Folding Dynamics of Single GCN-4 Peptides by Fluorescence Resonant Energy Transfer Confocal

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自组装多肽水凝胶的制备及其在细胞培养上的应用

- 124 -新技术开发０　引言随着科技的迅速发展，生活水平的提高，人们对于新型材料的需求日益迫切，现有的材料无法满足生产实践，于是人们把目光投向了生物材料，尤其是水凝胶。

水凝胶是一种以水为分散介质的凝胶，具有三维的空间结构。

多肽基水凝胶指的是以多肽大分子为主要构件元件的形成的凝胶，在检测、医疗、和食品等领域有广泛的应用。

多肽水凝胶的形成方式包括两种，一种是通过化学交联，其产物被称为化学凝胶。

这种结合方式形成的水凝胶强度大，但是对反应条件要求高，同时产物难以具有响应性。

另一种则是通过非共价相互作用如氢键、范德华力、静电作用等物理作用力进行结合，即大分子自组装，其产物通常被称为物理凝胶。

这种结合方式反应条件简单，无需复杂的设备，组装体系对于外界环境改变较为敏感，可以有效感知外界温度、光、pH 等条件，还具有一定的生物相容性。

本文系统地总结了自组装多肽水凝胶的制备方法，并对当前多肽水凝胶的应用热点做出了评述，以期对多肽水凝胶未来的研究提供参考。

１　自组装多肽水凝胶的制备方法１．１　多肽分子自身组装形成水凝胶通过对多肽分子的构成氨基酸序列进行理性设计，可以使得多肽分子之间通过氢键、范德华力、疏水作用力等等自发组装，当浓度达到一定程度时，便可形成多肽水凝胶。

比如2012年，康艳晶等人通过氨水挥发调整溶液pH 值的方法，将多肽链pal-RLRRLRARARA 自组装形成了pH 响应性的智能水凝胶。

在常温下，由于中性及酸性条件下的精氨酸带正电，上述多肽链由于静电斥力无法形成凝胶，当溶液呈碱性时多肽链间的斥力减小，从而形成凝胶；除此之外，2012年周庆翰等人，利用天然蚕丝纤维优良的力学性能及良好的生物相容性设计了多肽序列RAG-16 （Ac-RADAGAGARADAGAGA-NH2），并利用精氨酸和天冬氨酸之间的静电相互作用及链间疏水作用自组装制备了RAG-16水凝胶。

