pH7.9) overnight at4°C.DNA concentration was adjusted with the buffer to 3mg/mL.This stock solution was stored at4°C and was stable for several days.A solution of CT-DNA in water gave a ratio of UV absorbance at260and280nm,A260/A280of1.89–2.01,indicating that DNA was sufﬁciently free of protein.The concentration of DNA was determined from the UV absorbance at260nm using the extinction coefﬁcientε260=6600M−1cm−1[22].The absorbance titrations were performed at aﬁxed concentration of[Co2L2](NO3)2·2H2O·0.5C2H5OH complex and varying the concentration of double stranded CT-DNA.For an individual experiment,each DNA solution(1,2,3,4,5, 6,8,9,10,11,12,13,14and15μL of CT-DNA stock solution)was pipetted into a separate vial,to which10μL of a stock solution of compound were added and the volume adjusted to1mL with water. Reaction mixtures were incubated at37°C for2h with occasional vortexing.Primary spectra of all spectrometric measurements were imported into Microcal ORIGIN v8.0.2.9.DNA cleavage experimentsPlasmid pUC18was prepared by transformation of a clone from Escherichia coli RR1(pUC18,2686bp,purchased from Sigma-Aldrich, USA)into electrocompetent E.coli DH5αstrain cells according to the protocol for growing E.coli culture overnight in LB medium at37°C by electroporation with the“Gene Pulser”(Bio-Rad)[23].The plasmid DNA from E.coli clones were isolated by modiﬁed method of alkali lysis[24]and puriﬁed with the“JetStar”kit(Genomed)using anion-exchange column.Afterﬁnal washing step with ice-cold70%ethanol, DNA pellet was air-dried andﬁnally resuspended in150μL sterile H2O and stored at−20°C.The concentration of plasmid DNA(0.10nM of pUC18)was determined by measuring the absorbance of the DNA-containing solution at260nm.One optical unit corresponds to50μg/ mL of double stranded DNA.Plasmid DNA was incubated for4min at95°C,followed by incubation at4°C for the next20min.Then230ng of pUC18in a20μL reaction mixture in40mM sodium bicarbonate buffer,pH8.4,was incubated with different concentrations of Co(II),H2L and[Co2L2] (NO3)2·2H2O·0.5C2H5OH at37°C,for90min,or with10mM of [Co2L2](NO3)2·2H2O·0.5C2H5OH for2h.The reaction mixtures were vortexed from time to time.The reaction was terminated by short centrifugation at10,000rpm and adding7μL of loading buffer(0.25% bromophenol blue,0.25%xylene cyanol FF and30%glycerol in TAE buffer,pH8.24(40mM Tris-acetate,1mM EDTA)).The samples were subjected to electrophoresis on1%agarose gel(Amersham Pharma-cia-Biotech,Inc)prepared in TAE buffer pH8.24.The electrophoresis was performed at a constant voltage(80V)for about an hour(until bromophenol blue had passed through75%of the gel).After electrophoresis,the gel was stained for30min by soaking it in an aqueous ethidium bromide solution(0.5μg/mL),and after that was visualized under UV light.2.10.Antibacterial activityAntibacterial activity was evaluated using the standard agar-diffusion test,against four Gram positive(Bacillus subtilis,Bacillus cereus,Streptococcus pyogenes,Streptococcus agalactiae)and one Gram negative(Klebsiella pneumoniae)bacterial strain.All the strains were obtained from the Culture Collection of Extremophilic Microorgan-isms at the Department of Biology,Biotechnical Faculty,University of Ljubljana,Slovenia.The bacteria were allowed to grow overnight in LB medium,and their concentrations were determined by turbidimetric assay.After that,an appropriate volume of bacterial culture was added to LB nutrient agar previously cooled to42°C,to give theﬁnal concentrations of the bacteria approximately5×105CFU/ml.Twenty milliliters of inoculated medium were poured into Petri dishes and incubated at4°C for24h.The circles of agar(Ф=1cm)were cut out from the cooled medium.A stock solution of the complex in DMSO was gradually dissolved in deionized water.One hundred micro liters of the obtained solutions in various concentrations(1000,200,40,8, and0.16μg/mL)were pipetted into the holes cut in the inoculated1198R.Eshkourfu et al./Journal of Inorganic Biochemistry105(2011)1196–1203medium,and the dishes were incubated at37°C for24h.The diameters of the inhibition zone were then measured.As controls,the inhibitory activities of the pure solvents(appropriate aqueous solutions of DMSO)were checked as well.Every measurement was repeated at least in three parallel samples.3.Results and discussion3.1.GeneralStarting from the ethanolic solution of Co(NO3)2·6H2O and H2L in the presence of2,2′-dipyridilamine,a dinuclear complex of cobalt(III) with two molecules of ligand L coordinated to each cobalt atom,and two nitrate ions in the outer sphere was obtained.Elemental analysis showed that the crystal substance contains0.5molecule of ethanol and2molecules of water per complex cation.The molar conductivity data showed that the complex is an electrolyte of1:2type.2,2′-Dipyridylamine was added into the reaction mixture during synthesis in order to change dielectric constant of the solution from which the complex crystallizes so that crystals suitable for X-ray analysis could be obtained.During the reaction,oxidation of Co(II)to Co(III)by air occurred,affording a diamagnetic complex,as shown by magnetic moment measurement.3.2.Description of the crystal structureCompound[Co2L2](NO3)2·2H2O·0.5C2H5OH crystallizes in mono-clinic P crystal system with two independent and slightly different ionic complexes[Co2L2]2+in the structure,one of which is shown in Fig.1.The complex cation[Co2L2]2+possesses a dinuclear double-stranded helicate structure in which each Co(III)center is coordinated by two tridentate N2O chelating units from different ligands.Each Co(III) center thus occupies a six-coordinated pseudooctahedral environment surrounded by two chelating units coordinated to Co(III)atom in mer conﬁguration with pairs of carbonyl O atoms and pyridine N atoms in a cis relationship,whereas the hydrazide N atoms are trans to each other. The average Co\N bond distances(1.889Å)are slightly shorter thanaverage Co\O bond distances(1.902Å)but all are in the range usually reported.The C(O)\N and C(CH3)\N bond distances(av.1.310and av.1.295Å,respectively),together with_N\N\bond distances(av.1.393Å)suggest that the protons on hydrazide nitrogen atoms are lost and L acts as a dianionic bis-tridentate ligand during the coordination. The chelating ligands form dihedral angles between planes py\C(CH3) N_NC(O)at Co1of87.57and89.57°for the two independent molecules.The dihedral angles at Co2are89.23and86.15°for both ionic complexes in the structure.The separation distances between the metal ions are5.749and5.664Å,respectively.The bis(bidentate)ligand strands wrap around the cobalt centers,possessing pseudo two-fold rotation axis that passes through the metal ions.Interestingly,despite the presence of aromatic ligands in the crystal structure,the complex shows no obvious interactions between the aromatic rings.Each of the independent ionic complexes[Co2L2]2+in asymmetric unit is inherently chiral and possesses P for right-hand and M for left-hand helicity(Fig.2). Based on the fact that the complex[Co2L2](NO3)2·2H2O·0.5C2H5OH crystallized in achiral space group,the unit cell accommodates four molecules(both independent ionic complexes along with their enantiomers),thus forming a racemic solid product.3.3.NMR studiesFor further structural support the l H-NMR spectrum(Table1)of the cobalt(III)complex was compared with the l H-NMR spectra of free H2L.The NH proton of free H2L,observed at10.86ppm was absent in the spectrum of the complex,suggesting that deprotonation occurred.In the complex there are large relative shifts of all other protons,compared to ligand,indicating strong tridentate coordination to Co(III).The above statement is further substantiated by comparing the 13C-NMR spectrum of free H2L with that of the complex,indicating N, N,O coordination(Table2).The NMR spectra are in full accordance with the crystalstructure.Fig. 1.View of one of the two independent ionic complexes[Co2L2]2+found in asymmetric unit,with the atomic numbering scheme.Selected bond lengths(Å)and angles(°):Co1\N11.923(4),Co1\N21.851(4),Co1\N71.919(4),Co1\N81.851(4), Co1\O11.904(3),Co1\O31.907(3),Co2\N51.864(4),Co2\N61.923(4),Co2\N11 1.850(4),Co2\N12 1.931(4),Co2\O2 1.891(3),Co2\O4 1.905(3);N1\Co1\N2 83.11(19),N1\Co1\N792.92(16),N1\Co1\N898.39(18),N1\Co1\O1165.17(17), N1\Co1\O3165.17(17),N5\Co2\N682.35(17),N5\Co2\N11172.58(17), N5\Co2\N12103.02(17),N5\Co2\O282.50(16),N5\Co2\O492.04(16).Fig.2.Space-ﬁlling representation of the two independent ionic complexes[Co2L2]2+ found in asymmetric unit with P-and M-helicity.In both representations the two strands in each complex are differently colored(green(light color)and blue(dark color))for clarity.Hydrogen atoms are omitted for clarity.1199R.Eshkourfu et al./Journal of Inorganic Biochemistry105(2011)1196–12033.4.Biological activityBiological activity of the complex and the corresponding ligand was tested by the brine shrimp test(toxicity to A.salina).This test is in good correlation with cytotoxic activity[25].The complex showed a moderate activity,similar to that of the respective ligand(H2L)and Co (NO3)2·6H2O(Table3).3.5.Cytotoxic activityCytotoxic potential of newly synthesized complex[Co2L2] (NO3)2·2H2O·0.5C2H5OH as well as corresponding ligand(H2L)and salt was investigated on six cell lines including human epithelial breast cancer(MDA-361),human cervical carcinoma(HeLa),murine melanoma(B16),human leukemia cells(K562),human endothelial cells(EA.hy926)and human lungﬁbroblasts(MRC5).The compounds were applied in range of concentrations1–100μM for MDA-361,HeLa,K562and3–300μM for B16,EA.hy926and MRC5, and cells left for48h.The activities of the compounds were determined by means of Sulphorhodamine B and MTT tests and the results were expressed in terms of IC50values.In the investigated range of concentration,cytotoxic potential was observed for the complex on the epithelial breast cancer(MDA-361) cell line,where the IC50was50.9μM(Table4).The weak cytotoxic activity of the complex was observed with EA.hy926and B16cell line, reaching IC50values at258μM and221μM,respectively.The complex revealed low cytotoxicity against MRC5cells which are considered as normal,noncancer cell.The corresponding H2L ligand also showed moderate cytotoxic activity against all cell lines except K562and MDA-361cells(Table4). The ligand reached IC50value on most of the cell lines including HeLa, EA.hy926,MRC5with the highest potency on the murine melanoma B16cell line.The corresponding salt showed weak cytotoxic activity only on human leukemia K562cells,EA.hy926,B16cells,and the weakest activity was observed with the MRC5cell line(Table4). K562cells were resistant to both complex and ligand,the complex even stimulating cells to grow.Fig.3represents the survival curves of the cells treated with complex[Co2L2](NO3)2·2H2O·0.5C2H5OH.B16,EA.hy926and MRC5cells were treated with the complex in the range of concentration of3–300μM,whereas MDA-361,HeLa cells and K562cells were treated with the concentration of1–100μM.All cells showed dose-dependent inhibition of cell growth,except for the leukemic K562cells,where growth-stimulatory effect was observed.Since the complex showed strong cytotoxic potential against epithelial breast cancer cell line MDA-361,we continued our research towards mechanism of the observed cytotoxicity.3.6.Cell cycle and apoptosisResults of cytotoxic activity provide only the information on the restriction of the population of viable cells,but not the mechanism by which this restriction is performed.Since low IC50value on MDA-361 cell line was obtained,it was necessary to examine whether the observed cytotoxic potential is due to modulation of cell cycle progression or induction of cell death.For that purpose,inﬂuence of the complex[Co2L2](NO3)2·2H2O·0.5C2H5OH on apoptosis of the most sensitive MDA-361cell line was tested.The cells were treated with the concentration corresponding to IC50and2×IC50,and left for 24h,afterwards harvested,collected and analyzed for cell cycle perturbations.There was no signiﬁcant effect on cell cycle perturba-tions,as seen in the Fig.4.After24h of incubation of cells treated with the concentration of IC50and2×IC50,the investigated complex gave no signiﬁcant changes in the percentage of cells in the G1,S and G2/M phases.However,there is a clearly determined sub-G1peak in the histograms of treated cells,as shown in Fig.4.Those are hypodiploid cells,containing fragmentized DNA molecule,indicating that such cells are in late apoptotic phase.Therefore,the apoptotic potential of the complex[Co2L2]2+on MDA-361cell line was further examined, using double color staining with Annexin V–FITC/PI.The principle of this test lies in the exclusive characteristic of early apoptotic cells to expose phosphatidylserine(PS)molecule from the inner to the outer side of the cell membrane.The Annexin V is the Ca2+-protein dye with a strong preference towards negatively charged PS among other phospholipids.On the other hand,cells in late apoptosis and necrosis gain permeabilized cell membrane,which allows another dye used in this experimental procedure,propidium iodide,to enter the nucleus and bind DNA molecule.In this way,after analysis onﬂow cytometer, cells in early apoptotic stage can be clearly differentiated from necrotic and late apoptotic cells.The MDA-361cells were treated with the concentration of the complex corresponding to IC50and left for 24h,afterwards harvested and colored.The output histograms are shown in Fig.5.Obviously,the investigated complex induced a signiﬁcant percent of early apoptotic cells,giving28.18%comparing to 5.64%of control cells in the same phase.There was no signiﬁcant change in necrotic and late apoptotic phase cell population.Table11H NMR spectral data.Assignment(multiplicity,number of H-atoms,coupling constant,J in Hz)Chemical shift,δ(ppm)C-1_C-17(d,2H,3J=5.0)7.89C-2_C-16(t,2H,3J=6.5,6.0)7.49C-3_C-15(t,2H,3J=8.0,7.5)8.20C-4_C-14(d,2H,3J=7.5)8.13C-7_C-11(s,6H) 3.10C9(s,4H) 3.41 s—singlet;d—doublet;t—triplet.Table213C NMR spectral data.Assignment Chemical shift,δ(ppm)C-1_C-17152.16C-2_C-16128.80C-3_C-15141.86C-4_C-14126.55C-5_C-13159.81C-6_C-12162.54C-7_C-1115.05C-8_C-10183.74C934.44Table3Toxic effect(expressed as LC50values in mM)ofligand and complex on Artemia salina.Substance LC50[Co2L2]2+ 3.21H2L 3.97Co(NO3)2·6H2O 6.32Table4IC50values(μM)obtained with different cell lines.InvestigatedcompoundsCell lineEA.hy926MDA-361HeLa MRC5K562B16 [Co2L2]2+25850.9N100N300N100221 H2L124N10072.6107N10036.8 Co(NO3)2·6H2O108N100N10022262.11391200R.Eshkourfu et al./Journal of Inorganic Biochemistry105(2011)1196–1203。

U-POL环氧底漆技术数据表说明书

RAPTOR 2K EPOXY ANTI-CORROSIVE PRIMER 4:1 MIX••••••••Available Colours•Grey semi glossSubstrates••Mild Steel •E-coat••SMC• Polyester FillersBaseMixedViscosity 25 - 30 s/ DIN 4Solid content Specific gravity g/cm³1.35TECHNICAL DATA SHEETSuitable as a primer or as a primer filler for industrial refinish applications, the primer also provides excellent anti-rust protection.Excellent opacity and coverageEasy to applyChromate and Lead free Excellent anti-corrosion resistance PROPERTIESU-POL Raptor Epoxy primer is a 2K anti-corrosive VOC compliant primer with excellent salt spray resistance for application to most substrates.TECHNICAL DATAGood sandingExcellent topcoat gloss holdoutWet-on-wet process with a long open time for topcoat application 60.00%44.00%1.27surface preparation detailed belowGalvanizedAluminiumRAPTOR 2K EPOXY ANTI-CORROSIVE PRIMER 4:1 MIXTECHNICAL DATA SHEETSubstrate PreparationTo achieve best results, good preparation is essential. Degrease with U-POL Degreasers (S2000, S2001 or S2002). U-POL Epoxy Primer can be applied directly to suitably prepared rust without a requirement for chemical preparation.In all cases, loose and flaky material should be removed to ensure that the metal is returned to be a sound substrate before applying the primer.Sufficient abrasion / blasting must always to be employed to create a key to ensure adhesion of the primer.The degree of substrate preparation required depends on the severity of the prior corrosion and the exposure conditions anticipated during the coatings service life.For light corrosion / mild exposure conditions mechanical hand preparation (for example to the industry standard method SSPC-SP2 (SSI-St3)) or mechanical powertool preparation (for example to the industry standard method SSPC-SP3 (SSI-St3)) may be sufficient to prepare the surface.For heavy corrosion / severe exposure conditions, removal of rust by abrasive blasting (for example to the industry standard method SSPC-SP10 (SSI-SA2.5)) is recommended.For work which carries a warranty of performance this is the advised method, as lifetime of the coating in severe conditions is enhanced by more rigorous surface preparation. For all substrates, once abraded, clean once more with SYSTEM 20 Degreasers and dry thoroughly.Wood or plywood: dry sand with P180 -P240 to achieve a smooth surface free from loose fibresAbrade the surface as follows:For more details on surface preparation of corroded surfaces it is recommended that specialist advice is sought, eg from NACE or other similar industry body. See https:///home.aspxAluminium and Galvanized abrade with P180.U-POL Bodyfillers : dry sand with P180 - P240Please note this product is only compatible with the Epoxy Primer HardenerBare Metal (mild steel): abrade with P80.GRP, SMC, Glass fibre, Polyester Filler: dry flat with P180 P240.APPLICATION GUIDEHardener SelectionRAPTOR 2K EPOXY ANTI-CORROSIVE PRIMER 4:1 MIXTECHNICAL DATA SHEETMixing RatioStandard Application EPOXY PRIMER:Hardener25 - 30 Working Pot-Life @ 20°C2 hours4:1 by volume237.5g1080.0g 1350.0g47.5g 95.0g 142.5g 190.0g 270.0g 540.0g Viscosity DIN 4 / sec The time stated show effective life, however thematerial stays liquid for much longer but if used after this time will give unsatisfactory performanceQuantity Weight of PrimerWeight of Hardener250ml 500ml 750ml 810.0g 1000ml1250mlRAPTOR 2K EPOXY ANTI-CORROSIVE PRIMER 4:1 MIXTECHNICAL DATA SHEETCompliant HVLP Gravity GunGun tip size1.4 - 1.8mm Air Pressure at the gun1.8 -2.2 barGun tip size1.6 - 1.9mm Air Pressure at the gun3.5 -4.5 bar 2 - 3 coatsBetween coats10 mins Touch dry @ 20°C 2 hours Sandable @ 20°C 8 hours Sandable @ 60°C 30 mins Sandable @ 50°C45 minsSanding Wet Sanding by hand P500 - P600P800 - P1000Wet Sanding by machine P500 - P600P800 - P1000Dry Sanding by hand P280 - P360P400 - P500Dry Sanding by machineP280 - P360P400 - P500Wet Film Thickness approximately 140µ depending on applicationDry Film Thickness approximately 75µ depending on applicationBuild / micronsCoatsFlash-off @ 20°CDrying TimesTheoretical coverage @75µ Dry Film Thickness is approximately 5.88sq.m/ltrMethodInitial Roller ApplicationConventional spraygunFinal Apply 3 coats allowing 5 -10 minutes flash off time between coatsWet film thickness 45µ per coatDry film thickness 25µ per coatRAPTOR 2K EPOXY ANTI-CORROSIVE PRIMER 4:1 MIXTECHNICAL DATA SHEETShelf Life1 year from date of manufacture in sealed original containers.Recommended Storage Temperature5°C - 25°C Equipment CleaningImportant RemarksVOC InformationReady to use VOC g/lColourEpoxy PrimerGrey semi gloss499IMPORTANT:FOR PROFESSIONAL USE ONLY. Read full instructions before use.U-POL LtdUK SalesInternationalTechnical4th Floor, The Grange, 100 High Street T: +44 (0)20 8492 5920T: +44 (0)20 8492 5900T: +44 (0)1933 230 310London, N14 6BN F: +44 (0)20 8150 5605F: +44 (0)20 8150 5605F: +44 (0)1933 425 797**************************************************************Clean gun. Immediately after use with SYSTEM 20 thinner or gunwash.This product contains hazardous materials and therefore appropriate personal protective equipment should always be used. Please refer to the label and consult the safety data sheet for full handling instructions and personal protection information. U-POL disclaims any liability where the user does not wear the recommended personal protective equipment. The above data is for information only and may change without prior notice. It is the Buyers responsibility to ensure the suitability of the products for their own use and check the information is up to date. The recommendation of use of our products and application in our technical data sheets are based on our knowledge and experience. These data sheets are available via your local stockist or via the U-POL website at . U-POL is not responsible for the results obtained by others over whose methods we have no control and thereof U-POL is not liable for consequential or incidental damages including loss of profits.The EU limit for this product (product category: IIB.(c) in ready to use form is 540 g/litre. The VOC content of this product in ready to use form is 499 g/litre.Do not use activated product beyond pot life.Activated material should not be returned to the original can of non-activated material. After 10 minutes standing, the paint may need mild agitation to reliquify before 2nd coat.Do not apply when ambient temperature falls below 10°C or relative humidity exceeds 90%Apply only one coat of Epoxy Primer if a Polyester Filler or Polyester Spray Filler is to be applied over the Epoxy PrimerTypically the Epoxy Primer formulation is used in Industrial refinish where RAPTOR or a 2K topcoat is directly appliedThis Epoxy primer is not recommended for use with water based paint systemsAfter 60 minutes of the final coat and up to 7 days the epoxy primer can be directly overcoated with 2K polyurethanes such as U-POL RAPTOR without the need to reopen the surface by sanding. After 7 days the surface will need abrading as per the table above Wet - on - wet process @ 20°C air dryAfter 8 hours it can be overcoated with U-POL bodyfillersOver PaintingDry - on -dry process @ 20°C air dry。

布地格福与布地奈德福莫特罗治疗COPD_稳定期效果的对照研究

布地格福与布地奈德福莫特罗治疗COPD稳定期效果的对照研究贺李明（江西萍乡矿业集团有限责任公司总医院呼吸与危重症医学科，萍乡 337000）摘要目的：比较布地格福与布地奈德福莫特罗治疗慢性阻塞性肺疾病（COPD）稳定期的临床效果。

方法：收集2021年3月至2022年3月采用药物治疗的COPD稳定期患者60例，用随机数字表法分为观察组和对照组各30例。

观察组给予布地格福吸入气雾剂治疗，2揿/次，2次/d，共10 d。

对照组给予布地奈德福莫特罗吸入粉雾剂治疗，1吸/次，2次/d，共10 d。

比较两组治疗前后血气分析指标、肺功能指标、圣·乔治呼吸问题调查问卷（SGRQ）评分变化情况以及治疗期间药物相关不良反应发生情况。

结果：观察组治疗后血酸碱度（pH）、动脉血氧分压（PaO2）检测结果均高于对照组，动脉血二氧化碳分压（PaCO2）检测结果低于对照组（P＜0.05）。

观察组治疗后FEV1%、FEV1/FVC检测结果均高于对照组（P＜0.05）；症状评分、日常活动评分、心理状态评分及SGRQ总评分均低于对照组（P＜0.05）。

观察组和对照组治疗期间各有2例出现药物相关不良反应（P＞0.05）。

结论：布地格福与布地奈德福莫特罗治疗COPD 稳定期均有显著的疗效和良好的安全性，但布地格福效果更佳，可作为优选药物。

关键词慢性阻塞性肺疾病；布地格福；布地奈德福莫特罗；血气分析；肺功能中图分类号：R974 文献标志码：A 文章编号：1006-1533（2023）24-0037-04引用本文贺李明. 布地格福与布地奈德福莫特罗治疗COPD稳定期效果的对照研究[J]. 上海医药, 2023, 44(24): 37-40.A comparative study of the effect of budigfu and budesonide formoterol in thetreatment of chronic obstructive pulmonary disease in the stable stageHE Liming(Department of Respiratory and Critical Illness Medicine of General Hospital of Pingxiang Mining Group Co., Ltd, Jiangxi 337000, China) ABSTRACT Objective: To compare the clinical effects of budigfu and budesonide formoterol in the treatment of chronic obstructive pulmonary disease (COPD) in the stable phase. Methods: From March 2021 to March 2022, 60 patients with COPD in the stable stage were collected, and were divided into an observation group and a control group with 30 cases in each group with random number table method. The observation group was treated with budigfu inhalation aerosol, 2 presses/time, 2 times/day, for a total of 10 days, and the control group was treated with budesonide formoterol inhalation powder, 1 presses/time, 2 times/day, for a total of 10 days. The changes of blood gas analysis index, pulmonary function index, St. George’s respiratory questionnaire(SGRQ) score and the occurrence of drug-related adverse reactions were compared between the two groups before and after treatment.Results: After treatment, the detection results of blood pH and arterial partial pressure of oxygen(PaO2) in the observation group were higher than those in the control group, and the result of arterial partial pressure of carbon dioxide(PaCO2) was lower than that in the control group(P<0.05); the detection results of FEV1%, FEV1/FVC in the observation group were higher than those in the control group after treatment(P<0.05); the symptom score, daily activity score, mental state score and SGRQ total score in the observation group were lower than those in the control group(P<0.05). There were two incidence cases of drug-related adverse reactions in the both observation groups(P>0.05). Conclusion: Budigfu and budesonide formoterol have significant efficacy and good safety in the treatment of COPD in the stable stage, but budigfu is more effective and can be used as the preferred drug.通信作者：贺李明。

2024-2025学年福建省漳州市高一上学期期中英语试题与参考答案

2024-2025学年福建省漳州市英语高一上学期期中自测试题与参考答案一、听力第一节（本大题有5小题，每小题1.5分，共7.5分）1、What are the speakers mainly discussing?A. The importance of exercise.B. The benefits of a healthy lifestyle.C. A new fitness center in town.Answer: BExplanation: The conversation revolves around the benefits of maintaining a healthy lifestyle, with the speakers discussing the impact of diet and exercise on overall well-being.2、How does the woman describe her recent trip?A. It was a short but exciting trip.B. It was a relaxing and peaceful trip.C. It was a challenging and exhausting trip.Answer: CExplanation: The woman explicitly mentions that her recent trip was “a bit challenging” and “exhausting,” indicating that she found the trip physically demanding.3.You hear a conversation between two students discussing their weekend plans. Listen and answer the following question.Question: What does the girl suggest doing for the weekend?A. Going to the movies.B. Visiting a museum.C. Having a picnic in the park.D. Staying at home and reading.Answer: CExplanation: The girl mentions, “How about we have a picnic in the park this weekend?” which indicates her suggestion is to have a picnic.4.You listen to a weather report for a city. Listen and answer the following question.Question: What is the forecasted temperature for the city tomorrow?A. 25 degrees CelsiusB. 18 degrees CelsiusC. 30 degrees CelsiusD. 12 degrees CelsiusAnswer: BExplanation: The weather report states, “Tomorrow’s forecast is ex pected to be 18 degrees with partly cloudy skies.”5、What is the temperature forecast for the coming weekend?A)18℃ to 22℃B)20℃ to 25℃C)16℃ to 21℃D)17℃ to 24℃Answer: C) 16℃ to 21℃Explanation: The forecast for the coming weekend is given as a range of temperatures. The correct answer is option C, which indicates a forecast of 16℃ to 21℃ for the weekend. The other options do not match the given forecast.二、听力第二节（本大题有15小题，每小题1.5分，共22.5分）1、听力材料：W: Hi, John. How was your math test yesterday?M: Oh, it was okay. But I’m worried about my English midterm. I haven’t studied much for it.W: Don’t worry, John. I studied really hard and I think I did well. You can do it too!Q: What subject is John worried about?A. MathB. EnglishC. ScienceD. History答案：B解析：从对话中可以听到John提到他对英语期中考试感到担心，所以答案是B. English。

the tyndall effect thus implies

the tyndall effect thus implies“The Tyndall Effect”is a phenomenon often observed in everyday life, in which the scattering of light by suspended particles in a medium leads to the appearance of a visible beam of light. In this article, we will explore the underlying principles behind the Tyndall Effect and delve into its implications in various fields.Firstly, let us understand the basic concept of the Tyndall Effect. Named after the 19th-century physicist John Tyndall, this effect occurs when light encounters particles within a medium, causing some of the light rays to scatter in different directions. The scattered light is then reflected or refracted, creating a visible beam or cone of light. This phenomenon is most noticeable when a beam of light passes through a cloudy liquid or a dusty room, where suspended particles are abundant.To comprehend why the Tyndall Effect occurs, we must delve into the behavior of light waves. Light is composed of electromagnetic waves, which consist of alternating electric and magnetic fields. When light interacts with particles in a medium, such as smoke particles or water droplets, the electric and magnetic fields can induce a dipole moment within the particles. As a result of thisinteraction, the light waves are scattered in various directions.The intensity and color of the scattered light depend on the size of the particles and the wavelength of light. If the particles are larger than the wavelength of incident light, the scattered light will contain various colors, resulting in white light. However, if the particles are smaller than the wavelength of light, the scattering will be more pronounced for shorter wavelengths, such as blue and violet light. This explains why the scattered light appears blue, while the transmitted light through the medium appears yellow or red, as blue light is scattered more strongly in the atmosphere.Now that we have grasped the fundamental principles of the Tyndall Effect, let us explore its implications in various fields. One significant area where the Tyndall Effect is commonly observed is in atmospheric science. This phenomenon plays a crucial role in the scattering of sunlight in the Earth's atmosphere, giving rise to the blue color of the sky. As sunlight encounters tiny molecules and particles in the atmosphere, the shorter blue and violet wavelengths of light are scattered more efficiently, creating the appearance of a blue sky.Additionally, the Tyndall Effect has significant applications in the field of medical diagnostics. This effect is often exploited in technologies such as turbidimetry and nephelometry, which measure the concentration of suspended particles in a liquid sample. By analyzing the scattered light, these techniques allow healthcare professionals to identify abnormalities or monitor the progress of certain diseases, such as kidney disorders or bacterial infections.Furthermore, the Tyndall Effect has numerous applications in industrial processes. For instance, in the field of cosmetics, manufacturers use this phenomenon to create shimmering or sparkling effects in products. By incorporating finely suspended particles that scatter light, such as mica or titanium dioxide, cosmetics can enhance the perceived appearance of skin or add an iridescent quality to lipsticks or nail polishes.In conclusion, the Tyndall Effect is a fascinating phenomenon that arises from the scattering of light by suspended particles in a medium. This effect has implications in various fields, ranging from atmospheric science to medical diagnostics and industrialapplications. By understanding the underlying principles behind the Tyndall Effect, we can appreciate the beauty of everyday occurrences and harness its potential in diverse areas of research and development.。