其储能模量高达2723.9Pa，并且拥有良好的生物相容性。

J. Comput. Chem.

2D Depiction of Nonbonding Interactions forProtein ComplexesPENG ZHOU,1FEIFEI TIAN,2ZHICAI SHANG11Institute of Molecular Design&Molecular Thermodynamics,Department of Chemistry,Zhejiang University,Hangzhou310027,China2College of Bioengineering,Chongqing University,Chongqing400044,ChinaReceived7May2008;Revised25June2008;Accepted22July2008DOI10.1002/jcc.21109Published online22October2008in Wiley InterScience().Abstract:A program called the2D-GraLab is described for automatically generating schematic representation of nonbonding interactions across the protein binding interfaces.The inputﬁle of this program takes the standard PDB format,and the outputs are two-dimensional PostScript diagrams giving intuitive and informative description of the protein–protein interactions and their energetics properties,including hydrogen bond,salt bridge,van der Waals interaction,hydrophobic contact,p–p stacking,disulﬁde bond,desolvation effect,and loss of conformational en-tropy.To ensure these interaction information are determined accurately and reliably,methods and standalone pro-grams employed in the2D-GraLab are all widely used in the chemistry and biology community.The generated dia-grams allow intuitive visualization of the interaction mode and binding speciﬁcity between two subunits in protein complexes,and by providing information on nonbonding energetics and geometric characteristics,the program offers the possibility of comparing different protein binding proﬁles in a detailed,objective,and quantitative manner.We expect that this2D molecular graphics tool could be useful for the experimentalists and theoreticians interested in protein structure and protein engineering.q2008Wiley Periodicals,Inc.J Comput Chem30:940–951,2009Key words:protein–protein interaction;nonbonding energetics;molecular graphics;PostScript;2D-GraLabIntroductionProtein–protein recognition and association play crucial roles in signal transduction and many other key biological processes. Although numerous studies have addressed protein–protein inter-actions(PPIs),the principles governing PPIs are not fully under-stood.1,2The ready availability of structural data for protein complexes,both from experimental determination,such as by X-ray crystallography,and by theoretical modeling,such as protein docking,has made it necessary toﬁnd ways to easily interpret the results.For that,molecular graphics tools are usually employed to serve this purpose.3Although a large number of software packages are available for visualizing the three-dimen-sional(3D)structures(e.g.PyMOL,4GRASP,5VMD,6etc.)and interaction modes(e.g.MolSurfer,7ProSAT,8PIPSA,9etc.)of biomolecules,the options for producing the schematic two-dimensional(2D)representation of nonbonding interactions for PPIs are very scarce.Nevertheless,a few2D graphics programs were developed to depict protein-small ligand interactions(e.g., LIGPLOT,10PoseView,11MOE,12etc.).These tools,however, are incapable of handling the macromolecular complexes.Some other available tools presenting macromolecular interactions in 2D level mainly include DIMPLOT,10NUCPLOT,13and MON-STER,14etc.Amongst,only the DIMPLOT can be used for aesthetically visualizing the nonbinding interactions of PPIs. However,such a program merely provides a simple description of hydrogen bonds,hydrophobic interactions,and steric clashes across the binding interfaces.In this article,we describe a new molecular graphics tool, called the two-dimensional graphics lab for biosystem interac-tions(2D-GraLab),which adopts the page description language (PDL)to intuitively,exactly,and detailedly reproduce the non-bonding interactions and energetics properties of PPIs in Post-Script page.Here,the following three points are the emphasis of the2D-GraLab:(i)Reliability.To ensure the reliability,the pro-grams and methods employed in2D-GraLab are all widely used in chemistry and biology community;(ii)Comprehensiveness. 2D-GraLab is capable of handling almost all the nonbonding interactions(and even covalent interactions)across binding Additional Supporting Information may be found in the online version of this article.Correspondence to:Z.Shang;e-mail:shangzc@interface of protein complexes,such as hydrogen bond,salt bridge,van der Waals(vdW)interaction,hydrophobic contact, p–p stacking,disulﬁde bond,desolvation effect,and loss of con-formational entropy.The outputted diagrams are diversiform, including individual schematic diagram and summarized sche-matic diagram;(iii)Artistry.We elaborately scheme the layout, color match,and page style for different diagrams,with the goal of producing aesthetically pleasing2D images of PPIs.In addi-tion,2D-GraLab provides a graphical user interface(GUI), which allows users to interact with this program and displays the spatial structure and interfacial feature of protein complexes (see .Fig.S1).Identifying Protein Binding InterfacesAn essential step in understanding the molecular basis of PPIs is the accurate identiﬁcation of interprotein contacts,and based upon that,subsequent works are performed for analysis and lay-out of nonbonding mon methods identifyingprotein–protein binding interfaces include a Voronoi polyhedra-based approach,changes in solvent accessible surface area(D SASA),and various radial cutoffs(e.g.,closest atom,C b,andcentroid,etc.).152D-GraLab allows for the identiﬁcation of pro-tein–protein binding interfaces at residue and atom levels.Identifying Binding Interfaces at Residue LevelAll the identifying interface methods at residue level belong toradial cutoff approach.In the radial cutoff approach,referencepoint is deﬁned in advance for each residue,and the residues areconsidered in contact if their reference points fell within thedeﬁned cutoff ually,the C a,C b,or centroid are usedas reference point.16–18In2D-GraLab,cutoff distance is moreﬂexible:cutoff distance5r A1r B1d,where r A and r B are residue radii and d is set by users(as the default d54A˚,which was suggested by Cootes et al.19).Identifying Binding Interfaces at Atom LevelAt atom level,binding interfaces are identiﬁed using closestatom-based radial cutoff approach20and D SASA-basedapproach.21For the closest atom-based radial cutoff approach,ifthe distance between any two atoms of two residues from differ-ent chains is less than a cutoff value,the residues are consideredin contact;In the D SASA-based approach,the SASA is calcu-lated twice to identify residues involved in a binding interface,once for the monomers and once for the complex,if there is achange in the SASA(D SASA)of a residue when going from themonomers to the dimer form,then it is considered involved inthe binding interface.In2D-GraLab,three manners are provided for visualizing thebinding interfaces,including spatial structure exhibition,residuedistance plot,and residue-pair contact map(see .Figs.S2–S4).Analysis and2D Layout of NonbondingInteractionsThe inputﬁle of2D-GraLab is standard PDB format,and the outputs are two-dimensional PostScriptﬁle giving intuitive and informative representation of the PPIs and their strengths, including hydrogen bond,salt bridge,vdW interaction,desolva-tion effect,ion-pair,side-chain conformational entropy(SCE), etc.The outputs are in two forms as individual schematic dia-gram and summarized schematic diagram.The individual sche-matic diagram is a detailed depiction of each nonbonding proﬁle,whereas the summarized schematic diagram covers all nonbonding interactions and disulﬁde bonds across the binding interface.To produce the aesthetically high quality layouts,which pos-sess reliable and accurate parameters,several widely used pro-grams listed in Table1are employed in2D-GraLab to perform the core calculations and analysis of different nonbonding inter-actions.2D-GraLab carries out prechecking procedure for pro-tein structures and warns the structural errors,but not providing revision and reﬁnement functions.Therefore,prior to2D-GraLab analysis,protein structures are strongly suggested to be prepro-cessed by programs such as PROCHECK(structure valida-tion),27Scwrl3(side-chain repair),28and X-PLOR(structure reﬁnement).29Individual Schematic DiagramHydrogen BondThe program we use for analyzing hydrogen bonds across bind-ing interfaces is HBplus,23which calculates all possible posi-tions for hydrogen atoms attached to donor atoms which satisfy speciﬁed geometrical criteria with acceptor atoms in the vicinity. In2D-GraLab,users can freely select desired hydrogen bonds involving N,O,and/or S atoms.Besides,the water-mediated hydrogen bond is also given consideration.Bond strength of conventional hydrogen bonds(except those of water-mediated Table1.Standalone Programs Employed in2D-GraLab.Program FunctionReduce v3.0322Adding hydrogen atoms for proteinsHBplus v3.1523Identifying hydrogen bonds and calculatingtheir geometric parametersProbe v2.1224Identifying steric contacts and clashes at atomlevelMSMS v2.6125Calculating SASA values of protein atoms andresiduesDelphi v4.026Calculating Coulombic energy and reactionﬁeld energy,determining electrostatic energyof ion-pairsDIMPLOT v4.110Providing application programming interface,users can directly set and executeDIMPLOT in the2D-GraLab GUI9412D Depiction of Nonbonding Interactions for Protein ComplexesFigure1.(a)Schematic representation of a conventional hydrogen bond and a water-mediated hydro-gen bond across the binding interface of IGFBP/IGF complex(PDB entry:2dsr).This diagram was produced using2D-Gralab.The conventional hydrogen bond is formed between the atom N(at the backbone of residue Leu69in chain B)and the atom OE1(at the side-chain of residue Glu3in chain I);The water-mediated hydrogen bond is formed between the atom ND1(at the side-chain of residue His5in chain B)and the atom O(at the backbone of residue Asp20in chain I),and because hydrogen positions of water are almost never known in the PDBﬁle,the water molecule,when serving as hydrogen bond donor,is not yet determined for its H...A length and D—H...A angle,denoted as mark ‘‘????.’’In this diagram,chains,residues,and atoms are labeled according to the PDB format.(b)Spa-tial conformation of the conventional hydrogen bond.(c)Spatial conformation of the water-mediated hydrogen bond.hydrogen bonds)is calculated using Lennard-Jones 8-6potential with angle weighting.30D U HB¼E m 3d m 8À4d m6"#cos 4h ðh >90 Þ(1)where d is the separation between the heavy acceptor atom andthe donor hydrogen atom in angstroms;E m ,the optimum hydro-gen-bond energy for the particular hydrogen-bonding atoms con-sidered;d m ,the optimum hydrogen-bond length for the particu-lar hydrogen-bonding atoms considered.E m and d m vary accord-ing to the chemical type of the hydrogen-bonding atoms.The hydrogen bond potential is set to zero when angle h 908.31Hydrogen bond parameters are taken from CHARMM force ﬁeld (for N and O atoms)and Autodock (for S atom).32,33Figure 1a is the schematic representation of a conventional hydrogen bond and a water-mediated hydrogen bond across the binding interface of insulin-like growth factor-binding protein (IGFBP)/insulin-like growth factor (IGF)complex.In this dia-gram,abundant information about the hydrogen bond geometry and energetics properties is presented in a readily acceptant manner.Figures 1b and 1c are spatial conformations of the cor-responding conventional hydrogen bond and water-mediated hydrogen bond.Van der Waals InteractionThe small-probe approach developed in Richardson’s laboratory enables us to detect the all atom contact proﬁle in protein pack-ing.2D-GraLab uses program Probe 24to realize this method to identity steric contacts and clashes on the binding interfaces.Word et al.pointed out that explicit hydrogen atoms can effec-tively improve Probe’s performance.24However,considering calculations with explicit hydrogen atoms are time-consuming,and implicit hydrogen mode is also possibly used in some cases;therefore,in 2D-GraLab,both explicit and implicit hydrogen modes are provided for users.In addition,2D-GraLab uses the Reduce 22to add hydrogen atoms for proteins,and this programis also developed in Richardson’s laboratory and can be wellcompatible with Probe.According to previous deﬁnition,vdW interaction between two adjacent atoms is classiﬁed into wide contact,close contact,small overlap,and bad overlap.24Typically,vdW potential function has two terms,a repulsive term and an attractive term.In 2D-GraLab,vdW interaction is expressed as Lennard-Jones 12-6potential.34D U SI ¼E m d m d 12À2d md6"#(2)where E m is the Lennard-Jones well depth;d m is the distance at the Lennard-Jones minimum,and d is the distance between two atoms.The Lennard-Jones parameters between pairs of different atom types are obtained from the Lorentz–Berthelodt combina-tion rules.35Atomic Lennard-Jones parameters are taken from Probe and AMBER force ﬁeld.24,36Figure 2a was produced using 2D-GraLab and gives a sche-matic representation of steric contacts and clashes (overlaps)between the heavy chain residue Tyr131and two light chain res-idues Ser121and Gln124of cross-reaction complex FAB (the antibody fragment of hen egg lysozyme).By this diagram,we can obtain the detail about the local vdW interactions around the residue Tyr131.In contrast,such information is inaccessible in the 3D structural ﬁgure (Fig.2b).Desolvation EffectIn 2D-GraLab,program MSMS 25is used to calculate the SASA values of interfacial residues at atom level,and four atomic radii sets are provided for calculating the SASA,including Bondi64,Chothia75,Li98,and CHARMM83.32,37–39Bondi64is based on contact distances in crystals of small molecules;Chothia75is based on contact distances in crystals of amino acids;Li98is derived from 1169high-resolution protein crystal structures;CHARMM83is the atomic radii set of CHARMM force ﬁeld.Desolvation free energy of interfacial residues is calculated using empirical additive model proposed by Eisenberg andFigure 2.(a)Schematic representation of steric contacts and overlaps between the residue Tyr131in heavy chain (chain H)and the surrounding residues Ser121and Gln124in light chain (chain L)of cross-reaction complex FAB (PDB entry:1fbi).This diagram was produced using 2D-Gralab in explicit hydrogen mode.In this diagram,interface is denoted by the broken line;Wide contact,close contact,small overlap,and bad overlap are marked by blue circle,green triangle,yellow square,and pink rhombus,respectively;Moreover,vdW potential of each atom-pair is given in the histogram,with the value measured by energy scale,and the red and blue indicate favorable (D U \0)and unfav-orable (D U [0)contributions to the binding,respectively;Interaction potential 20.324kcal/mol in the center circle denotes the total vdW contribution by residue Tyr131;Chains,residues,and heavy atoms are labeled according to the PDB format,and hydrogen atoms are labeled in Reduce format.(b)Spatial conformation of chain H residue Tyr131and its local environment.Green or yellow stands forgood contacts (green for close contact and yellow for slight overlaps \0.2A˚),blue for wide contacts [0.25A˚,hot pink spikes for bad overlaps !0.4A ˚.It is revealed that Tyr131is in an intensive clash with chain L Gln124,while in slight contact with chain L Ser121,which is well consistent with the 2D schematic diagram.9432D Depiction of Nonbonding Interactions for Protein Complexes944Zhou,Tian,and Shang•Vol.30,No.6•Journal of Computational ChemistryFigure2.