修饰Sorption

ReviewRemoval of heavy metal ions from wastewater by chemicallymodiﬁed plant wastes as adsorbents:A reviewW.S.Wan Ngah *,M.A.K.M.HanaﬁahSchool of Chemical Sciences,Universiti Sains Malaysia,11800Penang,Malaysia Received 3April 2007;received in revised form 18June 2007;accepted 18June 2007Available online 27July 2007AbstractThe application of low-cost adsorbents obtained from plant wastes as a replacement for costly conventional methods of removing heavy metal ions from wastewater has been reviewed.It is well known that cellulosic waste materials can be obtained and employed as cheap adsorbents and their performance to remove heavy metal ions can be aﬀected upon chemical treatment.In general,chemically modiﬁed plant wastes exhibit higher adsorption capacities than unmodiﬁed forms.Numerous chemicals have been used for modiﬁcations which include mineral and organic acids,bases,oxidizing agent,organic compounds,etc.In this review,an extensive list of plant wastes as adsorbents including rice husks,spent grain,sawdust,sugarcane bagasse,fruit wastes,weeds and others has been compiled.Some of the treated adsorbents show good adsorption capacities for Cd,Cu,Pb,Zn and Ni.Ó2007Elsevier Ltd.All rights reserved.Keywords:Adsorption;Plant wastes;Low-cost adsorbents;Heavy metals;Wastewater treatment1.IntroductionHeavy metals have been excessively released into the environment due to rapid industrialization and have cre-ated a major global concern.Cadmium,zinc,copper,nickel,lead,mercury and chromium are often detected in industrial wastewaters,which originate from metal plating,mining activities,smelting,battery manufacture,tanneries,petroleum reﬁning,paint manufacture,pesticides,pigment manufacture,printing and photographic industries,etc.,(Kadirvelu et al.,2001a;Williams et al.,1998).Unlike organic wastes,heavy metals are non-biodegradable and they can be accumulated in living tissues,causing various diseases and disorders;therefore they must be removed before discharge.Research interest into the production of cheaper adsorbents to replace costly wastewater treatment methods such as chemical precipitation,ion-exchange,elec-troﬂotation,membrane separation,reverse osmosis,elec-trodialysis,solvent extraction,etc.(Namasivayam andRanganathan,1995)are attracting attention of scientists.Adsorption is one the physico-chemical treatment pro-cesses found to be eﬀective in removing heavy metals from aqueous solutions.According to Bailey et al.(1999),an adsorbent can be considered as cheap or low-cost if it is abundant in nature,requires little processing and is a by-product of waste material from waste industry.Plant wastes are inexpensive as they have no or very low eco-nomic value.Most of the adsorption studies have been focused on untreated plant wastes such as papaya wood (Saeed et al.,2005),maize leaf (Babarinde et al.,2006),teak leaf powder (King et al.,2006),lalang (Imperata cylindrica )leaf powder (Hanaﬁah et al.,2007),rubber (Hevea brasili-ensis )leaf powder (Hanaﬁah et al.,2006b,c ),Coriandrum sativum (Karunasagar et al.,2005),peanut hull pellets (Johnson et al.,2002),sago waste (Quek et al.,1998),salt-bush (Atriplex canescens )leaves (Sawalha et al.,2007a,b ),tree fern (Ho and Wang,2004;Ho et al.,2004;Ho,2003),rice husk ash and neem bark (Bhattacharya et al.,2006),grape stalk wastes (Villaescusa et al.,2004),etc.Some of the advantages of using plant wastes for wastewa-ter treatment include simple technique,requires little pro-0960-8524/$-see front matter Ó2007Elsevier Ltd.All rights reserved.doi:10.1016/j.biortech.2007.06.011*Corresponding author.Tel.:+6046533888;fax:+6046574854.E-mail address:wsaime@usm.my (W.S.Wan Ngah).Available online at Bioresource Technology 99(2008)3935–3948cessing,good adsorption capacity,selective adsorption of heavy metal ions,low cost,free availability and easy regen-eration.However,the application of untreated plant wastes as adsorbents can also bring several problems such as low adsorption capacity,high chemical oxygen demand(COD) and biological chemical demand(BOD)as well as total organic carbon(TOC)due to release of soluble organic compounds contained in the plant materials(Gaballah et al.,1997;Nakajima and Sakaguchi,1990).The increase of the COD,BOD and TOC can cause depletion of oxygen content in water and can threaten the aquatic life.There-fore,plant wastes need to be modiﬁed or treated before being applied for the decontamination of heavy metals. In this review,an extensive list of adsorbents obtained from plant wastes has been compiled and their methods of mod-iﬁcation were discussed.A comparison of adsorption eﬃ-ciency between chemically modiﬁed and unmodiﬁed adsorbents was also reported.2.Chemically modiﬁed plant wastesPretreatment of plant wastes can extract soluble organic compounds and enhance chelating eﬃciency(Gaballah et al.,1997).Pretreatment methods using diﬀerent kinds of modifying agents such as base solutions(sodium hydroxide,calcium hydroxide,sodium carbonate)mineral and organic acid solutions(hydrochloric acid,nitric acid, sulfuric acid,tartaric acid,citric acid,thioglycollic acid), organic compounds(ethylenediamine,formaldehyde,epi-chlorohydrin,methanol),oxidizing agent(hydrogen perox-ide),dye(Reactive Orange13),etc.for the purpose of removing soluble organic compounds,eliminating colour-ation of the aqueous solutions and increasing eﬃciency of metal adsorption have been performed by many research-ers(Hanaﬁah et al.,2006a;Reddy et al.,1997;Taty-Cos-todes et al.,2003;Gupta et al.,2003;Namasivayam and Kadirvelu,1997;Sˇc´iban et al.,2006a;Min et al.,2004; Kumar and Bandyopadhyay,2006;Baral et al.,2006;Acar and Eren,2006;Rehman et al.,2006;Abia et al.,2006; Shukla and Pai,2005a,Low et al.,1995;Azab and Peter-son,1989;Lazlo,1987;Wankasi et al.,2006).The types of chemicals used for modifying plant wastes and their maximum adsorption capacities are shown in Table1. 2.1.Rice husks/rice hullsRice husk consists of cellulose(32.24%),hemicellulose (21.34%),lignin(21.44%)and mineral ash(15.05%)(Rah-man et al.,1997)as well as high percentage of silica in its mineral ash,which is approximately96.34%(Rahman and Ismail,1993).Rice husk is insoluble in water,has good chemical stability,has high mechanical strength and pos-sesses a granular structure,making it a good adsorbent material for treating heavy metals from wastewater.The removal of heavy metals by rice husk has been extensively reviewed by Chuah et al.(2005).Among the heavy metal ions studied include Cd,Pb,Zn,Cu,Co,Ni and Au.Rice husk can be used to treat heavy metals in the form of either untreated or modiﬁed using diﬀerent modiﬁcation methods.Hydrochloric acid(Kumar and Bandyopadhyay, 2006),sodium hydroxide(Guo et al.,2003;Kumar and Bandyopadhyay,2006),sodium carbonate(Kumar and Bandyopadhyay,2006),epichlorohydrin(Kumar and Ban-dyopadhyay,2006),and tartaric acid(Wong et al.,2003a; Wong et al.,2003b)are commonly used in the chemical treatment of rice husk.Pretreatment of rice husks can remove lignin,hemicellulose,reduce cellulose crystallinity and increase the porosity or surface area.In general,chem-ically modiﬁed or treated rice husk exhibited higher adsorption capacities on heavy metal ions than unmodiﬁed rice husk.For example,Kumar and Bandyopadhyay (2006)reported that rice husk treated with sodium hydrox-ide,sodium carbonate and epichlorohydrin enhanced the adsorption capacity of cadmium.The base treatment using NaOH for instance appeared to remove base soluble mate-rials on the rice husk surface that might interfere with its adsorption property.Tarley et al.(2004)found that adsorption of Cd increase by almost double when rice husk was treated with NaOH.The reported adsorption capaci-ties of Cd were7and4mg gÀ1for NaOH treated and unmodiﬁed rice husk,respectively.Meanwhile,most of the acids used for treatment of plant wastes were in dilute form such as sulfuric acid, hydrochloric acid and nitric acid.According to Esteghla-lian et al.(1997),dilute acid pretreatment using sulfuric acid can achieve high reaction rates and improve cellulose hydrolysis.Concentrated acids are powerful agents for cel-lulose hydrolysis but they are toxic,corrosive and must be recovered(Sivers and Zacchi,1995).However,in some cases,hydrochloric acid treated rice husk showed lower adsorption capacity of cadmium than the untreated rice husk(Kumar and Bandyopadhyay,2006).When rice husk is treated with hydrochloric acid,adsorption sites on the surface of rice husk will be protonated,leaving the heavy metal ions in the aqueous phase rather than being adsorbed on the adsorbent surface.Wong et al.(2003a)carried out an adsorption study of copper and lead on modiﬁed rice husk by various kinds of carboxylic acids(citric acid,sali-cylic acid,tartaric acid,oxalic acid,mandelic acid,malic and nitrilotriacetic acid)and it was reported that the high-est adsorption capacity was achieved by tartaric acid mod-iﬁed rice husk.Esteriﬁed tartaric acid modiﬁed rice husk however signiﬁcantly reduced the uptake of Cu and Pb. The maximum adsorption capacities for Pb and Cu were reported as108and29mg gÀ1,respectively.Eﬀect of che-lators on the uptake of Pb by tartaric acid modiﬁed rice husk was also studied.It was reported that higher molar ratios of chelators such as nitrilotriacetic acid(NTA)and ethylenediamine tetraacetic acid(EDTA)caused signiﬁcant suppressing eﬀect on the uptake of Pb.Dyestuﬀtreated rice hulls using Procion red and Procion yellow for the removal of Cr(VI),Ni(II),Cu(II),Zn(II),Cd(II),Hg(II)and Pb(II) were studied by Suemitsu et al.(1986).More than80%of3936W.S.Wan Ngah,M.A.K.M.Hanaﬁah/Bioresource Technology99(2008)3935–3948W.S.Wan Ngah,M.A.K.M.Hanaﬁah/Bioresource Technology99(2008)3935–39483937 Table1Summary of modiﬁed plant wastes as adsorbents for the removal of heavy metal ions from aqueous solutionAdsorbent Modifying agent(s)Heavy metal Q max(mg gÀ1)SourceRice husk Water washed Cd(II)8.58Kumar and Bandyopadhyay(2006) Sodium hydroxide20.24Sodium bicarbonate16.18Epichlorohydrin11.12Rice husk Tartaric acid Cu(II)31.85Wong et al.(2003b)Pb(II)120.48Sawdust(cedrus deodar wood)Sodium hydroxide Cd(II)73.62Memon et al.(2007)Sawdust(S.robusta)Formaldehyde Cr(VI) 3.6Baral et al.(2006)Sawdust(Poplar tree)Sulfuric acid Cu(II)13.95Acar and Eren(2006)Sawdust(Dalbergia sissoo)Sodium hydroxide Ni(II)10.47Rehman et al.(2006)Sawdust(Poplar tree)Sodium hydroxide Cu(II) 6.92Sˇc´iban et al.(2006a)Zn(II)15.8Sawdust(Fir tree)Cu(II)12.7Zn(II)13.4Sawdust(Oak tree)Hydrochloric acid Cu(II) 3.60Argun et al.(2007)Ni(II) 3.37Cr(VI) 1.74Sawdust(Pinus sylvestris)Formaldehyde in Sulfuric acid Pb(II)9.78Taty-Costodes et al.(2003)Cd(II)9.29Walnut sawdust Formaldehyde in sulfuric acid Cd(II) 4.51Bulut and Tez(2003)Ni(II) 6.43Pb(II) 4.48Sawdust Reactive Orange13Cu(II)8.07Shukla and Pai(2005b)Ni(II)9.87Zn(II)17.09Peanut husk Sulfuric acid Pb(II)29.14Li et al.(2006a)Cr(III)7.67Cu(II)10.15Cr(VI)11.4Dubey and Gopal(2006) Groundnut husk Sulfuric acid followed by silverimpregnationCassava waste Thioglycollic acid Cd(II)NA Abia et al.(2006)Cassava tuber bark waste Thioglycollic acid Cd(II)26.3Horsfall Jr.et al.(2006)Cu(II)90.9Zn(II)83.3Wheat bran Sulfuric acid Cu(II)51.5O¨zer et al.(2004)Wheat bran Sulfuric acid Cd(II)101O¨zer and Pirinc¸c¸i(2006)Juniperﬁbre Sodium hydroxide Cd(II)29.54Min et al.(2004)Indian barks Hydrochloric acid Cu(II)Reddy et al.(1997)–sal51.4–mango42.6–jackfruit17.4Juteﬁbres Reactive Orange13Cu(II)8.40Shukla and Pai(2005a)Ni(II) 5.26Zn(II) 5.95Hydrogen peroxide Cu(II)7.73Ni(II) 5.57Zn(II)8.02Unmodiﬁed Cu(II) 4.23Ni(II) 3.37Zn(II) 3.55Banana pith Nitric acid Cu(II)13.46Low et al.(1995)Banana stem Formaldehyde Pb(II)91.74Noeline et al.(2005)Spent grain Hydrochloric acid Cd(II)17.3Low et al.(2000)Sodium hydroxide Pb(II)35.5(continued on next page)Cd(II),Pb(II)and Hg(II)ions were able to be removed by the two types of treated adsorbents,while Cr(VI)recorded the lowest percentage removal(<40%).2.2.Spent grainSpent grain obtained from brewery can be used to treat Pb(II)and Cd(II)ions as demonstrated by Low et al.,2000.Treatment of spent grain with NaOH greatly enhanced adsorption of Cd(II)and Pb(II)ions,whereas HCl treated spent grain showed lower adsorption than the untreated spent grain.The increase in adsorption of heavy metal ions after base treatment could be explained by the increase in the amount of galactouronic acid groups after hydrolysis of O-methyl ester groups.The best pH range for metal adsorption was4–6.Kinetic study reveals that the equilib-Table1(continued)Adsorbent Modifying agent(s)Heavy metal Q max(mg gÀ1)SourceCork powder Calcium chloride Cu(II)15.6Chubar et al.(2004)Sodium chloride19.5Sodium hydroxide18.8Sodium hypochlorite18.0Sodium iodate19.0Corncorb Nitric acid Cd(II)19.3Leyva-Ramos et al.(2005)Citric acid55.2Imperata cylindrica leaf powder Sodium hydroxide Pb(II)13.50Hanaﬁah et al.(2006a) Alfalfa biomass Sodium hydroxide Pb(II)89.2Tiemann et al.(2002)Azollaﬁliculoides (aquatic fern)Hydrogen peroxide–MagnesiumchloridePb(II)228Ganji et al.(2005)Cd(II)86Cu(II)62Zn(II)48Carrot residues Hydrochloric acid Cr(III)45.09Nasernejad et al.(2005)Cu(II)32.74Zn(II)29.61Sugarcane bagasse Sodium bicarbonate Cu(II)114Junior et al.(2006)Pb(II)196Cd(II)189Ethylenediamine Cu(II)139Pb(II)164Cd(II)189Triethylenetetramine Cu(II)133Pb(II)313Cd(II)313Sugarbeet pulp Hydrochloric acid Cu(II)0.15Pehlivan et al.(2006)Zn(II)0.18Bagasseﬂy ash Hydrogen peroxide Pb(II) 2.50Gupta and Ali(2004)Cr(III) 4.35Nipah palm shoot biomass Mercaptoacetic acid Pb(II)52.86Wankasi et al.(2006)Cu(II)66.71Groundnut shells Reactive Orange13Cu(II)7.60Shukla and Pai(2005b)Ni(II)7.49Zn(II)9.57Terminalia arjuna nuts ZnCl2Cr(VI)28.43Mohanty et al.(2005)Coirpith ZnCl2Cr(VI)NA Namasivayam and Sangeetha(2006)Ni(II)Hg(II)Cd(II)Kula et al.(2007)Sulfuric acid and ammonium persulphate Hg(II)154Namasivayam and Kadirvelu(1999) Cu(II)39.7Namasivayam and Kadirvelu(1997) Hg(II)NA Kadirvelu et al.(2001a)Pb(II)Cd(II)Ni(II)Cu(II)Ni(II)62.5Kadirvelu et al.(2001b)NA–not available.3938W.S.Wan Ngah,M.A.K.M.Hanaﬁah/Bioresource Technology99(2008)3935–3948rium time of adsorption was120min for both metal ions and adsorption followed pseudo-second-order model.The maximum adsorption capacity for lead was two times higher than cadmium.The eﬀect of organic ligands (EDTA,nitrilotriacetic acid and salicylic acid)on adsorp-tion eﬃciency was assessed and adsorption was greatly reduced by EDTA and nitrilotriacetic acid at molar ratio of1:1(metal:ligand).EDTA and nitrilotriacetic acid could chelate the heavy metal ions,therefore more metal ions would remain in the solutions rather than being adsorbed (Jeon and Park,2005).Salicylic acid on the other hand slightly reduced the percentage of cadmium adsorption but did not aﬀect adsorption of lead.2.3.Sugarcane bagasse/ﬂy ashJunior et al.(2006)reported the use of succinic anhy-dride modiﬁed sugarcane bagasse for treatment of Cu, Cd and Pb from aqueous solutions.Sugarcane bagasse consists of cellulose(50%),polyoses(27%)and lignin (23%).The presence of these three biological polymers causes sugarcane bagasse rich in hydroxyl and phenolic groups and these groups can be modiﬁed chemically to pro-duce adsorbent materials with new properties.The authors reported that the hydroxyl groups in sugarcane bagasse could be converted to carboxylic groups by using succinic anhydride.The carboxylic groups were later reacted with three diﬀerent chemicals mainly NaHCO3,ethylenediamine and triethylenetetramine to produce new properties of adsorbent materials which showed diﬀerent adsorption capacities for metal ions.It was found that sugarcane bagasse treated with ethylenediamine and triethylenetetra-mine shows a remarkable increase in nitrogen content com-pared to untreated sample,and triethylenetetramine modiﬁed sugarcane bagasse has a higher increasing extent. The presence of amide group was also detected in ethylene-diamine and triethylenetetramine modiﬁed sugarcane bag-asses as a result of the reaction between–COOH and–NH2groups.Kinetic studies showed that equilibrium time for adsorption of Cu,Cd and Pb onto tethylenediamine and triethylenetetramine modiﬁed sugarcane bagasses were slower than that for adsorbent modiﬁed with NaHCO3. Triethylenetetramine modiﬁed sugarcane bagasse was the best adsorbent material for removal of Cd and Pb since the adsorption capacities for both metals are two times higher than unmodiﬁed sugarcane bagasse.This was prob-ably caused by the higher number of nucleophilic sites introduced in triethylenetetramine modiﬁed sugarcane bagasse.When sugarcane bagasse was modiﬁed with meth-anol,however,the resulting adsorbent did not show a good uptake of cadmium as the maximum adsorption capacity was6.79mg gÀ1(Ibrahim et al.,2006).The performance of hydrogen peroxide treated bagasse ﬂy ash,a solid waste of sugar industry for removal of lead and chromium was explored by Gupta and Ali(2004). Hydrogen peroxide is a good oxidizing agent and used to remove the adhering organic matter on the adsorbent.It was found that hydrogen peroxide treated bagasseﬂy ash was able to remove chromium in a shorter period of time (60min)compared to lead(80min).The isotherm study also revealed maximum adsorption capacity for chromium was higher than lead.However,the recorded values of maximum adsorption capacities for both metals were low (2.50and4.35mg gÀ1for Pb and Cr,respectively).The detail mechanism of adsorption by the treated bagasseﬂy ash was not discussed,but it was thought that adsorption was controlled byﬁlm diﬀusion at lower metal concentra-tion and particle diﬀusion at higher concentration of metal ions.2.4.SawdustSawdust,obtained from wood industry is an abundant by-product which is easily available in the countryside at negligible price.It contains various organic compounds (lignin,cellulose and hemicellulose)with polyphenolic groups that could bind heavy metal ions through diﬀerent mechanisms.An experiment on the eﬃciency of sawdust in the removal of Cu2+and Zn2+ions was conducted by Sˇc´i-ban et al.(2006a).Two kinds of sawdust,poplar andﬁr wood were treated with NaOH(ﬁbre-swelling agent)and Na2CO3solutions and the adsorption capacities were com-pared with the untreated sawdusts.For unmodiﬁed saw-dust,both types of woods showed higher uptakes of Cu2+ions than Zn2+ions,and adsorption followed Lang-muir isotherm model.Equivalent amounts of adsorption capacities were recorded by both types of sawdust for Zn2+and Cu2+ions,although these two adsorbents have diﬀerent anatomical structure and chemical composition. After treating with NaOH,a marked increase in adsorption capacity was observed for both heavy metal ions,especially for Zn2+ions(2.5times for Cu2+and15times for Zn2+). The adsorption capacities shown by Langmuir model were 6.92mg gÀ1(poplar sawdust)and12.70mg gÀ1(ﬁr saw-dust)for Cu2+,and15.83mg gÀ1(poplar sawdust)and 13.41mg gÀ1for Zn2+(ﬁr sawdust),respectively.In another experiment,Sˇc´iban et al.(2006b)found that the leaching of coloured organic matters during the adsorption can be eliminated by pretreatments with formaldehyde in acidic medium,with sodium hydroxide solution after form-aldehyde treatment,or with sodium hydroxide only. According to Sˇc´iban et al.(2006a),NaOH improved the adsorption process by causing the liberation of new adsorption sites on the sawdust surface.An increase in the concentration of NaOH for modiﬁcation purpose how-ever did not cause a signiﬁcant increase of the adsorption capacity.The authors suggest that no greater than1%of concentration of NaOH solution should be used for mod-iﬁcation.The temperature of modiﬁcation was also not a signiﬁcant factor for the main increase of adsorption capacities of modiﬁed sawdusts.It was observed that only a slight increase in Cu2+and Zn2+adsorption occurred when theﬁr sawdust was treated with NaOH at higher tem-perature(80°C).The study on adsorption capacity byW.S.Wan Ngah,M.A.K.M.Hanaﬁah/Bioresource Technology99(2008)3935–39483939treatment with Na2CO3revealed the modiﬁed sawdusts had two times higher adsorption for Cu2+ions and six times higher for Zn2+ions compared to unmodiﬁed saw-dusts.The application of Na2CO3for chemical modiﬁca-tion is less eﬃcient than the use of NaOH.This is due to higher number of Na+ions in1g of NaOH compared to 1g of Na2CO3.In general,three possible reasons for the increase in adsorption capacities of heavy metal ions were given by the authors:(i)Changes on wood surface-increase in surface area,average pore volume and pore diameter after alkaline treatment.The surface area and average pore diame-ter increased about1.5–2times after modiﬁcation. (ii)Improvement in ion-exchange process especially with Na+ions.(iii)Microprecipitation of metal hydroxides–Cu(OH)2 and Zn(OH)2in the pores of sawdust.Although the work on adsorption of copper and zinc ions onto sawdust of poplar tree was reported by Sˇc´iban et al.(2006a),they did not carry out a detail experiment on the kinetic of adsorption.The eﬀect of sulfuric acid treatment on sawdust of pop-lar tree was studied by Acar and Eren(2006).Sulfuric acid poplar sawdust possessed good removal of92.4%Cu2+at pH5,while untreated sawdust could only removed47%. The kinetic of copper binding indicated that it is a rapid process and about70–80%of copper ions removed from the solution in10min.The percent of copper removal how-ever decreases as the metal concentration increases.The increase in percent of adsorption with adsorbent dose could be due to the increase in surface area and availability of more active sites.The treated poplar sawdust showed maximum adsorption capacity of13.945mg gÀ1against 5.432mg gÀ1for untreated sawdust which followed Lang-muir isotherm model.The maximum adsorption capacity for sulfuric acid treated poplar sawdust is higher than to the value recorded by NaOH treated poplar sawdust reported by Sˇc´iban et al.(2006a).Concentrated sulfuric acid was also used to modify coconut tree sawdust for removing mercury and nickel(Kadirvelu et al.,2003).It was reported that100%removal of mercury was achieved compared to81%for nickel and adsorption occurred in 1h.Rehman et al.(2006)reported the removal of Ni2+ions by using sodium hydroxide treated sawdust of Dalbergia sissoo,a byproduct of sawmills.The treatment of sawdust with NaOH results in the conversion of methyl esters which are the major constituents in cellulose,hemicellulose and lignin to carboxylate ligands.The adsorption time study revealed that nickel ions were removed fast in theﬁrst 20min due to extra-cellular binding.The maximum adsorp-tion capacity of Ni2+ions was found to be10.47mg gÀ1at 50°C.Adsorption was more favourable at higher tempera-ture and adsorption followed both Langmuir and Freund-lich isotherm models.A comparative study on the adsorption eﬃciency of untreated and NaOH treated sawdust of cedrus deodar wood was conducted by Memon et al.(2007).They reported that cedrus deodar sawdust mainly consists of acid detergentﬁbre(cellulose and lignin),hydroxyl groups (tannins)and phenolic compounds.The acidimetric–alkali-metric titration study revealed that sawdust has four major groups responsible for cadmium binding which were car-boxylic,phosphoric,amines and phenolic.Cadmium removal was more favoured by NaOH treated sawdust as the value of adsorption capacity was four times greater than untreated sawdust.Maximum removal of cadmium occurred at pH above4for both types of adsorbents.When the pH of the solution is greater than4,carboxylic groups will be deprotonated and the adsorbent surface will be neg-atively charged resulting in higher adsorption of cadmium. However;at pH less than3,carboxylic groups become pro-tonated and adsorption sites are unable to attract Cd2+ ions.NaOH treated sawdust also shows good settling prop-erty,making it easy toﬁlter or separate the adsorbent from the solution.Ion-exchange was considered as the predom-inant mechanism of cadmium adsorption as the values of adsorption energy(E)determined from Dubinin–Radushk-evic plots are in the range of9–16kJ molÀ1.Maximum adsorption capacity recorded at temperature of20°C was73.62mg gÀ1.A detail analysis on the ideal concentration of NaOH for modifying juniperﬁbre for adsorption of cadmium ions was carried out by Min et al.(2004).Sodium hydroxide treatment of lignocellulosic materials can cause swelling which leads to an increase in internal surface area,a decrease in the degree of polymerization,a decrease in crys-tallinity,separation of structural linkages between lignin and carbohydrates and disruption of the lignin structure. Sodium hydroxide is a good reagent for saponiﬁcation or the conversion of an ester group to carboxylate and alco-hol,as shown in the equation below:RCOOR0þH2O!OHÀRCOOÀþR0OHð1ÞBased on the FTIR analysis,it was found that as the con-centration of NaOH increases(from0to 1.0M),the amount of carboxylate was also increased.A maximum concentration of0.5M of NaOH was suitable to carry out saponiﬁcation process.After base treatment,the max-imum adsorption capacity of cadmium increased by about three times(from9.18to29.54mg gÀ1)compared to un-treated juniperﬁbre despite a decrease in speciﬁc surface area for the treated adsorbent.Data obtained from pseu-do-second-order kinetic study also revealed that base trea-ted juniperﬁbre had higher values of adsorption capacity, q e(mg gÀ1)and initial adsorption rate constant,h (mg gÀ1minÀ1)when compared to untreated adsorbent.Hexavalent chromium adsorption by formaldehyde treated sawdust was studied by Baral et al.,2006.Formal-dehyde is a common compound used to immobilize colour and water soluble compounds from sawdust(Garg et al.,3940W.S.Wan Ngah,M.A.K.M.Hanaﬁah/Bioresource Technology99(2008)3935–39482004).The adsorption capacity of Cr(VI)determined from Langmuir isotherm was low(3.60mg gÀ1)and equilibrium adsorption time took about5h.Adsorption process was strongly aﬀected by several physico-chemical parameters such as pH,adsorbent dose,temperature and initial con-centration of chromium solution.Maximum adsorption occurred at pH range3–6and reduced signiﬁcantly beyond pH6.The percentage adsorption of Cr(VI)increased with increase in adsorbent dose,but decreased with increase in metal concentration and temperature.Adsorption rate tends to increase with increase in adsorbent dose due to higher number of available adsorption sites.As concentra-tion of Cr(VI)increases withﬁxed amount of adsorbent dose,more Cr(VI)ions will remain in the aqueous phase, thus percentage adsorption will be small.The decrease in adsorption rate with increase in temperature indicates exo-thermic nature of adsorption,in which adsorption is more favourable at lower temperatures.According to Taty-Costodes et al.(2003),treatment with formaldehyde induces a stabilization of the hydrosol-uble compounds of adsorbent by creating covalent bonds on the constitutive units.This could eliminate the problem associated with the release of polyphenolic compounds which could cause an increase in COD in wastewater.A research on the adsorption of Pb(II)and Cd(II)onto form-aldehyde treated sawdust of Pinus sylvestris shows that the two metal ions were successfully removed in less than 20min at low concentrations(<10mg lÀ1).It was reported that metal ions could form complexes with the oxygen atom on carbonyl and hydroxyl groups(acting as a Lewis base).The maximum adsorption capacities of Pb(II)and Cd(II)were9.78and9.29mg gÀ1,respectively.Adsorption kinetic indicates that pseudo-second-order model was bet-terﬁtted than pseudo-ﬁrst-order and intraparticle diﬀusion is one of the rate determining steps.Nickel,cadmium and lead adsorption by walnut sawdust treated with formalde-hyde in sulfuric acid was studied and it was found that adsorption is dependent on contact time,metal concentra-tion and temperature(Bulut and Tez,2003).Equilibrium time was established in about60min for all heavy metals. The kinetic study reveals that adsorption followed pseudo-second-order model better than pseudo-ﬁrst-order.The maximum adsorption capacities were 6.43, 4.51and 4.48mg gÀ1for Ni,Cd and Pb,respectively.Based on tem-perature study,adsorption was favaourable at higher tem-peratures as the values of D G°become more negative.Chubar et al.(2004)studied the performance of various kinds of chemically treated cork powder obtained from cork oak tree for the removal of Cu,Zn and Ni.Treatment of cork powder with salts such as NaCl and CaCl2causes the conversion of active binding sites from the H+form to Na+and Ca2+form.The salt modiﬁed cork powder shows greater adsorption capacity than the unmodiﬁed cork especially at higher heavy metal concentrations.It was also noted that the Na+form cork recorded a higher adsorption capacity value than the Ca2+form.This obser-vation can be explained in terms of the diﬀerent charge of cations whereby the interaction of cork powder binding sites with divalent calcium ions is stronger than the mono-valent sodium ions.Hence,the biosorption reaction of cop-per will be hindered.Treatment of cork powder with an alkaline solution(NaOH)at high temperature increased the sorption capacity toward heavy metals by about33%.A high concentration of NaOH however causes a decrease in copper removal due to the destruction of the biomass. Besides NaCl,CaCl2and NaOH,modiﬁcation of cork powder could be carried out using commercial laundry detergent and the amount of copper removed was found to increase,probably due to the exposure of new binding sites.The use of NaClO and NaIO3will increase the num-ber of active binding sites by oxidation of the some of func-tional groups of cork to carboxylic groups,hence more copper ions could be sorbed.An increase of70–80%in cork capacity for copper was achieved after treating cork powder with NaClO containing7%of active chlorine. 2.5.Wheat branWheat bran,a by-product of wheat milling industries proved to be a good adsorbent for removal of many types of heavy metal ions such as Pb(II),Cu(II)and Cd(II).The application of a strong dehydrating agent like sulfuric acid (H2SO4)can have a signiﬁcant eﬀect on the surface area of the adsorbent,which eventually results in better eﬃciency of adsorption of copper ions as reported by O¨zer et al. (2004).It was found that upon treatment with sulfuric acid, wheat bran had a much higher surface area.The authors suggested that acid treatment caused changes in surface area by increasing the conversion of macropores to microp-ores.Maximum adsorption capacity for Cu(II)ions was reported as51.5mg gÀ1(at pH5)and equilibrium time of adsorption was achieved in30min.O¨zer and Pirinc¸c¸i (2006)conducted a study on the removal of lead ions by sulfuric acid treated wheat bran.It was reported that max-imum lead removal(82.8%)occurred at pH6after2h of contact time.Three isotherm models were analyzed for determining the maximum adsorption capacity of wheat bran particularly Langmuir,Freundlich and Redlich-Peter-son.Based on the non-linear plots,it was found that adsorptionﬁtted well to the Redlich-Peterson than Lang-muir and Freundlich models.The Langmuir plots indicate that maximum adsorption capacities increased with an increase in temperature(79.37mg gÀ1at60°C and 55.56mg gÀ1at25°C).The decrease in the values of D G°suggests that adsorption was more favourable at higher temperatures and adsorption was endothermic in nature. The kinetic study showed that lead adsorption could be described well with n th-order kinetic model.O¨zer(2006) also examined the sulfuric acid treated wheat bran for cad-mium ion removal from aqueous solution.After4h of con-tact time,the maximum adsorption capacity that could be achieved for cadmium was101mg gÀ1at pH5.Therefore, in general the order of maximum removal of the above three metals follows:Cd(II)>Pb(II)>Cu(II).W.S.Wan Ngah,M.A.K.M.Hanaﬁah/Bioresource Technology99(2008)3935–39483941。