(Legend on page943.)Maclachlam,40and the conformation of interfacial residues is assumed to be invariant during the binding process.D G dslv¼Xic i D A i(3)where the sum is over all the atoms;c i and D A i are the atomic solvation parameter(ASP)and the changes in solvent accessible surface area(D SASA)of atom i,respectively.Juffer et al.41 found that although desolvation free energies calculated from different ASP sets are linear correlation to each other,the abso-lute values are greatly different.In view of that,2D-GraLab pro-vides four ASP sets published in different periods:Eisenberg86, Kim90,Schiffer93,and Zhou02.40,42–44As shown in Figure3,the D SASA and desolvation free energy of interfacial residues in chain A of HLA-A*0201pro-tein complex during the binding process are reproduced in a rotiform diagram form using2D-GraLab.In this diagram,the desolvation free energy contributed by chain A is28.056kcal/ mol,and moreover,the D SASA value of each interfacial residue is also presented clearly.Ion-PairThere are six types of residue-pairs in the ion-pairs:Lys-Asp, Lys-Glu,Arg-Asp,Arg-Glu,His-Asp,and ually,ion-pairs include three kinds:salt bridge,NÀÀO bridge,and longer-range ion-pair,and found that most of the salt bridges are stabi-lizing toward proteins;the majority of NÀÀO bridges are stabi-lizing;the majority of the longer-range ion-pairs are destabiliz-ing toward the proteins.45The salt bridge can be further distin-guished as hydrogen-bonded salt bridge(HB-salt bridge)and nonhydrogen-bonded salt bridge(NHB-salt bridge or salt bridge).46In2D-GraLab,the longer-range ion-pair is neglected, and for short-range ion-pair,four kinds are deﬁned:HB-salt bridge,NHB-salt bridge or salt bridge,hydrogen-bonded NÀÀO bridge(HB-NÀÀO bridge),and nonhydrogen-bonded N-O bridge (NHB-NÀÀO bridge or NÀÀO bridge).Although both the N-terminal and C-terminal residues of a given protein are also charged,the large degree ofﬂexibility usually experienced by the ends of a chain and the poor structural resolution resulting from it.47Therefore,we preclude these terminal residues in the 2D-GraLab.A modiﬁed Hendsch–Tidor’s method is used for calculating association energy of ion-pairs across binding interfaces.48D G assoc¼D G dslvþD G brd(4)where D G dslv represents the sum of the unfavorable desolvation penalties incurred by the individual ion-pairing residues due to the change in their environment from a high dielectric solvent (water)in the unassociated state;D G brd represents the favorable bridge energy due to the electrostatic interaction of the side-chain charged groups.We usedﬁnite difference solutions to the linearized Poisson–Boltzmann equations in Delphi26to calculate the D G dslv and D G brd.Centroid of the ion-pair system is used as grid center,with temperature of298.15K(in this way,1kT50.593kcal/mol),and the Debye-Huckel boundary conditions are applied.49Considering atomic parameter sets have a great inﬂu-ence on the continuum electrostatic calculations of ion-pair asso-ciation energy,502D-GraLab provides three classical atomic parameter sets for users,including PARSE,AMBER,and CHARMM.51–53Figure4is the schematic representation of four ion-pairs formed across the binding interface of penicillin acylase enzyme complex.This diagram clearly illustrates the information about the geometries and energetics properties of ion-pairs,such as bond length,centroid distance,association energy,and angle. The ion-pair angle is deﬁned as the angle between two unit vec-tors,and each unit vector joins a C a atom and a side-chain charged group centroid in an ion-pairing residue.54In this dia-gram,the four ion-pairs,two HB-salt bridges,and two HB-NÀÀO bridges formed across the binding interface are given out. Association energies of the HB-salt bridges are both\21.5 kcal/mol,whereas that of the HB-NÀÀO bridges are all[20.5 kcal/mol.Therefore,it is believed that HB-salt bridge is more stable than HB-NÀÀO bridge,which is well consistent with the conclusion of Kumar and Nussinov.45,46Side-Chain Conformational EntropyIn general,SCE can be divided into the vibrational and the con-formational.55Comparison of several sets of results using differ-ent techniques shows that during protein folding process,the mean conformational free energy change(T D S)is1kcal/mol per side-chain or0.5kcal/mol per bond.Changes in vibrational entropy appear to be negligible compared with the entropy change resulted from the loss of accessible rotamers.56SCE(S) can be calculated quite simply using Boltzmann’s formulation.57S¼ÀRXip i ln p i(5)where R is the universal gas constant;The sum is taken over all conformational states of the system and p i is the probability of being in state i.Typical methods used for SCE calculations, include self-consistent meanﬁeld theory,58molecular dynam-ics,59Monte Carlo simulation,60etc.,that are all time-consum-ing,thus not suitable for2D-GraLab.For that,the case is sim-pliﬁed,when we calculate the SCE of an interfacial residue,its local surrounding isﬁxed(adopting crystal conformation).In this way,SCE of each interfacial residue is calculated in turn.For the20coded amino acids,Gly,Ala,Pro,and Cys in disulﬁde bonds are excluded.57For other cases,each residue’s side-chain conformation is modeled as a rotamer withﬁnite number of discrete states.61The penultimate rotamer library used was developed by Lovell et al.,62as recommended by Dun-brack for the study of SCE.63For an interfacial residue,the potential E i of each rotamer i is calculated in both binding state and unbinding state,and subsequently,rotamer’s probability dis-tribution(p)of this residue is resulted by Boltzmann’s distribu-tion law,then the SCE in different states are solved out using eq.(5).The situation of rotamer i is deﬁned as serious clash or nonclash:serious clash is the clash score of rotamer i more than a given threshold value,and then E i511;whereas for the9452D Depiction of Nonbonding Interactions for Protein Complexes946Zhou,Tian,and Shang•Vol.30,No.6•Journal of Computational ChemistryFigure3.Schematic representation of desolvation effect for interfacial residues in chain A of HLA-A*0201complex(PDB entry:1duz).This diagram was produced using2D-GraLab.In this diagram,the pie chart is equally divided,with each section indicates an interfacial residue in chain A;In a sec-tor,red1blue is the SASA of corresponding residue in unbinding state,the blue is in binding state,and the red is thus of D SASA;The green polygonal line is made by linking desolvation free energy ofeach interfacial residue,and at the purple circle,desolvation free energy is0(D U50),beyond thiscircle indicates unfavorable contributions to binding(D U[0),otherwise is favorable(D U\0);Inthe periphery,residue symbols are colored in red,blue,and black in terms of favorable,unfavorable,and neutral contributions to the binding,respectively;The SASA and desolvation free energy for eachinterfacial residue can be measured qualitatively by the horizontally black and green scales.[Colorﬁgure can be viewed in the online issue,which is available at .]Figure4.Four ion-pairs formed across the binding interface of penicillin acylase enzyme complex (PDB entry:1gkf).In thisﬁgure,left is2D schematic diagram produced using2D-GraLab,and posi-tively and negatively charged residues are colored in blue and red,respectively;Bridge-bonds formed between the charged atoms of ion-pairs are colored in green,blue,and yellow dashed lines for the hydrogen-bonded bridge,nonhydrogen-bonded bridge,and long-range interactions,respectively;The three parameters in bracket are ion-pair type,angle,and association energy.The right in thisﬁgure is the spatial conformations of corresponding ion-pairs.[Colorﬁgure can be viewed in the online issue, which is available at .]Figure5.(a)Loss of side-chain conformational entropy of chain B interfacial residues in HIV-1 reverse transcriptase complex(PDB entry:1rt1).This diagram was produced using2D-GraLab.In this diagram,the pie chart is equally divided,with each section indicates an interfacial residue in chain B; In a sector,side-chain conformational entropies in unbinding and binding state are colored in yellow and blue,respectively;The green polygonal line is made by linking conformational free energy of each interfacial residue;The conformational entropy and conformational free energy for each interfa-cial residue can be measured qualitatively by the horizontally black and green scales,respectively;In the periphery,residue symbols are colored in yellow,blue,and black in terms of favorable,unfavora-ble,and neutral contributions to binding,respectively.(b)The rotamers of chain B interfacial residues Lys20,Lys22,Tyr56,Asn136,Ile393,and Trp401in HIV-1reverse transcriptase complex.These rotamers were generated using2D-GraLab.[Colorﬁgure can be viewed in the online issue,which is available at .]9472D Depiction of Nonbonding Interactions for Protein Complexes948Zhou,Tian,and Shang•Vol.30,No.6•Journal of Computational ChemistryFigure5.(Legend on page947.)Figure6.The summarized schematic diagram of nonbonding interactions and disulﬁde bond across the interface of AIV hemagglutinin H5complex(PDB entry:1jsm).Length of chain A and chain B are321and160,represented as two bold horizontal lines.Interface parts in the bold lines are colored in orange,and residue-pairs in interactions are linearly linked;Conventional hydrogen bond,water-mediated hydrogen bond,ionpair,hydrophobic force,steric clash,p–p stacking,and disulﬁde bond are colored in aqua,bottle green,red,blue,purple,yellow,and brown,respectively;In the‘‘dumbbell shape’’symbols,residue-pair types and distances are also presented.[Colorﬁgure can be viewed in the online issue,which is available at .]9492D Depiction of Nonbonding Interactions for Protein Complexescase of nonclash,four potential functions are used in2D-Gra-Lab:(i)E i5E0,a constant61;(ii)statistical potential,the poten-tial energy E i of rotamer i is calculated from database-derived probability61;(iii)coarse-grained model,E i of rotamer i is esti-mated by atomic contact energies(ACE)64;and(iv)Lennard-Jones potential.58Loss of binding entropy of chain B interfacial residues in HIV-1reverse transcriptase complex is schematically repre-sented in Figure5a.Similar to desolvation effect diagram,loss of binding entropy is also presented in a rotiform diagram form. This diagram reveals that during the process of forming HIV-1 reverse transcriptase complex,the total loss of conformational free energy of chain B is9.14kcal/mol,indicating a strongly unfavorable contribution to binding(D G[0),and the average loss of conformational free energy for each residue is about0.3 kcal/mol,much less than those in protein folding(about1kcal/ mol56).Figure5b shows the rotamers of six interfacial residues in chain B.Summarized Schematic DiagramFigure6illustrates nonbonding interactions and disulﬁde bond formed across the binding interface of avian inﬂuenza virus (AIV)hemagglutinin H5.This protein is a dimer linked by a disulﬁde bond.In this diagram,conventional hydrogen bond, water-mediated hydrogen bond,ion-pair,hydrophobic force, steric clash,p–p stacking,and disulﬁde bond are represented in different colors.Hydrogen bonds,colored in aqua,are calculated by program HBplus.23Data in this diagram are the separation between the acceptor atom and the heavy donor atom.Water-mediated hydrogen bonds are colored in bottle green, also calculated by HBplus.23Ion-pairs,colored in red,include salt bridge and NÀÀO bridge,determined by the Kumar’s rule.45,46Data in this dia-gram are centroid distance of ion-pair.Hydrophobic forces are colored in blue.According to the D SASA rule,if the two apolar and/or aromatic interfacial resi-dues(Leu,Ala,Val,Ile,Met,Cys,Pro,Tyr,Phe,and Trp)are within the distance d\r A1r B12.8(r A and r B are side-chain radii,2.8is the diameter of water molecule),they are considered in hydrophobic contact.Data in this diagram are centroid–cent-roid separation between the two residues.Steric clashes are colored in purple.Here,only bad overlaps calculated by Probe24are presented.In2D-GraLab,explicit and implicit hydrogen modes are provided,hydrogen atoms in explicit hydrogern mode are added using Reduce.22Data in this diagram are the centroid–centroid separation when the two atoms are badly overlapped.p–p stacking are colored in yellow.Presently,studies on pro-tein stacking interactions are in lack.In2D-GraLab,p–p stack-ing is identiﬁed using the McGaughey’s rule,65i.e.,if the cent-roid–centroid separation between two aromatic rings is within 7.5A˚,they are regarded as p–p stacking(aromatic residues are Phe,Tyr,Trp,and His).This rule has been successfully adopted to study the p–p stacking across protein interfaces by Cho et al.66Besides,2D-GraLab also sets the constraints of stacking angle(dihedral angel between the planes of two aromatic rings).Data in this diagram are centroid–centroid separations between two aromatic rings in stacking state.Disulﬁde bonds are colored in brown,taken from the PDB records.Data in this diagram are the separations of two sulﬁde atoms.ConclusionsMost,if not all,biological processes are regulated through asso-ciation and dissociation of protein molecules and essentially controlled by nonbonding energetics.67Graphically-intuitive vis-ualization of these nonbonding interactions is an important approach for understanding the mechanism of a complex formed between two proteins.Although a large number of software packages are available for visualizing the3D structures,the options for producing schematic2D summaries of nonbonding interactions for a protein complex are comparatively few.In practice,the2D and3D visualization methods are complemen-tary.In this article,we have described a new2D molecular graphics tool for analyzing and visualizing PPIs from spatial structures,and the intended goal is to schematically present the nonbonding interactions stabilizing the macromolecular complex in a graphically-intuitive manner.We anticipate that renewed in-terest in automated generation of2D diagrams will signiﬁcantly reduce the burden of protein structure analysis and make insights into the mechanism of PPIs.2D-GraLab is written in C11and OpenGL,and the output-ted2D schematic diagrams of nonbinding interactions are described in PostScript.Presently,2D-GraLab v1.0is available to academic users free of charge by contacting us. References1.Chothia,C.;Janin,J.Nature1974,256,705.2.Jones,S.;Thornton,J.M.Proc Natl Acad Sci USA1996,93,13.3.Luscombe,N.M.;Laskowski,R.A.;Westhead,D.R.;Milburn,D.;Jones,S.;Karmirantzoua,M.;Thornton,J.M.Acta Crystallogr D 1998,54,1132.4.DeLano,W.L.The PyMOL Molecular Graphics System;DeLanoScientiﬁc:San Carlos,CA,2002.5.Petrey,D.;Honig,B.Methods Enzymol2003,374,492.6.Humphrey,W.;Dalke,A.;Schulten,K.J Mol Graphics1996,14,33.7.Gabdoulline,R.R.;Wade,R.C.;Walther,D.Nucleic Acids Res2003,31,3349.8.Gabdoulline,R.R.;Hoffmann,R.;Leitner,F.;Wade,R.C.Bioin-formatics2003,19,1723.9.Wade,R. C.;Gabdoulline,R.R.;De Rienzo, F.Int J QuantumChem2001,83,122.10.Wallace, A. C.;Laskowski,R. A.;Thornton,J.M.Protein Eng1995,8,127.11.Stierand,K.;Maaß,P.C.;Rarey,M.Bioinformatics2006,22,1710.12.Clark,A.M.;Labute,P.J Chem Inf Model2007,47,1933.13.Luscombe,N.M.;Laskowski,R. A.;Thorntonm J.M.NucleicAcids Res1997,25,4940.14.Salerno,W.J.;Seaver,S.M.;Armstrong,B.R.;Radhakrishnan,I.Nucleic Acids Res2004,32,W566.15.Fischer,T.B.;Holmes,J.B.;Miller,I.R.;Parsons,J.R.;Tung,L.;Hu,J.C.;Tsai,J.J Struct Biol2006,153,103.950Zhou,Tian,and Shang•Vol.30,No.6•Journal of Computational Chemistry。