描述电影英语作文

When it comes to describing a movie in an English essay,there are several key elements to consider.Heres a stepbystep guide to help you craft a wellstructured and engaging movie description:1.Introduction:Begin your essay with a brief introduction to the movie.Mention the title, director,main actors,and the year of release.You might also want to include a general statement about the movies genre and its appeal to audiences.Example:Directed by Christopher Nolan and released in2010,Inception is a science fiction film that stars Leonardo DiCaprio alongside an ensemble cast.The movie has captivated audiences worldwide with its unique blend of action,drama,and mindbending concepts.2.Plot Summary:Provide a concise summary of the movies plot without giving away any major spoilers.Focus on the main storyline and avoid going into too much detail about subplots or minor characters.Example:The story revolves around Dom Cobb,a skilled thief who specializes in the art of extraction,stealing secrets from peoples subconscious while they dream.Cobb is offered a chance at redemption by performing inception,planting an idea in someones mind,which is considered to be nearly impossible.3.Character Analysis:Discuss the main characters and their roles in the movie.Describe their personalities,motivations,and how they contribute to the story.Example:Leonardo DiCaprios portrayal of Dom Cobb is both complex and compelling. His character is haunted by his past and driven by the desire to return to his family,which adds depth to his performance and makes the audience empathize with him.4.Cinematography and Visual Effects:Comment on the movies visual aspects,including the cinematography,special effects,and overall aesthetic.Discuss how these elements contribute to the storytelling and the audiences experience.Example:The cinematography in Inception is stunning,with each dream layer having its own distinct visual style.The visual effects are groundbreaking,especially the scenes where the cityscape folds in on itself,creating a surreal and unforgettable cinematic experience.5.Soundtrack and Score:Mention the movies soundtrack and score,and how they enhance the mood and atmosphere of the film.Example:Hans Zimmers score for Inception is powerful and evocative,perfectly complementing the films intense and emotional moments.The music adds another layer of depth to the narrative,making the audience feel as if they are part of the dream world.6.Themes and Messages:Discuss the themes and messages conveyed in the movie. Analyze how these themes are explored through the story,characters,and visual elements.Example:One of the central themes in Inception is the power of the subconscious mind and the impact of our memories on our reality.The movie raises questions about the nature of reality and the consequences of manipulating it,prompting viewers to reflect on their own perceptions and experiences.7.Conclusion:End your essay with a conclusion that summarizes your analysis and provides your personal opinion on the movie.You might also want to mention its impact on the film industry or its cultural significance.Example:In conclusion,Inception is a cinematic masterpiece that challenges the boundaries of storytelling and filmmaking.Its complex narrative,captivating performances,and innovative visual effects have left a lasting impression on audiences and the film industry alike.It is a thoughtprovoking film that invites viewers to question the nature of reality and the power of the human mind.Remember to maintain a formal tone throughout your essay and use proper grammar and punctuation.By following these steps,you can create a comprehensive and engaging description of a movie in your English essay.。

广东省茂名市英语中考试卷及答案指导(2025年)