溶液空间限域法制备有机-无机杂化卤化铅钙钛矿单晶薄膜及其器件应用研究进展

第53卷第4期2024年4月人㊀工㊀晶㊀体㊀学㊀报JOURNAL OF SYNTHETIC CRYSTALS Vol.53㊀No.4April,2024溶液空间限域法制备有机-无机杂化卤化铅钙钛矿单晶薄膜及其器件应用研究进展张庆文,单东明,张㊀虎,丁㊀然(吉林大学电子科学与工程学院,集成光电子学国家重点实验室,长春㊀130012)摘要:近年来,有机-无机杂化卤化铅钙钛矿材料因其出色的光电特性在国际上备受瞩目,并已成功应用于太阳能光伏㊁光电探测㊁电致发光等多个领域㊂目前绝大部分器件研究都集中在钙钛矿多晶材料上,但钙钛矿单晶材料拥有更低的缺陷态密度㊁更高的载流子迁移率㊁更长的载流子复合寿命㊁更宽的光吸收范围,以及更高的稳定性等优异的性质,可有效减少载流子传输过程中的散射损失,以及在晶界处的非辐射复合,并抑制离子迁移所引起的迟滞效应㊂采用钙钛矿单晶薄膜作为器件有源层有望制备性能更高效且更稳定的钙钛矿光电器件㊂目前,已报道的多种钙钛矿单晶薄膜制备方法包括溶液空间限域法㊁化学气相沉积法㊁自上而下加工法等,其中溶液空间限域法的发展和应用最为广泛㊂本文聚焦利用溶液空间限域法制备高质量钙钛矿单晶薄膜的相关方法,以及钙钛矿单晶薄膜在光电探测器㊁太阳能电池㊁场效应晶体管和发光二极管等相关器件应用中的研究进展,并对钙钛矿单晶薄膜及其光电器件的未来发展趋势进行了展望㊂关键词:钙钛矿半导体材料;溶液空间限域法;钙钛矿单晶薄膜;光电子器件;单晶薄膜生长中图分类号:O78;O484;TN36㊀㊀文献标志码:A ㊀㊀文章编号:1000-985X (2024)04-0572-13Research Progress on Preparation of Organic-Inorganic Hybrid Lead Halide Perovskite Single-Crystalline Thin-Films by Solution-Processed Space-Confined Method and Their Device ApplicationsZHANG Qingwen ,SHAN Dongming ,ZHANG Hu ,DING Ran(State Key Laboratory of Integrated Optoelectronics,College of Electronic Science and Engineering,Jilin University,Changchun 130012,China)㊀㊀收稿日期:2023-11-20㊀㊀基金项目:国家重点研发计划青年科学家项目(2022YFB3607500);国家自然科学基金(62274076)㊀㊀作者简介:张庆文(1999 ),男,山东省人,硕士研究生㊂E-mail:zhangqw1012@ ㊀㊀通信作者:丁㊀然,教授,博士生导师㊂E-mail:dingran@Abstract :In recent years,organic-inorganic hybrid lead halide perovskite materials have attracted much attention in the world because of their excellent photoelectric properties,and have been successfully applied in many fields such as solar photovoltaic,photoelectric detection,electroluminescence and so on.At present,most of the device research focuses on perovskite polycrystalline materials,but perovskite single crystal materials have excellent properties such as lower defect state density,higher carrier mobility,longer carrier recombination lifetime,wider light absorption range and higher stability,which can effectively reduce the scattering loss during carrier transport and non-radiative recombination at the grain boundary,and inhibit the hysteresis effect caused by ion ing perovskite single crystal thin film as the active layer of the device is expected to produce more efficient and stable perovskite photoelectric devices.At present,many preparation methods of perovskite single crystal films have been reported,mainly including solution-processed space-confined method,chemical vapor deposition method,top-down processing method,etc.Among them,solution-processed space-confined method is the most widely developed and applied.This paper focuses on the preparation of high-quality perovskite single crystal thin films by solution-processed space-confined method,and the research progress of perovskite single crystal thin films in photodetectors,solar cells,field effect transistors,light-emitting diodes and other related devices,and prospects the future development trend of perovskite single crystal thin films and photoelectric devices.㊀第4期张庆文等:溶液空间限域法制备有机-无机杂化卤化铅钙钛矿单晶薄膜及其器件应用研究进展573㊀Key words:hybrid perovskite semiconductor;solution-processed space-confined method;perovskite single-crystalline thin-film;optoelectronic device;growth of single crystal thin film0㊀引㊀㊀言近年来,有机-无机杂化卤化铅钙钛矿材料因高的光吸收系数[1]㊁高的载流子迁移率[2-3]㊁长的载流子扩散距离[4]㊁带隙可调谐[5-7]等优异的光电性能,引起了科研界和产业界的广泛关注㊂尤其是在光伏器件领域,钙钛矿电池的功率转换效率(power conversion efficiency,PCE)从最初的3.8%[8]攀升到目前的25.9%[9],发展速度出人意料且远超其他光伏材料体系㊂理论计算得到单结钙钛矿电池的最高转换效率可达33%,这一效率优于晶体硅的理论极限效率29.4%㊂除光伏领域外,钙钛矿材料在光电探测[5,10-15]㊁电致发光[16-19]㊁光泵激光[20-23]和辐射探测[24-26]等诸多光电领域也展现出巨大的应用前景㊂有机-无机杂化卤化铅钙钛矿材料化学结构式通常为ABX3,一般为立方体或八面体结构[27],对于典型的三维钙钛矿材料,其中A代表一价阳离子(如MA+㊁FA+等),B代表二价Pb2+阳离子,X为一价卤素阴离子(如Cl-㊁Br-㊁I-等)㊂在钙钛矿材料中,B离子位于立方晶胞的中心[28],被6个X离子包围形成配位立方八面体结构㊂钙钛矿光电器件有源层材料以多晶薄膜为主,多晶材料虽然在器件应用方面已展现出卓越的性能,但是内部存在大量晶界,且在晶界处存在高密度的晶格位错,以及无序的晶粒生长,从而导致薄膜内存在大量的晶格缺陷和可自由移动的离子㊂多晶膜内大量晶粒㊁晶界㊁空隙和表面缺陷等,会显著增大非辐射复合过程并诱使激子猝灭,严重限制光电及电光转换效率[29-30]㊂同时,在外场作用下钙钛矿多晶膜中会产生明显的离子迁移现象,移动的离子会抑制自由载流子的感生㊁积累与传输,也将极大影响器件的光电性能[31]㊂相比之下,钙钛矿单晶拥有更低的缺陷态密度㊁更长的载流子扩散长度㊁更长的载流子复合寿命㊁更宽的光吸收范围,以及更高的稳定性等[32-33]㊂这些优秀的本征特性为克服以上挑战提供了良好的载体,有望制备性能更高效且更稳定的钙钛矿光电器件㊂从晶体形态学角度区分,钙钛矿单晶材料主要可分为块体[34-35]和薄膜两种类型[36-38]㊂相比于单晶块体材料,单晶薄膜更易于与传统半导体工艺相集成,并有望制备性能更加优越的光电器件,更因其突出的柔性[39]和机械性,在未来柔性电子器件领域也展现出良好的应用前景㊂目前,已报道的钙钛矿单晶薄膜制备方法中,主要包括溶液空间限域法[36-37,40]㊁化学气相沉积法[41-44]㊁自上而下加工法[13,45-48]等,其中溶液空间限域法的发展和应用最为广泛㊂由于单晶各向异性生长,为了有效控制单晶薄膜厚度,抑制薄膜沿垂直纵向方向生长,并且提高水平横向方向的生长速率㊁增大薄膜的表面积,常引入空间结构限制策略,实现可控制备钙钛矿单晶薄膜㊂本文聚焦利用溶液空间限域法制备高质量钙钛矿单晶薄膜的相关技术方法,以及钙钛矿单晶薄膜在光电探测器㊁太阳能电池㊁场效应晶体管和电致发光器件等相关器件应用中的研究进展㊂同时,对未来钙钛矿单晶薄膜材料的发展及其应用所面临的难题提出可行的解决方案㊂1㊀钙钛矿单晶薄膜生长策略目前,溶液法生长钙钛矿单晶块体技术较为成熟,包括冷却结晶法[4,49-52]㊁逆温结晶法[46,53-57]㊁反溶剂扩散法[58-62]等方法,但单晶块体的厚度较厚,展现出较高的光吸收损耗和较长的激子扩散距离,不适于垂直结构型光电器件的应用㊂为了进一步扩展钙钛矿单晶材料在光电器件领域的应用,急需开发厚度和形貌可控㊁重复性高的钙钛矿单晶薄膜制备方法㊂2016年,陕西师范大学刘生忠教授团队报道采用空间限域结合动态流反应系统的生长方法,通过控制两个玻璃片之间的间隙大小,确保钙钛矿单晶薄膜在预设的限域空间结构内生长,达到单晶薄膜厚度可控的目的,如图1(a)所示[37]㊂利用蠕动泵驱动空隙中溶液流动,为单晶薄膜生长提供源源不断的前驱体溶液,最终实现一系列厚度约为150μm的MAPbI3单晶薄片㊂然而,微米厚度的钙钛矿单晶薄膜依然无法满足垂直结构型器件的需求,通过施加外部压力的方式来控制几何限域空间的间隙距离,达到进一步减薄钙钛矿单晶薄膜的作用㊂2016年,中国科学院化学研究所胡劲松研究员团队设计如图1(b)所示装置,实现可控制备厚度均匀的钙钛矿单晶薄膜生长方法[36]㊂实验具体流程是将两个平面衬底夹在一起,通过控制夹具的压力来限制几何限域空间间隙,再垂直浸入钙钛矿前驱体溶液中,在毛细力的作用下溶液会填充满整个限域空间,然后加热底部前驱体溶液,控制溶剂挥发速率,形成底部饱和㊁顶部过574㊀综合评述人工晶体学报㊀㊀㊀㊀㊀㊀第53卷饱和的溶液环境,由于温度差引起的热对流,底部的溶液不断向顶部流动补充,为限域空间内生长钙钛矿单晶薄膜提供充足的前驱体溶液㊂制备的单晶薄膜具有厚度从纳米至微米可调㊁表面积达到亚毫米尺寸㊁横纵比可达~105等特点㊂同时,该方法可将钙钛矿单晶薄膜制备在各种衬底(如玻璃㊁石英㊁氧化铟锡(indiumtin oxide,ITO)㊁氟掺杂氧化锡(F-doped tin oxide,FTO))上,其厚度只取决于两个衬底之间的间隙距离,不同厚度的薄膜呈现出多彩均匀的颜色㊂图1㊀溶液空间限域法中厚度可控策略制备钙钛矿单晶薄膜㊂(a)溶液空间限域结合动态流反应系统生长法[37];(b)溶液空间限域法生长厚度可调的钙钛矿单晶薄膜[36]Fig.1㊀Strategies for the growth of thickness-controlled perovskite single-crystalline thin-films.(a)Schematic diagram of the geometry-confined dynamic-flow reaction system[37];(b)schematic diagram of the solution-processed space-confined growthmethod for perovskite single-crystalline thin-films[36]为了扩大钙钛矿单晶薄膜的横向尺寸,从晶体成核动力学角度出发,降低溶液空间限域法中衬底的表面能,将有助于提高溶剂中离子的扩散速度和扩散距离,诱导晶体沿横向方向加速生长㊂2017年,美国北卡罗来纳大学教堂山分校黄劲松教授团队提出对衬底表面进行疏水处理,在ITO衬底表面旋涂疏水的聚[双(4-苯基)(2,4,6-三甲基苯基)胺](Poly[bis(4-phenyl)(2,4,6-trimethylphenyl)amine,PTAA)空穴传输层材料,再用两片PTAA修饰后的ITO衬底构建限域空间,在空间内滴加MAPbBr3前驱体溶液后,将衬底结构置于㊀第4期张庆文等:溶液空间限域法制备有机-无机杂化卤化铅钙钛矿单晶薄膜及其器件应用研究进展575㊀110ħ热台上[1]㊂对比PTAA处理和未处理的衬底所构建限域空间内前驱体溶液的扩散差异,从图2(a)不难发现,由于疏水材料处理的衬底表面具有较低的表面能,将加速前驱体溶液中离子的扩散速率,解决生长过程中离子长程输运差的问题,有助于减少多晶成核结晶概率,同时增大单晶薄膜的横向生长尺寸㊂基于该衬底修饰方法,实现MAPbBr3单晶薄膜厚度可控制在10~20μm,横向截面尺寸可达数十mm2,该工作证明了对衬底表面进行合理改性对于控制钙钛矿单晶薄膜横向生长至关重要㊂2020年,北京大学马仁敏教授团队采取对衬底表面进行特异性处理的策略[63]㊂具体方式是对玻璃衬底进行不同的亲疏水处理,由于具有特异性的亲疏水能力,衬底展现出大小不同的溶液接触角㊂在观测亲疏水能力与单晶成核密度之间的关系后,发现从亲水到疏水的转变过程中,衬底表面的成核密度显著降低㊂分析其原因是亲水表面的成核自由能垒相对低于疏水条件下的表面成核自由能垒,从而拥有较快速的成核速率;并且亲水表面更易于吸附和捕获前驱体溶液中的离子,而降低了离子的扩散速率,导致单晶结晶速率较为缓慢㊂因此,疏水处理的衬底可有效降低单晶成核密度,并且加快单晶生长速率,更易于制备大尺寸的钙钛矿单晶薄膜㊂制得的MAPbBr3单晶薄膜边长尺寸达到1cm,厚度控制在10μm,同时展现出较好的结晶质量,薄膜陷阱态密度仅为1011cm-3,载流子迁移率超过60cm2/(V㊃s)㊂除了衬底修饰策略,衬底自身独特的表面特征也有助于钙钛矿单晶薄膜的生长㊂2020年,天津理工大学吴以成教授团队以云母作为溶液空间限域法的生长衬底[64],如图2(b)所示,将含有适量油酸(oleic acid,OA)的钙钛矿前驱体溶液滴加到两片云母组成的间隙中,旋转云母衬底去除多余的前驱体溶液,然后放置于热板上加热,最终获得超薄的MAPbBr3单晶薄膜㊂该方法是基于云母表面的钾原子与钙钛矿中卤素原子之间会产生较强的相互作用,导致界面能降低并促进钙钛矿单晶薄膜在云母表面横向生长,同时油酸作为表面改性剂附着在钙钛矿表面,抑制钙钛矿单晶薄膜沿纵向方向的生长,最终成功制备出厚度仅为8nm㊁横向尺寸可达数百微米的MAPbBr3单晶薄膜㊂图2㊀溶液空间限域法中衬底修饰策略制备钙钛矿单晶薄膜㊂(a)PTAA处理和未处理的ITO衬底结构中前驱体溶液扩散速度对比图[1];(b)云母衬底上生长钙钛矿单晶薄膜流程示意图[64]Fig.2㊀Substrate modification for the growth of perovskite single-crystalline thin-films.