2025年广东省茂名市英语中考自测试卷及答案指导一、听力部分（本大题有20小题，每小题1分，共20分）1、Listen to the following dialogue and choose the best answer to the question you hear.A. The man is buying a book at a bookstore.B. The woman is trying to find a book for her friend.C. The man is helping the woman find a book.Answer: BExplanation: The dialogue begins with the woman saying, “I need to find a book for my friend’s birthday. Do you know where I can find it?” This indicates that she is looking for a book, making option B the correct answer.2、Listen to the following short passage and answer the question.What is the main topic of the passage?A. The benefits of reading books.B. The history of the library.C. The importance of technology in education.Answer: CExplanatio n: The passage mentions that “in today’s world, technology plays a crucial role in education.” It goes on to discuss how technology has changed the way students learn and access information. Therefore, option C, “Theimportance of technology in education,” is the main topic of the passage.3、Listen to the conversation and choose the best answer to the question you hear. (Conversation will be played twice.)Question: What does the man suggest the woman should do with her old laptop?A. Sell it online.B. Give it to her brother.C. Donate it to charity.Correct Answer: A. Sell it online.Explanation: In the conversation, the man suggests that the woman could sell her old laptop on an online marketplace because it’s still in good condition and someone else might find it useful.4、Listen to the dialogue and determine what the relationship between the speakers is. (Dialogue will be played twice.)Question: What is the relationship between the speakers?A. Teacher and student.B. Boss and employee.C. Doctor and patient.Correct Answer: B. Boss and employee.Explanation: The dialogue indicates that one person is giving instructions about completing a project by the end of the week and discussing performance reviews, which suggests a professional relationship where one individual has authority over the other, such as a boss and employee scenario.5.Listen to the conversation and choose the best answer for each question.A. Man: How was your trip to New York last week?Woman: It was amazing! I visited the Statue of Liberty and Central Park.Question: What did the woman visit in New York?A) The White HouseB) The Statue of Liberty and Central ParkC) Times SquareD) The Empire State BuildingAnswer: B) The Statue of Liberty and Central ParkExplanation: The woman mentions that she visited the Statue of Liberty and Central Park, which are the correct answers.6.Listen to the following dialogue and answer the question.Man: Hi, Sarah. Are you ready for the school sports day?Woman: Not quite. I’m still trying to decide what sport to participate in. Do you have any suggestions?Question: What is the woman trying to decide?A) What to wearB) What to eat for lunchC) What sport to participate inD) What to bring to the school sports dayAnswer: C) What sport to participate inExplanation: The woman explicitly states that she is trying to decide whatsport to participate in, which is the correct answer.7、What does the speaker say about the weather forecast for tomorrow?A)It’s going to be sunny all day.B)There will be light rain in the morning.C)The weather will be partly cloudy with a chance of showers.Answer: CExplanation: In the recording, the speaker mentions that while there will be some sunshine, clouds will roll in bringing a possibility of showers throughout the day.8、Where did the man say he lost his keys?A)In the parkB)At the supermarketC)On the busAnswer: BExplanation: The man clearly states in the dialogue that he thinks he must have dropped his keys while shopping at the supermarket because that was the last place he remembered having them in his hand.9.You will hear a short conversation between two friends. Listen and choose the best answer to the question.A)They are discussing a movie they saw.B)They are planning a vacation.C)They are comparing their book collections.Answer: BExplanation: The conversation indicates that they are talking about their upcoming trip, which suggests they are planning a vacation. The mention of “packings” and “flight” supports this choice.10.You will hear a monologue about the importance of exercise. Listen and answer the question.What does the speaker say is one of the benefits of regular exercise?A)Improved memoryB)Increased risk of injuryC)Decreased energy levelsAnswer: AExplanation: The speaker in the monologue explicitly mentions that regular exercise can improve memory, which is a recognized benefit of physical activity. The other options, increased risk of injury and decreased energy levels, are not mentioned as benefits.11.You will hear a conversation between two friends, discussing their weekend plans. Listen carefully and choose the best answer to the question that follows.Question: What does the woman plan to do on Saturday afternoon?A. Go shopping with her friends.B. Attend a concert.C. Visit her grandparents.D. Stay at home and watch movies.Answer: CExplanation: In the conversation, the woman says, “I’m thinking ofvisiting my grandparents this weekend.” This indicates that her plan for Saturday afternoon is to visit her grandparents.12.You will hear a news report about a recent environmental event. Listen carefully and choose the best answer to the question that follows.Question: What is the main cause of the environmental problem mentioned in the report?A. Overfishing in the ocean.B. Deforestation in the Amazon.C. Air pollution in a major city.D. Water pollution in a river.Answer: DExplanation: The news report states, “A recent study shows that water pollution in the river has reached alarming levels due to industrial waste and agricultural runoff.” This indicates that the main cause of the environmental problem is water pollution in the river.13.You will hear a conversation between two friends discussing their weekend plans. Listen carefully and answer the question.Question: What does the woman plan to do on Saturday morning?A. Go shopping.B. Visit a friend.C. Attend a sports event.Answer: B. Visit a friend.Explanation: In the conversation, the woman mentions that she is planning to visit a friend on Saturday morning, which is why the correct answer is B.14.You will hear a news report about a recent discovery in the field of science. Listen carefully and answer the question.Question: According to the report, what is the significance of the discovery?A. It could lead to a new medical treatment.B. It will improve energy efficiency.C. It has no immediate practical application.Answer: A. It could lead to a new medical treatment.Explanation: The news report highlights the potential medical benefits of the discovery, suggesting that it could lead to a new treatment. Therefore, the correct answer is A.15.You hear a conversation between two students discussing their weekend plans.Student A: “Are you going to the beach this weekend?”Student B: “No, I think I’ll stay in and catch up on some reading.”Question: What does Student B plan to do this weekend?A) Go to the beachB) Stay in and readC) Go hikingD) Visit a friendAnswer: B) Stay in and readExplanation: In the conversation, Student B explicitly states that they will stay in and catch up on some reading, which means they plan to stay home and read books.16.You hear a news report about a new initiative in the city.News A nchor: “The city council has just announced a new recycling program aimed at reducing waste. According to the mayor, the program will involve the installation of more recycling bins in public areas and an educational campaign to encourage residents to recy cle more effectively.”Question: What is the main purpose of the new recycling program?A) To increase tourismB) To reduce wasteC) To improve city infrastructureD) To promote local businessesAnswer: B) To reduce wasteExplanation: The news report clearly states that the purpose of the new recycling program is to reduce waste, as mentioned by the mayor. The other options are not mentioned in the context of the report.17.You will hear a conversation between two students, Alex and Sarah, discussing their weekend plans. Listen carefully and answer the following question.Question: What activity does Sarah plan to do on Saturday morning?A. Go to the gymB. Visit her grandparentsC. Attend a music concertAnswer: B. Visit her grandparentsExplanation: In the conversation, Sarah mentions that she is planning to visit her grandparents on Saturday morning, so the correct answer is B.18.You will hear a short interview with a chef, discussing his favorite dish to cook. Listen carefully and answer the following question.Question: What is the chef’s favorite dish to prepare?A. PizzaB. SushiC. Italian pastaAnswer: C. Italian pastaExplanation: The chef explicitly states in the interview that Italian pasta is his favorite dish to prepare, so the correct answer is C.19.You will hear a conversation between two friends discussing their weekend plans. Listen and answer the following question.What does the woman suggest doing for the weekend?A. Going to the beach.B. Attending a music festival.C. Staying in and watching movies.Answer: AExplanation: The woman suggests going to the beach because she mentions, “I was thinking of going t o the beach this weekend. How about you?”20.Listen to a short lecture about the benefits of exercise. Identify the main point the speaker makes.What is the primary benefit of regular exercise according to the speaker?A. Improved sleep quality.B. Increased energy levels.C. Weight loss.Answer: BExplanation: The speaker emphasizes the increase in energy levels as a result of regular exercise, stating, “One of the most noticeable benefits of regular exercise is the boost it gives to your energy levels.”二、阅读理解（30分）Reading ComprehensionPassage:The advancements in technology have revolutionized the way we live and work. One such innovation is the rise of artificial intelligence (AI). AI has become an integral part of various industries, from healthcare to transportation. This article explores the impact of AI on different sectors and its potential future developments.Artificial intelligence refers to the simulation of human intelligence inmachines that are programmed to think like humans and mimic their actions. AI systems are designed to perform tasks that typically require human intelligence, such as visual perception, speech recognition, decision-making, and language translation.The healthcare industry has seen significant benefits from AI applications. For instance, AI algorithms can analyze medical images, such as X-rays and MRI scans, to detect early signs of diseases like cancer. This early detection can lead to timely interventions, improving patient outcomes. Additionally,AI-powered chatbots can provide 24/7 patient support and advice, reducing the workload on healthcare professionals.In the transportation sector, AI is transforming the way we travel. Autonomous vehicles, powered by AI, are being developed to reduce accidents and traffic congestion. These vehicles use sensors and algorithms to navigate roads safely, making driving a more efficient and less stressful experience. Furthermore, AI can optimize traffic flow by analyzing data from traffic cameras and GPS devices, leading to better road management.The potential of AI is not limited to these sectors. Education is another area where AI is making an impact. AI-powered tutoring systems can adapt to individual learning styles, providing personalized educational experiences. This can help students learn more effectively and improve their academic performance.Despite its numerous benefits, AI also raises ethical and societal concerns.Issues such as job displacement, data privacy, and decision-making transparency are at the forefront of discussions. As AI continues to evolve, it is crucial for policymakers, industry leaders, and the general public to address these challenges and ensure that AI is used responsibly.Questions:1.What is the primary purpose of AI systems according to the passage?A) To replace human intelligence completely.B) To mimic human intelligence and perform tasks requiring human intelligence.C) To only perform tasks that humans find enjoyable.D) To operate independently of human input.2.How is AI expected to impact the healthcare industry?A) By increasing the number of healthcare professionals.B) By reducing the need for medical imaging.C) By improving early detection of diseases and patient support.D) By eliminating the need for hospitals and clinics.3.What are some of the ethical and societal concerns mentioned in the passage related to AI?A) Increased traffic congestion and job displacement.B) Privacy issues and the potential for bias in decision-making.C) The decline in the number of students attending schools.D) The rise of illegal activities due to increased AI capabilities.Answers:1.B) To mimic human intelligence and perform tasks requiring human intelligence.2.C) By improving early detection of diseases and patient support.3.B) Privacy issues and the potential for bias in decision-making.三、完型填空（15分）Passage:The Internet has become an integral part of our daily lives, impacting the way we communicate, work, and even entertain ourselves. One of the most significant changes brought about by the Internet is the ease with which we can access information. However, this convenience comes with its own set of challenges. Below are some statements about the impact of the Internet. Choose the best option for each blank from the given choices.1.The Internet has revolutionized the way we access information, making it ( ) than ever before.a) expensiveb) complicatedc) quickerd) limited2.While the Internet offers a wealth of information, it also poses a significant risk of ( ) information.a) misrepresentingb) misplacingc) losingd) misusing3.Many argue that the Internet has made communication ( ) due to the various social media platforms.a) slowerb) more efficientc) less personald) less frequent4.Despite its many benefits, some people believe that the Internet has led to a ( ) of attention spans.a) declineb) extensionc) changed) shift5.To navigate the vast amount of information available on the Internet, it is crucial to develop strong ( ) skills.a) cookingb) mathematicalc) researchd) cookingAnswers:1.c) quicker2.d) misusing3.c) less personal4.a) decline5.c) research四、语法填空题（本大题有10小题，每小题1分，共10分）1、The teacher asked the students to submit their homework_______(by) the end of the week.答案：by解析：本题考查介词的用法。

Fluorescent biosensors of intracellular targets from genetically encoded reporters

REVIEW ARTICLEFluorescent Biosensors of Intracellular Targets from Genetically Encoded Reporters to Modular Polypeptide ProbesMay C.MorrisPublished online:17November 2009ÓHumana Press Inc.2009Abstract With the escalation of drug discovery pro-grammes,it has become essential to visualize and monitor biological activities in healthy and pathological cells,with high spatial and temporal resolution.To this aim,the development of probes and sensors,which can report on the levels and activities of speciﬁc intracellular targets,has become essential.Together with the discovery of the Green Fluorescent Protein (GFP),and the development of GFP-based reporters,recent advances in the synthesis of small molecule ﬂuorescent probes,and the explosion of ﬂuores-cence-based imaging technologies,the biosensor ﬁeld has witnessed a dramatic expansion of ﬂuorescence-based reporters which can be applied to complex biological samples,living cells and tissues to probe protein/protein interactions,conformational changes and posttranslational modiﬁcations.Here,we review recent developments in the ﬁeld of ﬂuorescent biosensor technology.We describe different varieties and categories of ﬂuorescent biosensors together with an overview of the technologies commonly employed to image biosensors in cellulo and in vivo.We discuss issues and strategies related to the choice of syn-thetic ﬂuorescent probes,labelling,quenching,caging and intracellular delivery of biosensors.Finally,we provide examples of some well-characterized genetically encoded FRET reporter systems,peptide and protein biosensors and describe biosensor applications in a wide variety of ﬁelds.Keywords Biosensor ÁFluorescence ÁImaging ÁPeptide ÁEnzyme ÁCell cycleAbbreviations CDK Cyclin-dependent kinase CPP Cell-penetrating peptide CRS Cytoplasmic retention sequence FACS Fluorescence-activated cell sorting FCS Fluorescence correlation spectroscopy FP Fluorescent proteinFRET Fluorescence resonance energy transfer FLIM Fuorescence life-time imaging GFP Green ﬂuorescent protein IRFP Infra-red ﬂuorescent protein Mant MethylanthraniloylMMP Matrix metalloproteinase NIRF Near-infrared ﬂuorescence NLS Nuclear localization sequence PBD p21-Derived binding domain PTD Protein transduction domain RBD RhoA-Binding domainIntroductionWith the escalation of drug discovery programmes,it has become essential to visualize biological activities in heal-thy and pathological cells,and to monitor their spatial and temporal dynamics for a comprehensive understanding of their behaviour.Together with the development of ﬂuo-rescent probes and evergrowing advances in imaging technologies,biosensor systems capable of probing and reporting on the presence and activity of speciﬁc targets and biomarkers have come of age.M.C.Morris (&)Interactions and Molecular Mechanisms regulating Cell CycleProgression,Universite´de Montpellier,CRBM-CNRS UMR5237,1919Route de Mende,IFR122,34293Montpellier,Francee-mail:may.morris@rs.frCell Biochem Biophys (2010)56:19–37DOI 10.1007/s12013-009-9070-7Biosensors:Deﬁnition and General ConceptsBiosensors are deﬁned as ‘‘analytical devices’’which combine a biological component derived from biological or biomimetic material (e.g.tissue,microorganism,receptor,enzyme,antibody,nucleic acid,etc.)with a physicochem-ical transducer (optical,electrochemical,thermometric,piezoelectric,magnetic or micromechanical).Whilst the biological component is responsible for sensing a speciﬁc analyte or target,the physicochemical transducer converts recognition of the analyte into a detectable and measurable signal,a suitable quantitative output (Fig.1)[1].Biosensors offer a convenient,rapid,speciﬁc and sensitive means of monitoring analytes and of reporting on the presence of speciﬁc target substrates or enzymes,through a signal which is generally proportional to the concentration or activity of the target.Whilst the type of biosensor will really be adapted to meet the needs of an individual application,its speciﬁcity and sensitivity is paramount for its utility and applicability within a complex physiological ensemble or solution.Although such criteria are often inherently deﬁned by the biological molecules,the sensors derive from,they may be improved to optimize the selectivity of target recognition thanks to molecular biology and protein engineering approaches.Over the years the development of biosensors has evolved together with technologies available and the applications requiring this type of device.The ﬁrst bio-sensor can undoubtedly be traced down to the invention of the electrochemical oxygen biosensor by Clark [2],closely followed by the development of a glucose biosensor through combination of glucose oxidase as an enzyme transducer to an oxygen electrode in 1962[3].In 1969,the ﬁrst potentiometric enzyme electrode was developed by Guilbault and Montalvo [4],the urea sensor,based on immobilization of urease onto an ammonium-selective liquid membrane electrode.Thermal enzyme probes also known as thermistors were next developed by Mosbach et al.[5,6].In 1975,the ﬁrst ‘‘microbial electrodes’’,basedon the use of bacterial microorganisms were developed for alcohol measurement by Divis [7],whilst Lubbers and Oppitz [8,9]developed the ﬁrst ﬁbre-optic sensors or ‘‘optodes’’which were applied to measure carbon dioxide,oxygen and alcohol,respectively.In 1976,Clemens et al.[10]introduced an electrochemical glucose biosensor into an artiﬁcial pancreas,which would later be improved and commercialized for generalized use in the clinic.A real breakthrough for in vivo applications was the development of needle-type enzyme electrodes for subcutaneous implantation by Shichiri et al.[11].In 1983,an altogether novel type of biosensor was developed by Liedberg et al.[12],the immunosensor,precursor of the widely used BIAcore system,based on immobilization of antibodies to a piezoelectric or potentiometric transducer,thereby enabling immuno-recognition of analytes by surface plas-mon resonance.Ever since biosensor technology has expanded to meet the needs of a variety of applications and analytical problems in medicine,environment,food and process industries,security and defence [13].Over the past decade,a number of optical biosensors based on surface plasmon resonance,waveguides and resonant mirrors have been developed to monitor a variety of biomolecular interactions between label-free targets in vitro,with important applications in the ﬁeld of drug discovery [14].In parallel,thanks to signiﬁcant advances in the develop-ment of ﬂuorescent probes,in particular green ﬂuorescent protein (GFP)-based systems by Roger Tsien,and more recently of small ﬂuorescent dyes with enhanced spectral properties for in vivo imaging,and the concomitant explosion of ﬂuorescent imaging technologies,the bio-sensor ﬁeld has witnessed a dramatic expansion of ﬂuo-rescence-based biosensors for probing protein activities,conformational changes and postranslational modiﬁcations in living cells with high spatial and temporal resolution [15–27].Fluorescence-Based BiosensorsFluorescence-based biosensors are useful tools for detec-tion of biomolecules both in vitro and in vivo.Fluores-cence is sensitive,speciﬁc and lends itself to non-destructive imaging.As such high-resolution imaging and real-time measurements of ﬂuorescent biosensors have provided precious information on the spatial and the tem-poral localization of a wide variety of intracellular targets and have proven a successful means of visualizing dynamic processes and of monitoring enzymatic activities in living cells,as well as useful tools for drug discovery [23,24,28–36].In order to transduce ligand binding into a detectable optical signal,ﬂuorescence-based biosensors bear a sensing moiety that is either genetically or chemically coupled toTransducerSensitive MoietyTransduction and Analyte Target(a)ﬂuorescent probe (s)whose spectral properties undergo measurable variations upon target recognition.Fluorescent biosensors come in all sorts of ﬂavours,which are commonly distinguished based on their chem-ical nature,i.e.genetically encoded,peptide-,protein,or nucleotide-based,or on the ﬂuorescence technology applied to monitor target recognition,i.e.ﬂuorescence resonance energy transfer (FRET)-based or environmentally sensitive.Moreover,ﬂuorescent biosensors may be designed to probe an enzymatic activity,or to recognize a speciﬁc target or conformation,and are consequently referred to as activity-,ligand-or afﬁnity-based,respectively.These different families of biosensors overlap;as such activity-based bio-sensors may be either genetically encoded,peptide-or nucleotide-based;peptide-based biosensors may be FRET-based or environmentally sensitive,activity-or afﬁnity-based.The characteristics of some of the most common ﬂuorescent biosensors are brieﬂy described below and schematized in Fig.2.Activity-based reporters are amongst the most common ﬂuorescent biosensors developed for monitoring speciﬁc enzymatic activities.They consist of enzyme substrates,typically polypeptide sequences which undergo a speciﬁc physico-chemical modiﬁcation,such as phosphorylation,methylation,ubiquitinylation or enzymatic cleavage by the target enzyme,which in turn affects the spectral properties of the associated ﬂuorescent probe(s),leading to measur-able changes in ﬂuorescence (Fig.2a–c).Typical examples include genetically encoded biosensors that monitor kinase activity through phosphorylation-induced intramolecular FRET (Fig.2a),peptide-based biosensors that monitor proteolytic activities through cleavage-induced abrogation of intramolecular FRET (Fig.2b),and peptide-based bio-sensors that monitor phosphorylation-induced enhance-ment of a ﬂuorescent probe adjacent to the phosphorylated residue (Fig.2c).Nucleotide-based sensors consist of ﬂuorescently label-led nucleotide analogs whose ﬂuorescent properties are environmentally sensitive,and modiﬁed upon incorporation into the nucleotide-binding pocket of target enzymes,such as methylantraniloyl-GTP or -ATP (Fig.2d).In particular,this strategy has been widely applied to study ATPase and GTPase activities,the dynamics and conformational chan-ges of protein kinases and motor proteins.Although nucleotide-based probes remain very useful for in vitro studies of enzyme activities,their application to living cellsKinaseSubstrateFRETP Substrate(a)(b)FRETSubstrateProteaseKinasePSubstrateGTPaseNNGTPase(d)Inactive TargetInterface(c)(e)Active TargetActive TargetFig.2Common examples of ﬂuorescence-based biosensors.Sche-matic representation of different varieties of ﬂuorescence-based biosensors.a ,b ,c Activity-based reporters consist of polypeptide sequences which undergo a speciﬁc physico-chemical modiﬁcation by the target enzyme which affects ﬂuorescence of the probe.a FRET-based genetically encoded single-chain biosensor of protein kinase A activity through phosphorylation-induced intramolecular FRET,b peptide-based biosensor of proteolytic activity through cleavage-induced abrogation of intramolecular FRET,c peptide-basedbiosensor of protein phosphorylation through enhancement of adjacent probe ﬂuorescence,d Nucleotide-based sensors consist of ﬂuores-cently labelled nucleotide analogs whose ﬂuorescence is generally enhanced upon incorporation into the nucleotide-binding pocket of target enzymes,e interfacial biosensors consist of polypeptide domains or sequences that recognize unique interfaces or bind a speciﬁc conformation of the target,thereby generating a measurable variation of associated probe ﬂuorescenceis limited,due to an inherent lack of speciﬁcity,as they cannot discriminate between different enzymes requiring these nucleotide cofactors for their activity.Afﬁnity-based or interfacial biosensors can be described as reporters harbouring a polypeptide sequence or domain that recognizes a unique interface or which binds a speciﬁc conformation of a target protein,thereby generating a measurable variation of associated probeﬂuorescence (Fig.2e).Afﬁnity-based biosensors can provide a high degree of speciﬁcity for target recognition,and are excel-lent alternatives to activity-based probes for monitoring active enzyme conformations in vitro and in cellulo. Genetically Encoded Versus Non-Genetic BiosensorsTheﬁrstﬂuorescent biosensors of protein function were derived from proteins,peptides or nucleotides which were chemically labelled[28].Although some of these were introduced into living cells,essentially through microin-jection or electroporation,relatively few were applied to monitor dynamic activities in cellulo,let alone in vivo,due to limitations associated with internalization,biodistribu-tion and imaging technologies.The discovery of GFP and the development of GFP-based systems by Roger Tsien truly revolutionizedﬂuorescence technology,allowing for genetic engineering of cell lines that report on the spatio-temporal localization and dynamics of ectopically expres-sed GFP-fusion products[15–25].Moreover,this technol-ogy has provided a solid basis for developing genetically encodedﬂuorescent reporters,to probe enzymatic activi-ties,protein interfaces or conformational changes directly in living cells with high spatial and temporal resolution [29–33].The GFPﬂuorophore and its variants are excellent tools for probing biological components and activities in living cells,and genetically encoded GFP-based biosensor technology provides unique opportunities of engineering constructs to suit the speciﬁcity of the target.However,this technology is subject to certain limitations,notably the control over the timing and levels of expression within cells,as well as the behaviour of the GFP-fusions,whose size may affect the behaviour of the sensing element,its localization and ability to recognize the intracellular target. As such,there is a growing demand for non-genetic bio-sensors designed to report on speciﬁc biological activities, with maximal resolution,speciﬁcity and sensitivity. Genetically Encoded FRET BiosensorsGenetically encoded single-chain FRET biosensors con-stitute one of the most widespread classes ofﬂuorescent biosensors.They consist of plasmid constructs engineered to express a target reporter sequence(typically a peptide substrate),ﬂanked by a FRET pair ofﬂuorescent proteins derived from GFP(FPs),typically CFP and YFP.Modiﬁ-cation of the target sequence upon activation of the target enzyme induces a conformational change that modiﬁes the proximity of the FPs,consequently favouring or disrupting FRET between the donor and the acceptor.Theﬁrst reporters to provide a valid proof-of-concept of this tech-nology were theﬂuorescent Ca2?indicators,dubbed ‘‘cameleons’’,consisting of tandem fusions of GFP variants with calmodulin and the calmodulin-binding peptide M13 [37,38].Binding of Ca2?to calmodulin promotes its binding to the M13domain,thereby bringing together the FRET pair of FPs and favouring their intramolecular FRET (Fig.3a).Ever since a wide variety of genetically encoded single-chain FRET biosensors have been developed. Genetically encoded FRET-based biosensors of kinase activity have been generated through incorporation of a kinase-speciﬁc substrate sequence and a matching phos-phoaminoacid-binding domain in a plasmid construct, together with a FRET pair of GFP variants(Fig.3b)[39–47].A similar approach has been employed to design ﬂuorescent reporters of histone methylation by incorpo-rating a sequence encoding a substrate peptide from the N-terminus of histone H3fused to a matching chromodo-main,between a FRET pair of GFP variants[48].More recently a biosensor of histone acetylation‘‘Histac’’has been developed,consisting of an acetylation-binding bromodomain and a substrate histone H4[49].Several elegant FRET-based biosensors have been developed to monitor GTPase activation,bearing a GTPase together with a binding domain which only recognizes the con-formationally active form of the enzyme[50,51](Fig.3c). The converse strategy has been applied to generateﬂuo-rescent protease reporters to probe proteolytic activities,by sandwiching speciﬁc substrate sequences between GFP variants,whose ability to FRET is disrupted by proteolytic cleavage[52–55](Fig.3d)(further details are provided in ‘‘Biosensor applications’’).Peptide-and Polypeptide-Based Biosensors and Probes Together with the development of new generations ofﬂuo-rescent probes with enhanced photostability and optimal photophysical properties for cellular applications,quench-ing and caging strategies for reducing basalﬂuorescence,as well as technologies enabling direct enzymatic labelling of speciﬁc sequences in living cells,the improvement of chemical strategies to synthesize membrane-permeant ana-logs and the development of suitable and efﬁcient cellular internalization technologies,non-genetically encoded pep-tide-and polypeptide-based reporters have become attrac-tive alternatives to GFP-based genetically encoded systems (Fig.4).Peptide-and polypeptide biosensors are generated by chemical coupling or enzymatic incorporation of (a)ﬂuo-rescent label(s)into a peptide or polypeptide sensing sequence,generally derived from a substrate or a unique-binding interface.In principle,a peptide or polypeptide derived from a protein substrate or a partner-bindingCa2+FRET(a)(b)FRETProteaseKinaseSubstrateFRETP SubstrateCamodulinCamodulinCa2+M 13M13(c)activator FRETGTPaseSubstrate(d)Active GTPaseFig.3Genetically encoded single-chain FRET-based biosensors consist of plasmid constructs engineered to express a target reporter sequence,ﬂanked by a FRET pair of ﬂuorescent proteins (FPs)derived from GFP,typically CFP and YFP.Modiﬁcation of the target sequence upon activation of the target enzyme induces a conforma-tional change that modiﬁes the proximity of the FPs,consequently favouring or abrogating FRET between the donor and the acceptor.a Fluorescent Ca2?indicators or ‘‘cameleons’’,consisting of tandem fusions of GFP variants with calmodulin and the calmodulin-binding peptide M13,b FRET-based biosensors of kinase activity consist of tandem fusions of FPs with a kinase-speciﬁc substrate sequence and a matching phosphoaminoacid-binding domain,c ﬂuorescent reporters of histone methylation bear a sequence encoding a substrate peptide from the N-terminus of histone H3and a matching chromodomain,between a FRET pair of GFP variants,d FRET-based biosensors of GTPase activation bear a GTPase together with a binding domain which only recognizes the conformationally active form of the enzyme,e FRET-based protease reporters harbour the speciﬁc substrate sequence between GFP variants,whose ability to FRET is abrogated upon proteolytic cleavage(b)Binding InterfaceTarget(a)Binding InterfaceTarget(c)PSubstrate Sequence(d)Binding InterfaceQuencherTargethv(e)CageTargetFig.4Peptide-and polypeptide biosensors.Non-genetically encoded biosensors are generated by chemical coupling or enzymatic incor-poration of a ﬂuorescent label into a peptide or polypeptide sensing sequence,generally derived from a substrate or a unique-binding interface.Target binding (a ),target-induced conformational change (b )and/or posttranslational modiﬁcation (c )will affect the spectral properties of environmentally sensitive ﬂuorescent probes.Moreover,probes can be internally quenched to minimize noise to signal (d )or caged,so as to control release of an active biosensor through selective photoactivation (e )interface can function as a sensor,reporting on its interac-tion with the target enzyme thanks to an ‘‘environmentally sensitive’’ﬂuorescent probe coupled to a neighbouring amino acid,whose spectral properties will be affected by changes in the local environment,whether these involve interactions with the target,enzyme-induced conforma-tional change,or a chemical modiﬁcation,such as a phos-phorylation.As for genetically encoded systems,the speciﬁcity of protein/substrate or protein/partner interac-tions can be reﬁned,so as to achieve optimal selectivity for target recognition in a complex solution.In addition,bili-gand strategies can be devised,in which multiple interfaces are combined to recognize a single target,or a heterodimeric complex.Moreover,this technology allows for the use of a wide variety of small molecule ﬂuorescent labels,which can be internally quenched to minimize noise to signal,or caged,so as to control the release of the active biosensor through selective photoactivation.Although a wide variety of peptide-and protein-biosensors have been developed to probe activities of kinases,phosphatases and proteases (see ‘‘Biosensor applications’’for a more detailed description),few have actually been applied to living cells or in vivo,due to cellular permeability issues associated with their chemi-cal nature.However,advances in the ﬁeld of cell-penetrat-ing peptides (CPPs)and protein transduction domains (PTDs)have provided efﬁcient means of introducing these biosensors into living cells and in vivo.Synthetic Fluorescent Probes and Labelling Strategies In order to obtain a high signal-to-noise ratio in living cells,and to limit the autoﬂuorescence of biological samples,it is important to use ﬂuorophores with long-wave excitation and emission spectral properties.Over the recent years efforts have been made to generate novel generations of ﬂuorescent probes with enhanced photostability and photophysical properties suited to live-cell imaging (for review [26,27,56,57]).In particular,infra-red ﬂuorescent proteins (IRFPs)and near-infra-red ﬂuorescent probes (NIRFs)are of particular interest for cellular and in vivo application as they yield far less tissular damage upon in vivo imaging [57–59]Moreover,the development of(a)(b)(d)(c)PCNAPM TargetingNLSGemininCdt1Accumulation in S/G2/MAccumulation in G1DNA LigaseCyclin B(e)(f)cdk1Cyclin B1S-phase MarkerDnmt1Fig.5Cell cycle sensors.(a )GFP-PCNA (proliferating cell nuclear antigen)was proposed as a marker of S-phase,as it accumulates in DNA replication foci during S-phase [175–177],b the combination of RFP-DNA ligase with GFP-Dnmt1DNA methyltransferase have been employed to distinguish all cell cycle phases [178],c GFP-cyclin B uses functional elements from cyclin B1including its endogenous promoter (CCNB1)allowing for expression from S phase through G2and mitosis,its destruction box (D-box),leading to its degradation at the end of mitosis,and its cytoplasmic retention signal (CRS)inactivation of which promotes cyclin B1translocation into the nucleus [181,182],d genetically encoded YFP associated with a plasma membrane (PM)targeting domain and a nuclear localization sequence (NLS)allows to visualize nuclear envelope breakdown at mitosis [179],e the FUCCI system combines GFP-Geminin which accumulates in S/G2/M and is degraded in G1and RFP-Cdt1with accumulates in G1[180],f the biligand peptide biosensor of cdk/cyclins recognizes heterodimeric cdk/cyclin complexes [186]quantum dots,semi-conductor nanocrystals with excep-tional optical and spectroscopic properties,have provided an attractive alternative to both small-molecule and protein ﬂuorophores for the high-resolution imaging of intracellu-lar processes and in vivo imaging[60–64].Quantum dots have been successfully coupled to peptides,proteins and biosensors,through standard chemical approaches,can serve as excellent FRET donors,and have been delivered into cells for intracellular imaging applications thanks to cell-penetrating peptides[62–65].Smallﬂuorescent labels may be incorporated into the peptide or polypeptide sensor during synthesis or covalently coupled postsynthesis through classical chemical coupling to amino,sulfhydryl, hydroxy or carboxy groups.Alternative approaches have been developed for site-speciﬁcﬂuorescent labelling of recombinant proteins in living cells.The FlAsH system, developed by Tsien and coworkers[66,67],involves incorporation of a membrane-permeant biarsenicalﬂuo-rescein derivative into a polypeptide sequence at a speciﬁc tetracysteine motif.The transglutaminase-based strategy, developed by Weiss and coworkers[68],enables enzy-matic labelling of a reactive glutamine in a speciﬁc rec-ognition sequence.The SNAP-tag and the CLIP-tag, developed by Johnsson et al.[69–71]are derived from the human DNA repair protein O6-alkylguanine-DNA alkyl-transferase(AGT)and are used to label proteins in living cells withﬂuorescent derivatives of O6-benzylguanine and O6-benzylcytosine,respectively.Finally incorporation of genetically encodedﬂuorescent amino acids during poly-peptide synthesis in E.coli has been proposed as an alter-native to postsynthetic coupling ofﬂuorescent labels[72]. Quenching and Caging StrategiesOne of the major limitations of mostﬂuorescent probes is their generalﬂuorescence both in solution and when bound to a target,which largely restricts overall changes in intensity upon recognition of the target.Therefore in an attempt to control and optimize the sensitivity and selec-tivity of the response so as to obtain a robustﬂuorescent signal upon target recognition,strategies have been devised to quench and/or cage theﬂuorophore.Strategies of intramolecularﬂuorophore quenching have proven to be effective in reducing backgroundﬂuorescence prior to target binding toﬂuorescent peptide biosensors both in vitro and in living cells[73–76].Molecular caging allows the conversion of a biomolecule into an inert form through chemical modiﬁcation with a photolabile protective group. Selective photoactivation of the caged molecule allows for its release into a form which is readily accessible for its biological function,enzymatic activity or ability to bind a partner.In the case of a biosensor,this provides a means of controlling biosensor/target recognition in space and in time.This approach has been largely developed for selec-tive activation of enzymes and peptides,and provides a means ofﬁne-tuning new generations ofﬂuorescent bio-sensors[77–82].An alternative strategy involves direct modiﬁcation of probes,as exempliﬁed by the esterase-reactiveﬂuorophores developed by Raines and coworkers [83],whoseﬂuorescence is masked by‘‘trimethyl locks’’, which can be hydrolyzed to yield a robustﬂuorescent signal.Another noteworthy example is the application of the photoreactive light oxygen voltage(LOV)domain from phototropin which has recently been used to sterically block interactions of the Rac1GTPase until photoactiva-tion,in an entirely reversible fashion[84].Intracellular Delivery StrategiesIntroduction of molecules with biological activity into liv-ing cells,tissues and animals is a major challenge for basic research,as well as for therapeutic and for diagnostic applications.For application in living cells,both genetically encoded reporters and non-genetically encodedﬂuorescent probes must be delivered through the cell membrane in a non-invasive fashion and be able to reach their intracellular target at the appropriate subcellular location.Whilst genetically encoded biosensors may be introduced into cells thanks to standard transfection,electroporation or micro-injection procedures,peptides and polypeptides may rely on a different set of approaches for efﬁcient internalization into cells and tissues,which will preserve their integrity and stability.Theﬁrst approaches classically employed for introduction of peptides and proteins into cells relied on mechanical or physical techniques,such as microinjection, electroporation or‘‘cell-loading’’techniques,which apply a mechanical stress that affects the plasma membrane per-meability[85].Although microinjection may be applied to introduce of polypeptides into a subset of cultured cells,this approach is not applicable to larger populations of cells, tissues or animals.As such,several alternative strategies for delivery of polypeptides and peptides into living cells and animal models have been developed,including lipid-based formulations,cell-penetrating peptides and carbon-based nanoparticles or nanotubes.Although each of these approaches presents speciﬁc advantages and limitations,the critical issue is to ensure maximal uptake,whilst preserving cargo integrity and stability.Uptake efﬁciency is directly related to the chemical nature of the cargo,to the physico-chemical characteristics of the carrier and to its mechanism of cellular internalization,and dependent on the cell type, due to differences in membrane components.PTDs and CPPs constitute a family of polypeptide domains and peptides which can insert into and cross-bio-logical membranes to penetrate into living cells whose。

人教版英语高三上学期期中试题及答案指导(2024-2025学年)

2024-2025学年人教版英语高三上学期期中模拟试题及答案指导一、听力第一节（本大题有5小题，每小题1.5分，共7.5分）1、Listen to the conversation and answer the question.What is the man’s main concern about the weather?A. It’s too hot.B. It’s too cold.C. It’s too humid.D. It’s not sunny enough.Answer: BExplanation: The man says, “I can’t stand this cold weather,” indicating that he is worried about the cold weather.2、 Listen to the short passage and complete the sentence with the missing word.The lecture was so informative that the audience was completely _1__.A. interestedB. boredC. surprisedD. confusedAnswer: AExplanation: The phrase “so informative” suggests that the audience found the lecture very interesting, thus making option A the correct answer.3.You will hear a conversation between two students discussing their weekend plans. Listen and answer the following question:What activity do both students agree to do together on Saturday?A)Go shoppingB)Visit a museumC)Go to the moviesD)Have a picnicAnswer: B) Visit a museumExplanation: The students discuss their plans for the weekend, and they both mention visiting the museum as something they would like to do together.4.You will hear a professor giving a lecture on environmental issues. Listen and answer the following question:What is the professor’s main concern about the current state of the environment?A)The rapid increase in populationB)The depletion of natural resourcesC)The pollution of water sourcesD)The loss of biodiversityAnswer: B) The depletion of natural resourcesExplanation: The professor discusses various environmental issues, but themain concern expressed is about the depletion of natural resources, such as water, forests, and minerals.5、Question: How much time will the man spend on the road trip?A) 4 hoursB) 6 hoursC) 8 hoursD) 10 hoursAnswer: B) 6 hoursExplanation: According to the conversation, the man mentions that they will drive for about 3 hours, then take a break for 1 hour, and then continue for another 2 hours. Therefore, the total time spent on the road trip is 3 + 1 + 2 = 6 hours.二、听力第二节（本大题有15小题，每小题1.5分，共22.5分）1、What does the man suggest they should do about the weather forecast?A. Stay inside and watch TV.B. Go for a walk despite the rain.C. Prepare for a sunny day.D. Cancel the outdoor plans.Answer: DExplanation: In the conversation, the man mentions, “It looks like it’s going to rain all day, so we might as well cancel the picnic plans.” This impliesthat he suggests they should not go ahead with the outdoor plans due to the weather forecast.2、Why does the woman say she has to go to the library?A. She needs to do research for her assignment.B. She has a meeting with her professor.C. She is returning a borrowed book.D. She wants to borrow some books for her friends.Answer: AExpl anation: The woman responds to the man’s question about her plans with, “I actually need to go to the library. I have to do some research for my history assignment.” This indicates that her reason for going to the library is to conduct research for her assignment.3、You will hear a conversation between two students discussing their study plans. Listen and answer the question.Question: What subject do the students agree to study together?A)MathematicsB)HistoryC)ScienceD)LiteratureAnswer: A) MathematicsExplanation: In the conversation, the students mention that they both have a math class on Wednesday afternoon, indicating their agreement to study mathematics together.4、You will hear a short lecture about the importance of exercise. Listen and answer the question.Question: What is the main benefit of regular exercise according to the lecture?A)Improved memoryB)Increased productivityC)Better sleep qualityD)Faster metabolismAnswer: C) Better sleep qualityExplanation: The lecture specifically states that regular exercise can lead to better sleep quality, which is the main benefit mentioned.5.You will hear a conversation between two friends, Alice and Bob, discussing their plans for the weekend. Listen carefully and answer the question.Question: What does Bob suggest doing for the weekend?A. Going to the movies.B. Visiting a museum.C. Going hiking.Answer: C. Going hiking.Explanation: Bob suggests going hiking, as he mentions, “Hey, why don’t we go hiking this weekend? It’ll be great exercise and we can enjoy the nature.”6.You will hear a dialogue between a teacher and a student, Sarah, discussing her progress in her math class. Listen carefully and answer the question.Question: What does the teacher say about Sarah’s understanding of the math concepts?A. She needs more practice.B. She’s doing very well.C. She’s struggling with the material.Answer: B. She’s doing very well.Explanation: The teacher says, “Sarah, you’ve really improved since the last test. You’re doing very well with the math concepts.” This indicates that Sarah is doing well in the class.7.You are listening to a conversation between two students discussing their weekend plans.Question: What does the woman suggest doing for their weekend activity?A. Visiting the art museum.B. Going to the beach.C. Having a picnic in the park.D. Attending a concert.Answer: B解析：The woman mentions that she heard there’s a great beach nearby and suggests they go there for the weekend. This indicates that the correct answer is B. Going to the beach.8.You are listening to a lecture about the importance of exercise.Question: According to the lecturer, what is one of the benefits of regularexercise?A. Improved memory.B. Increased stress levels.C. Decreased immune system function.D. Higher blood pressure.Answer: A解析：The lecturer explicitly states that regular exercise can improve memory and cognitive function. Therefore, the correct answer is A. Improved memory. The other options are mentioned as negative effects of lack of exercise or stress, not as benefits.9.W: Have you ever traveled to New York City?M: Yes, I have. I visited it last summer. The city is fascinating, and I especially love its diverse culture.Q: What did the man do last summer?A: He traveled to New York City.B: He visited the city for business.C: He moved to New York City.D: He worked in New York City.Answer: AExplanation: The man explicitly states that he visited New York City last summer, which is option A.10.W: I heard you’re going to graduate soon. Do you have any plans for aftergraduation?M: Not yet. I’m considering studying abroad, but I’m not sure which country to choose. I’m thinking about the UK or Canada.Q: What is the man currently considering for after graduation?A: He plans to study in the UK.B: He wants to move to Canada.C: He is thinking about studying abroad.D: He plans to work in the UK.Answer: CExplanation: The man mentions that he is considering studying abroad, which is option C. He doesn’t explicitly state his preference for the UK or Canada, so options A and B are incorrect. Option D is also not mentioned, making optionC the correct answer.11.You will hear a conversation between two students discussing their study plans. Listen to the conversation and answer the question.Q: What subject are the students planning to study together?A: 1. ScienceB. MathC. EnglishD. HistoryAnswer: BExplanation: The students mention that they need to study for their math testtogether, indicating that they are planning to study math. Therefore, the correct answer is B. Math.12.You will hear a short lecture about the importance of exercise. Listen to the lecture and answer the question.Q: According to the lecture, which of the following benefits does exercise NOT provide?A. Improved moodB. Enhanced cognitive functionC. Increased risk of injuryD. Better sleep qualityAnswer: CExplanation: The lecture discusses various benefits of exercise, such as improved mood, enhanced cognitive function, and better sleep quality. The option that is NOT mentioned as a benefit is increased risk of injury. Therefore, the correct answer is C. Increased risk of injury.13.You will hear a conversation between two students discussing their weekend plans. Listen carefully and answer the question.Question: What does the student suggest they do on Saturday?A)Go to the movies.B)Visit a museum.C)Have a picnic.Answer: C) Have a picnic.Explanation: The student suggests having a picnic because they mention thatit’s a beautiful day and they can enjoy the weather outdoors.14.You will hear a short lecture about the impact of social media on teenagers. Listen carefully and answer the question.Question: According to the lecture, what is one of the main concerns regarding social media use by teenagers?A)Decreased physical activity.B)Negative effects on self-esteem.C)Increased academic pressure.Answer: B) Negative effects on self-esteem.Explanation: The lecture discusses how social media can negatively affect teenagers’ self-esteem, particularly due to the constant comparison with others’ lives.15.How much money did the speaker spend on the new book?A)35 dollars.B)45 dollars.C)55 dollars.D)65 dollars.Answer: A) 35 dollars.Explanation: The speaker mentions that the new book cost him 35 dollars, so the correct answer is A) 35 dollars.三、阅读第一节（第1题7.5分，其余每题10分，总37.5分）第一题Passage:The global pandemic has had a profound impact on the way we live and work. One of the most significant changes has been the shift to remote work, which has become a permanent fixture in many industries. This shift has brought both challenges and opportunities. Here is a look at how remote work is transforming the modern workplace.Paragraph 1:Remote work allows employees to enjoy a better work-life balance. With the flexibility to work from home, employees can reduce their commuting time and save on transportation costs. Additionally, working from home can provide a more comfortable and personalized work environment, which can lead to increased productivity.Paragraph 2:However, remote work also poses several challenges. One of the main concerns is the potential for decreased communication and collaboration among team members. Without face-to-face interactions, it can be more difficult to build trust and maintain a strong team dynamic. Moreover, remote workers may struggle with maintaining a consistent work schedule and managing distractions at home.Paragraph 3:To mitigate these challenges, companies are implementing various strategies.Some organizations are investing in video conferencing tools to facilitate regular team meetings and ensure that everyone stays connected. Others are providing training and support to help employees develop effective remote work habits. Additionally, many companies are reevaluating their performance metrics to focus more on outcomes rather than the number of hours worked.Paragraph 4:The rise of remote work has also led to a shift in the traditional office space. Many companies are downsizing their office spaces or even closing them altogether, as they realize that employees can be just as productive from home. This shift has resulted in significant cost savings for businesses and has also reduced the carbon footprint associated with commuting.Questions:1、What is one of the benefits of remote work mentioned in the passage?A. Increased communication among team members.B. Reduced commuting time and transportation costs.C. Higher productivity due to a more comfortable work environment.D. Improved team dynamics through face-to-face interactions.2、According to the passage, what is a challenge that remote workers may face?A. Having access to advanced video conferencing tools.B. Maintaining a consistent work schedule.C. Building trust and collaboration among team members.D. Reducing the carbon footprint associated with commuting.3、How are companies addressing the challenges of remote work?A. By downsizing office spaces and closing them.B. By providing training and support to develop effective work habits.C. By focusing on outcomes rather than the number of hours worked.D. By investing in video conferencing tools and regular team meetings.4、What is the main impact of the rise of remote work on traditional office spaces?A. Increased carbon footprint due to more commuting.B. Higher transportation costs for employees.C. Significant cost savings for businesses.D. Improved communication and collaboration among team members.Answers:1、B2、C3、B4、CSecond QuestionReading Section 1Passage:In the small coastal town of Oceanview, the local community has always been close-knit. The townsfolk rely heavily on each other for support and assistance.One such event that brought the community together was the annual Oceanview Beach Cleanup. This event was not only a way to clean up the beach but also a time for people to socialize and bond.The Oceanview Beach Cleanup was organized by the local environmental club, which had been actively promoting environmental conservation in the area for years. The club, led by the passionate and dedicated president, Emily Johnson, had managed to gather a large group of volunteers from all walks of life. The volunteers ranged from school children to senior citizens, all united by a common goal of preserving the beauty of their hometown.The morning of the cleanup began with a brief assembly where Emily Johnson addressed the volunteers. She emphasized the importance of the cleanup, highlighting the impact of pollution on marine life and the local ecosystem. She also encouraged everyone to take personal responsibility for the environment.As the volunteers split into groups, they set off to different parts of the beach. The younger volunteers focused on picking up litter and debris along the shoreline, while the older ones ventured further into the tidepools to remove trash that had been buried by the sand. Everyone worked diligently, and within a few hours, the beach looked significantly cleaner.The event was not without its challenges. The weather turned unexpectedly windy, making it difficult for some volunteers to stay on their feet. However, the group spirit remained strong, and everyone pulled together to ensure thecleanup’s success.As the day came to a close, the volunteers gathered for a group photo to mark the occasion. They were all proud of what they had accomplished and the positive impact they had made on the community. Emily Johnson, the club president, summed up the day’s events with a heartfelt speech, thanking everyone for their hard work and dedication.Questions:1、What is the main purpose of the Oceanview Beach Cleanup?A. To promote tourism in Oceanview.B. To raise funds for local charities.C. To clean up the beach and preserve the environment.D. To provide a social event for the community.2、Who is Emily Johnson in the passage?A. A volunteer at the beach cleanup.B. The president of the local environmental club.C. A teacher at the local school.D. A journalist covering the event.3、What was the weather like on the day of the cleanup?A. Sunny and warm.B. Windy and chilly.C. Rainy and wet.D. Overcast and cool.4、What did Emily Johnson say in her speech at the end of the cleanup?A. She congratulated the volunteers on their hard work.B. She criticized the community for being too lazy.C. She demanded more funding for the environmental club.D. She announced that there would be no more cleanups in the future.Answers:1、C2、B3、B4、A第三题Reading Passage:In the bustling city of New York, there’s a peculiar place called the “Reading Room”. This is no ordinary library; it’s a place where people come to read, think, and socialize. The room is filled with a wide array of books on different subjects, from literature to science, history, and more. The atmosphere is calm and serene, making it an ideal spot for those seeking intellectual stimulation and a break from the hustle and bustle of city life.One sunny afternoon, a young woman named Emily enters the Reading Room. She is looking for a book that will inspire her. As she strolls through the aisles, she notices a group of people gathered around a table, deep in conversation. Curious, she joins them and learns that they are discussing a recently publishednovel.1、What is the main purpose of the Reading Room in New York?A. To provide a quiet place for people to read.B. To sell books.C. To host book readings.D. To serve as a community center.2、What does the author describe about the atmosphere of the Reading Room?A. Noisy and chaotic.B. Calm and serene.C. Busy and crowded.D. Dark and eerie.3、Why does Emily enter the Reading Room?A. To sell books.B. To find a job.C. To find a place to sleep.D. To find a book that will inspire her.4、What is Emily doing when she enters the Reading Room?A. Reading a book.B. Writing a book.C. Joining a conversation.D. Looking for a friend.Answers:1、A. To provide a quiet place for people to read.2、B. Calm and serene.3、D. To find a book that will inspire her.4、C. Joining a conversation.第四题Passage:It was a chilly autumn morning when Alex decided to visit the local museum. He had always been fascinated by history and was excited to explore the new exhibit on ancient civilizations. As he walked through the museum, he encountered a series of interactive displays that allowed visitors to learn about different aspects of ancient life. One particular exhibit caught his attention: a reconstructed marketplace from the Roman Empire.The marketplace was bustling with activity, and Alex could almost imagine the sounds and smells of the past. Merchants displayed their goods, from fresh produce to exotic spices, while customers haggled over prices. A sign near the entrance read, “Try Your Hand at Being a Roman Merchant!” Curious, Alex approached a table where a museum volunteer was waiting.The volunteer handed Alex a small, leather pouch containing various goods, including olive oil, wine, and bread. “Here, you have 100 denarii to start with,” the volunteer explained. “Your goal is to buy as much as you can with your money and then sell it at a profit to make a profit of at least 50 denarii.”Alex quickly began to make purchases, choosing items that he thought wouldbe in high demand. He bought several loaves of bread, a jar of olive oil, and a few bunches of grapes. As he walked around the marketplace, he noticed that other visitors were also trying their hand at being merchants.After a few minutes, Alex decided to sell his goods. He set up a small stall and began to call out to potential customers. “Fresh bread, the best olive oil, and delicious grap es!” he shouted. The other merchants around him were also trying to attract customers, and the marketplace became a lively scene.After some time, Alex managed to sell all his goods and even made a profit of 60 denarii. He was quite pleased with his success and decided to visit the other interactive exhibits in the museum. The experience had given him a new appreciation for the complexities of ancient trade and the skills of the merchants of the Roman Empire.Questions:1、What was Alex’s main reason for vis iting the local museum?A) To see the new exhibit on ancient civilizations.B) To learn about the museum’s history.C) To attend a special event.D) To meet the museum volunteers.2、How much money did Alex start with in the marketplace simulation?A) 50 denariiB) 100 denariiC) 200 denariiD) 500 denarii3、What was one of the items Alex bought to sell at a profit?A) Silk fabricsB) Olive oilC) PotteryD) Gold coins4、How much profit did Alex make from selling his goods?A) 20 denariiB) 30 denariiC) 40 denariiD) 60 denariiAnswers:1、A2、B3、B4、D四、阅读第二节（12.5分）Title: The Art of MeditationReading Passage:Meditation has been practiced for thousands of years, and it is still a popular form of relaxation and stress reduction today. The practice involvessitting quietly and focusing the mind on a single object, thought, or activity. It is believed that meditation can have numerous benefits, including improved concentration, reduced anxiety, and increased self-awareness.In recent years, scientific studies have begun to support these claims. A study published in the Journal of Psychosomatic Medicine found that regular meditation can lead to a significant reduction in stress levels. Another study, conducted by the University of California, Los Angeles (UCLA), showed that meditation can alter the brain’s structure, making it more adaptable to stress.One of the most common types of meditation is mindfulness meditation, which involves paying attention to the present moment without judgment. This form of meditation can help individuals become more aware of their thoughts and emotions, leading to better mental health.Below is a short excerpt from a mindfulness meditation session:“Close your eyes and take a deep breath in. Hold it for a moment. Now, exhale slowly. As you breathe, notice the sensation of the air entering and leaving your body. Feel the rise and fall of your chest. With each breath, let go of any tension you may be holding. Focus on the present moment, on the sounds around you, the feel of your clothing, the temperature of the air. Do not judge these experiences; simply observe them. Continue to breathe deeply and slowly, allowing yourself to become fully present in this moment.”Questions:1.What is the primary purpose of meditation according to the passage?A) To improve concentrationB) To reduce stressC) To increase self-awarenessD) To enhance physical strength2.Which of the following benefits of meditation is mentioned in the passage?A) Increased muscle massB) Reduced stress levelsC) Improved cardiovascular healthD) Enhanced immune system3.What is the main focus of mindfulness meditation?A) To control the mind’s thoughtsB) To become more aware of the present momentC) To visualize positive outcomesD) To practice deep breathing exercises4.According to the passage, how can meditation alter the brain’s structure?A) By increasing the size of the brainB) By making it more adaptable to stressC) By reducing the size of the prefrontal cortexD) By increasing the amount of gray matter5.What is the main activity described in the meditation excerpt?A) Mindfulness meditationB) Physical exerciseC) Breathing exercisesD) Guided imageryAnswers:1.B2.B3.B4.B5.A五、语言运用第一节 _ 完形填空（15分）Passage:After years of hard work, John finally graduated from university and secured a job as an engineer at a prestigious company. He was excited to start his new career, but little did he know that it would bring him unexpected challenges.John’s first day at work was filled with excitement and nervousness. He met his team, who seemed to be quite experienced and knowledgeable. However, John felt that he had a lot to learn. He spent the first few weeks observing his colleagues, trying to understand their work processes and the company culture.One afternoon, John’s manager called him into her office. She explained that they were about to start a new project and she needed John to lead the team. John was both thrilled and apprehensive. He knew he had to prove himself to his colleagues and the company.As the project progressed, John faced numerous difficulties. He often foundhimself struggling with complex technical issues and feeling overwhelmed. Despite these challenges, he refused to give up. He spent extra hours at the office, researching and seeking advice from his more experienced colleagues.One day, as John was working late, he received a message from his girlfriend, Sarah. She had heard about the project and wanted to support him. She offered to help him by providing background information on the industry and sharing relevant articles. John was grateful for her support and felt more confident in tackling the challenges ahead.Weeks passed, and finally, the day of the presentation arrived. John stood in front of the board, presenting his team’s findings. He was nervous but determined. As he finished, he received a round of applause from his colleagues. The project was a success, and John was proud of his achievements.The experience taught John that success is not just about having the right skills and knowledge; it’s also about perseverance and determination. He realized that with hard work and support, he could overcome any challenge.Questions:26.What was John’s first reaction to his new job?A)He was excited and eager to learn.B)He felt nervous and uncertain.C)He was disappointed because the job was not what he expected.D)He wanted to leave the company as soon as possible.Answer:A)He was excited and eager to learn.六、语言运用第二节 _ 语法填空（15分）文章内容：In recent years, the concept of “slow living” has gained popularity among people seeking a more balanced and fulfilling life. This movement encourages individuals to take a break from the fast-paced world and focus on the present moment. By slowing down, people can appreciate the beauty of nature, enjoy simple pleasures, and build stronger relationships with family and friends.One of the main reasons for the rise of slow living is the increasing awareness of the negative impacts of a fast-paced lifestyle. Stress, burnout, and mental health issues are on the rise, and many people are looking for ways to reduce their stress levels and improve their well-being. Slow living offers a solution by promoting activities such as mindfulness, exercise, and spending time in nature.Moreover, slow living is not just about reducing our pace but also about consuming less and living more sustainably. It encourages us to buy local, support small businesses, and reduce our carbon footprint. This shift in mindset has a positive impact on the environment and helps create a more sustainable future.Many people have already embraced the slow living philosophy and have seen significant improvements in their lives. For example, Sarah, a 35-year-old lawyer, decided to leave her stressful job and start her own business. She now spends more time with her family, practices yoga, and takes long walks in thecountryside. “I’ve never felt so relaxed and happy,” she says.However, the transition to slow living can be challenging for some. It requires a co nscious effort to change habits and prioritize what’s truly important. But with determination and patience, anyone can adopt a slower, more fulfilling lifestyle.语法填空题目：61.In recent years, the concept of “slow living” has [] popularity among people seeking a more balanced and fulfilling life.A. gainedB. gained inC. gained fromD. gained on答案：B. gained in七、写作第一节 _ 应用文写作（15分）Writing Section 1: Application WritingTask:Write a letter to the editor of your school newspaper expressing your concerns about the recent increase in littering around the school campus. Suggest at least two practical solutions that the school administration can implement to address this issue. Be sure to include a clear introduction, body, and conclusion.Example:。

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Enhanced ORR activity of cobalt porphyrin co-deposited with transition metal oxides on Au and C electrodes.The ORR thresholddataNiancai Cheng,Robert Kutz,Christopher Kemna,Andrzej Wieckowski ⇑Department of Chemistry,University of Illinois at Urbana-Champaign,Urbana,IL 61801,USAa r t i c l e i n f o Article history:Received 19March 2013Received in revised form 28June 2013Accepted 1July 2013Available online 20July 2013Keywords:Cobalt porphyrines Oxygen reductionTransition metal nanoparticles Fuel cella b s t r a c tWe co-deposited cobalt porphyrins (CoP)and transition metal oxides on gold and carbon/graphene elec-trodes as catalysts for the oxygen reduction reaction (ORR).Porphyrins were adsorbed spontaneously,and the transition metal oxides (CoO x and NiO x )were deposited using spontaneous deposition tactics or an electrochemical deposition method.The electrodes were characterized in acidic media by cyclic vol-tammetry (CV),by broad-band sum frequency generation (BB-SFG)and –in vacuum –by the Auger elec-tron spectroscopy (AES).The ORR activity data indicate that the activity of cobalt porphyrin towards ORR is enhanced by co-deposited transition metal oxides.The detailed reasons for this behavior are being interrogated.Ó2013Elsevier B.V.All rights reserved.1.IntroductionIt is a paramount importance to develop non-platinum metal catalysts to replace expensive platinum-based catalysts.This is one of the major approaches being explored to reduce high costs of fuel cells [1,2].Other metals than Pt [3–5],have been extensively studied as alternative catalysts.However,these metals present lower catalytic activity towards the oxygen reduction reaction (ORR),and lower stability in electrolytic media,compared to plat-inum.Transition metal N 4-macrocycles (such as metalloporphyrins and/or metallophthalocyanines)have been investigated for several decades as promising candidates for a fuel cell oxygen reduction reaction (ORR)catalyst due to lower costs compared to noble met-als,displaying –at the same time –a reasonable activity [6–11].To improve the macrocycles activity and durability even further,macrocycles were supported on carbon support and pyrolyzed [2,12–18].However,the ORR mechanisms and active sites in those pyrolyzed catalysts still remain under debate [19,20].In the above context,understanding oxygen reduction mecha-nistic pathways and active sites are important in development of the design of efﬁcient and stable cathode catalysts [21].Jahnke et al.[22]compared various organic dyestuff,and found that the N 4-compounds are active in catalyzing reduction of oxygen due to the interaction between oxygen and the central metal ion based on the model of molecular orbital theory.Alt et al.[23]found that the N 4-complexes have a conjugated p -electron system which seems to be a prerequisite for activation of the oxygen molecule.To preserve the high cathodic activity and,at the same time,to keep the macrocyclic structure intact,Wieckowski and co-workers [24]prepared carbon supported cobalt porphyrin catalysts without pyrolysis and found that the transition metal oxide addition to the porphyrin-pretreated surface improves the ORR activity of cobalt porphyrin (and some other metalloporphyrins)in an acid medium [24].The cited authors used the Campbell catalyst synthetic meth-od [24].The rigor of this method and,in particular,the use non-aqueous solvents and relatively high temperature needed for the catalyst preparation,were here avoided.In this paper,the cobalt porphyrin (CoP,Fig.1[25])and the transition metal oxides were co-deposited on gold and carbon/graphene electrodes,and such co-deposited CoP electrodes were used to study their catalytic activity towards ORR in acid medium.We were looking for the effect of metal oxides addition obtained spontaneously or under the electrochemical treatment.In agree-ment with result of Ref.[24],the effects of co-deposition on oxygen reduction are clearly measurable.Electrochemistry,broad-band sum frequency generation (BB-SFG),and the Auger electron spec-troscopy (AES)were used to characterize the co-deposited cata-lysts.The BB-SFG data (on the porphyrin alone)indicate that the porphyrin is adsorbed non-dissociatively,most likely through a physical adsorption route.2.Experimental 2.1.Electrode preparationThree types of electrodes:a gold disk electrode,a glassy carbon electrode,and the GC plus graphene (6mm)were used.The1572-6657/$-see front matter Ó2013Elsevier B.V.All rights reserved./10.1016/j.jelechem.2013.07.001Corresponding author.E-mail address:awieckow@ (A.Wieckowski).electrode surface was polished to a mirrorﬁnish with alumina slurry(0.3l m,and successively with0.05l m),was ultrasonically cleaned in high purity water,and it was washed with water and dried in argon.For spontaneous deposition of porphyrins,the elec-trode was next immersed in toluene(for5min)containing 0.3mmol dmÀ3CoP,and thoroughly washed off with toluene and water.Transition metal oxides were added to the electrodes,cov-ered by CoP,using spontaneous deposition[26]or the electro-chemical deposition method[27–29].In spontaneous deposition: (a)the CoP covered electrode was immersed in a10mmol CoCl2 solution(deairated by argon)for5min,and washed with water.(b)(to conform to previous procedure[24]),the electrode was im-mersed in an aqueous solution containing0.1mmol dmÀ3NaBH4 and0.2mmol dmÀ3NaOH for5s,washed off with water and dried. (The procedure to co-deposit nickel oxide with surface CoP was the same as that for CoO x.)In the electrochemical deposition method [27–29]:the cobalt oxides were obtained by anodic deposition of cobalt ions by the voltammetric cycling(50mV sÀ1)between0.0 and1.1V(SCE).The deposition was performed using10mM CoCl2 in0.1M sodium acetate solutions buffered at pH7.3.2.2.Graphene electrode preparationGraphene(XG sciences,the average diameters of particles is 5mm and$5–8nm thickness)was dispersed in ethanol(2mg/ mL)with ultrasonication for30min.Two microliters of the graph-ene suspension was cast on the surface of clean glassy carbon,and dried in air.Prior to use,the modiﬁed electrode was carefully rinsed with water to remove the loosely attached graphene,and dried in air.2.3.Catalysts characterization in vacuumThe Auger electron spectroscopy was carried out using a Phys-ical Electronics model PHI660Scanning Auger Microprobe(SAM). 5keV primary electron beam energy was used with the emission current of20.9nA.For the AES analyses,all samples were depos-ited on a gold foil substrate(4mmÂ4mm).The samples were prepared as follows:(1)the clean gold foil was immersed in the Co-porphyrin solution,then washed off with toluene and water.(2)For the oxides co-deposition,the CoP foil was immersed in 10mmol CoCl2(or NiCl2)solution,and again washed with the water.Then the foil was immersed in an aqueous solution contain-ing0.1mmol dmÀ3NaBH4and0.2mmol dmÀ3NaOH for5s, washed off with water and dried for use in argon environment.2.4.Electrochemical testElectrochemical and electrocatalytic properties of the catalysts were examined using cyclic voltammetry(CV)in a three-electrode cell at room temperature.Gold wire was used as the counter elec-trode,and the Ag/AgCl electrode(3.0M KCl)was a reference.(All potentials are reported versus a reversible hydrogen electrode (RHE)).2.5.BB-SFG characterizationThe BB-SFG method was described previously in a number of papers from this laboratory.The cobalt-porphyrin system had been identiﬁed for the BB-SFG study,as there was an interest in deter-mining whether spectroscopic characterization of the porphyrin as a function of applied potential could provide insight into its sta-bility and/or mechanism of operation.The results show the capa-bility of SFG spectroscopy to examine a wide variety of systems with unique results.3.Results and discussionThe gold electrode was cycled between0and1.6V with a scan rate of100mV sÀ1in the de-aerated0.1M HClO4solution until a stable CV was obtained(prior to electrochemical measurements and/or the CoP co-deposition).Fig.2A shows cyclic voltammo-grams of CoP/Au electrode with different immersing time and dif-ferent concentration of the CoP solution on the Au surface(the CoP coverage).When CoP was adsorbed,the Au oxide reduction peaks decreases,compared to the clean Au electrode.The oxide reduction peaks of CoP/Au decreases with an increase in the CoP coverage,as expected(Fig.2A).The data in Fig.2B indicate that the activity of surface(CoP/Au)towards ORR increases with the increase in the CoP coverage.The data in Fig.3A refer to a static Au electrode(the threshold oxygen reduction data)obtained in the O2-saturated0.1M per-chloric acid.They show the effect of transition metal oxides addi-tion on the activity of CoP towards ORR.The data indicate that the CoO x and NiO x themselves do not possess their own(enhanc-ing)activity towards ORR[24].However,as shown in Fig.3,they activate surface porphyrins;the enhancing effect is sensitive to the nature of co-adsorbed transition metal oxides.For instance, the CoO x–CoP/Au electrode displays better ORR activity than the NiO x–CoP/Au,in agreement with results of Reference[24] (Fig.3A).In order to further explore the effect of transition metal oxides on the activity of CoP towards oxygen reduction,the cata-lytic activity for CoP,CoP–NiO x and CoP–CoO x catalysts were stud-ied using a rotating disk electrode(RDE).Fig.4B presents the catalytic activity of CoP,CoP–NiO x and CoP–CoO x to oxygen reduc-tion on RDE at1600rpm.The limiting current of Au electrode is ca.3.0mA cmÀ2,which is in agreement with the literature data[30]. Recent work on Carbon modiﬁed by porphyrines would indicate that this is a4electron process[31].On pure Au it is a2electron process[30].The results of porphyrine modiﬁcation of the Au on the number of electrons exchanged needs to be determined by fur-ther research.The half wave potential of each catalyst was esti-mated by determining the maxima of the derivatives of the reduction current.The E1/2value of Au is0.22V.The CoP–CoO x/ Au is the most active catalyst that yields the highest E1/2value of 0.28V,which is higher than CoP/Au(E1/2=0.26V)and CoP–NiO x/ Au(E1/2=0.27V).For CoP and oxides supported on carbon,the E1/2value of CoP–CoO x/C is0.60V.Clearly,the co-deposited transi-tion metal oxides on Au electrode improve the activity of CoP towards ORR.structure of cobalt porphyrin:2,3,7,8,12,13,17,18-Octaethyl-21H,23H-porphine cobalt(II),the CoP.Chemistry705(2013)8–129The Auger electron spectroscopy was used to prove that the transition metal oxides were co-deposited on the electrodes.Fig.4shows the AES of CoP/Au,CoO x –CoP/Au and NiO x –CoP/Au.The CoO x –CoP/Au shows stronger cobalt signal compared with CoP/Au,which indicates that the cobalt oxides were co-deposited on the CoP/Au electrode.Equivalent data were obtained with the electrode.It is known that the electrochemical deposition delivers metal oxides to metal electrodes,and that the amount of the deposit can be controlled by a number of voltammetric deposition cycles.[27–29,32].Fig.5shows cyclic voltammograms of CoO x /Au elec-trode prepared by the electrochemical deposition in 0.1M NaOH solution [27,29,33].Fig.6shows the activity of CoP co-deposited CoO x on Au electrode prepared by the electrodeposition method.We found that the CoO x –CoP/Au has the best activity towards ORR when the CoO x was added to the CoP/Au electrode using two voltammetric cycles.Overall,the data show that the cobalt oxides prepared by the electrochemical method (Fig.6)[11]has similar effect on the por-phyrins as the cobalt oxides prepared by the spontaneous deposi-tion method (Fig.2A).The half wave potential of electrodes obtained from Fig.6is summarized in Table 1.Fig.2.(A)Cyclic voltammograms (CV)of Au electrode and CoP/Au electrodes with different immersing time and different concentration of CoP solution in the de-aerated 0.1M HClO 4solution and (B)Current–potential curves for oxygen reduction on a static Au electrode and CoP/Au with different CoP coverage in O 2-saturated 0.1M HClO 4solution (the ORR activity threshold).Fig. 3.In (A)static Au data (the ORR threshold).Current–potential curves for oxygen reduction on a static Au electrode,CoO x /Au,NiO x /Au,CoP/Au electrode,CoO x –CoP/Au electrode and NiO x –CoP/Au electrode in O 2-saturated 0.1M HClO solution.In (B)the rotating disk electrode data.The current–potential curves for oxygen reduction on Au electrode,CoP/Au RDE electrode,CoO x –CoP/Au electrode and NiO x –CoP/Au electrode in O 2-saturated 0.1M HClO 4solution and at 1600rpm.Fig.4.The Auger electron spectroscopy analysis of Au,CoP/Au,CoO x –CoP/Au and NiO x –CoP/Au.(The 4mm Â4mm Au strip was cleaned up and used to deposit CoP and transition metal oxides).because the oxide can increase the number of peroxide electrore-duction/chemical decomposition sites[34].Wieckowski and co-workers suggested that ORR proceeds via a single active site mechanism on N4–Co center[24].The recent BB-SFG data are shown in Fig.9.The consecutive ser-ies of potentiodynamic SFG spectra of the Co-porphyrin deposited onto an the Au electrode in0.1M HClO4indicate that there is noFig.5.Cyclic voltammograms(20th cycle)of the electrodeposited cobalt oxides the Au electrode in0.1M NaOH solution with the scan rate of50mV SÀ1.Fig. 6.The ORR threshold Au data using electrochemical deposition method. Current–potential curves for oxygen reduction on a static Au electrode,CoP/Au electrode and CoO x-CoP/Au electrode in O2-saturated0.1M HClO4solution.[11] with permission by Springer.Table1The half wave potential data of different electrodes and the data obtained from Fig.Electrodes Au CoP/Au CoP-CoO x/Au Half wave potential(V)0.340.420.47Fig.7.Current–potential curves for oxygen reduction of cobalt oxide and CoP on static glassy carbon electrode,GS(A)and grapheme electrode(B)in O2-saturated 0.1M HClO4solution.Fig.8.The Auger electron spectroscopy analysis of CoP/GC and CoP/Graphene.spectrum change after surface interaction(the nondissociative adsorption)and that there is no Stark effect due to the deposition. This discovery shows that the porphyrin surface attachment occurs via a physical adsorption route.4.ConclusionsThe activity of cobalt porphyrin towards oxygen reduction reac-tion(ORR)was enhanced by co-deposited transition metal oxides. (The presence of transition metal oxide near the N4–Co center im-proved the O2reduction activity).The enhancing effect was found sensitive to the nature of the co-adsorbed(with CoP)transition metal oxides.The CoO x–CoP/Au catalyst showed better activity to facilitate O2reduction than NiO x–CoP/Au.More work need to be done to improve the metal oxides durability and to increase under-standing of mechanisms of ORR on metalloporphyrin catalysts. From the BB-SFDG data we might conclude on weak forces that operate between the metal and the porphyrin(the forces are still strong enough to keep the porphyrin on the Au and C surface). The present work provides a promising way for the design and syn-thesis of non-precious metal electrocatalyst for ORR.AcknowledgementsThis work was supported by the National Science Foundation (under the grant CHE06-51083)and by the Army Research Ofﬁce (under the grant W911NF-08-10309).The authors also thank Dr. Haasch for the AES analyses carried out in the Frederick Seitz Mate-rials Research Laboratory Central Facilities,University of Illinois, which are partially supported by the US Department of Energy un-der grants DE-FG02-07ER46453and DE-FG02-07ER46471. 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