(a)Comparison of the diffusion rate of precursor solution within the PTAA treated and untreated ITO substrates[1];(b)growth of perovskite single-crystalline thin-films on mica substrates[64]钙钛矿单晶薄膜的生长开始于成核阶段,考虑到处于复杂溶液环境中,晶体将发生各向异性生长,容易形成多个晶核,并诱使出现晶畴㊁晶界等结构,严重影响钙钛矿单晶成膜的结晶质量[65]㊂为解决这一问题,科研人员提出了一种晶种法技术策略,首先生长钙钛矿单晶种子,再将种子转移到目标衬底,最后在合适的溶液环境中再结晶生长形成高质量的钙钛矿单晶薄膜㊂2018年,中国科学院化学研究所宋延林研究员团队提出了一种溶液空间限域结合晶种印刷法的生长策略,通过晶种再生长的方式,实现了厚度可控㊁重复性好㊁576㊀综合评述人工晶体学报㊀㊀㊀㊀㊀㊀第53卷结晶质量高的钙钛矿单晶薄膜[66]㊂如图3(a)所示,首先使用喷墨打印技术将钙钛矿前驱体溶液选择性滴加在目标衬底上,随着前驱体溶液的挥发,形成规则排布的钙钛矿单晶种子㊂获得的钙钛矿单晶种子将有效抑制无序成核结晶现象㊂然后,将载有钙钛矿单晶种子的衬底转移并浸入到钙钛矿前驱体饱和溶液中,置于热台上加热结晶后,通过控制钙钛矿单晶种子的数量和尺寸,最终制备出批量的毫米级钙钛矿单晶薄膜㊂2021年,韩国首尔大学Lee教授团队进一步拓展了晶种生长法,结合种子转移技术,如图3(b)所示[67]㊂首先在两片玻璃片中注入前驱体溶液,玻璃片之间由厚度为25μm的聚四氟乙烯(polytetrafluoroethylene,PTFE)薄膜隔开,在110ħ的加热温度下,过饱和的钙钛矿前驱体溶液成核结晶,形成厚度为23μm㊁尺寸为100~200μm 的MAPbBr3单晶种子㊂然后,挑选出单个种子转移至一个密封式液体池腔体中,随着浓度为1mol/L的MAPbBr3前驱体溶剂以5μL/min速率源源不断地流入液体池腔体内,基于逆温结晶法,MAPbBr3单晶薄膜将匀速生长,最终制得了高质量㊁大尺寸的MAPbBr3单晶薄膜,其厚度为40μm,表面积可达16.23mm2,表面粗糙度为0.51nm,缺陷态密度仅有7.61ˑ108cm-3㊂图3㊀溶液空间限域法中晶种法策略制备钙钛矿单晶薄膜㊂(a)溶液空间限域结合晶种印刷法制备钙钛矿单晶薄膜技术流程示意图[66];(b)晶种生长法结合晶种转移技术制备钙钛矿单晶薄膜技术流程示意图[67]Fig.3㊀Seed-induced methods for the growth of perovskite single-crystalline thin-films.(a)Technical flow diagram of preparation of perovskite single crystal film by solution-processed space-confined combined with seed printing[66];(b)process flow diagram of preparation of perovskite single crystal thin film by seed growth and seed transfer technology[67]图案化生长钙钛矿单晶薄膜对于推动钙钛矿单晶材料面向集成化光电器件应用至关重要㊂其主要思路是通过引入周期性的模板,构建结构化限域空间用于生长图案化钙钛矿单晶[68-74]㊂2021年,合肥工业大学罗林保教授团队利用高密度数字视频光盘(digital video disc,DVD)上的沟道作为结构化限域空间用于溶液空间限域法,如图4(a)所示[71]㊂首先,将聚二甲基硅氧烷(polydimethylsiloxane,PDMS)溶液旋涂在准备好的DVD磁盘上,固化后形成与磁盘沟道结构和形貌一致的PDMS模板㊂然后,在亲水性衬底上滴加钙钛矿前驱体溶液,溶液在亲水衬底上形成一层均匀的液膜,再将表面具有周期性沟道结构的PDMS模板覆盖其上,前驱体溶液便被重新分配并限制在PDMS模板与亲水性衬底形成的纳米沟道之间㊂放置于热台上加热之后,晶体沿着纳米沟道不断生长,最终形成规则且均匀的钙钛矿单晶阵列,得到的钙钛矿单晶阵列的结构完全与磁盘沟道形貌相一致,并可实现在不同衬底上生长大规模钙钛矿单晶阵列结构㊂2022年,苏州大学揭建胜教授团队开发了类似的三维限制结晶方法,在三维结构化的微通道模板上方利用一个三角形PDMS 基板协助溶液剪切过程,用于生长钙钛矿单晶阵列,PDMS模板紧密地附着在微通道表面,避免了溶液剪切㊀第4期张庆文等:溶液空间限域法制备有机-无机杂化卤化铅钙钛矿单晶薄膜及其器件应用研究进展577㊀过程中对微通道的破坏,同时利用PDMS模板表面的疏水性,可以有效防止溶液黏附在三角形PDMS基板上,如图4(b)所示[72]㊂在底部进行加热的情况下,缓慢移动三角形玻璃基板,钙钛矿前驱体溶液逐渐挥发结晶,最终形成与模板结构相同的MAPbI3单晶阵列㊂为了进一步提高钙钛矿单晶阵列横向尺寸,韩国汉阳大学Sung教授团队引入滚筒印刷技术,如图4(c)所示[73]㊂首先,钙钛矿前驱体溶液加在180ħ加热的基板衬底上,通过旋转图案化的PDMS模具包裹的圆柱形金属滚轮,PDMS模具上具有宽度为10mm㊁深度为200nm的周期性阵列,前驱体溶液被限制在模具和基板衬底之间,随着前驱体溶液的迅速蒸发而结晶,最终制得的钙钛矿单晶薄膜阵列与滚筒图案完全一致㊂成功实现了总宽度为10mm,周期尺寸为400nm,厚度为200nm的MAPbI3单晶薄膜阵列㊂利用该方法不仅可以在横向方向上约束钙钛矿单晶的生长,并且实现滚筒印刷制备大尺度钙钛矿单晶薄膜阵列的目的㊂通过上述总结,围绕溶液空间限域法制备大尺寸㊁高质量钙钛矿单晶薄膜,详细阐述了从厚度可控㊁衬底修饰㊁晶种生长㊁图案化生长等几个主要方面的生长和制备方法,相关性能参数如表1所示,对于未来实现可控制备钙钛矿单晶薄膜材料,进一步扩展其在光电器件领域的应用至关重要㊂图4㊀溶液空间限域法中图案化生长策略制备钙钛矿单晶薄膜㊂(a)磁盘沟道模板生长钙钛矿单晶阵列的技术流程图[71];(b)三维限制结晶方法生长钙钛矿单晶阵列装置示意图[72];(c)滚筒印刷技术制备大尺度钙钛矿单晶阵列的装置流程图[73] Fig.4㊀Periodic structures for the growth of perovskite single-crystalline thin-films.(a)Digital channel template for the growth of perovskite single-crystalline arrays[71];(b)schematic diagram of apparatus for growing perovskite single crystal array by a three-dimensional restricted crystallization method[72];(c)flow chart of device for preparing large-scale perovskite singlecrystal array by roller printing technology[73]578㊀综合评述人工晶体学报㊀㊀㊀㊀㊀㊀第53卷表1㊀溶液空间限域法及其改进策略制备钙钛矿单晶薄膜的相关性能参数Table1㊀Performance parameters of the perovskite single-crystalline thin-films prepared by solution-processedspace-confined method and its improvement strategySolution-processed space-confined method and its improvement strategy Perovskitematerial type Thickness/μmDensity of defectstates/cm-3Carrier mobility/(cm2㊃V-1㊃s-1)Surface dimension ReferenceDynamic-flow reaction system MAPbI3~1506ˑ10839.6 5.84mmˑ5.62mm[37] Thickness controlledgrowth method MAPbBr30.01~1 4.8ˑ101015.7Hundreds of microns[36]Substrate treatment MAPbI310~40Electron:36.8ʃ3.7Hole:12.1ʃ1.5Tens of square millimeters[1] Substrate specific processing MAPbBr3~10 1.6ˑ1011>601cm[63] Mica substrate MAPbX30.008~0.01436.5Hundreds of microns[64] Seed printing method MAPbX3,CsPbBr30.1~10 2.6ˑ101014000μm2[66] Seed transfer technology MAPbBr3407.61ˑ10816.23mm2[67] Digital channeltemplate method MAPbI3~0.065cycle:760nm[71] Three-dimensional confinedcrystallization method MAPbI30.5~58.5ˑ1010cycle:8μm[72] Rolling mould printingtechnology MAPbI30.2or0.545.64cycle:400nm[73] 2㊀钙钛矿单晶薄膜器件应用钙钛矿单晶薄膜因其高的光吸收系数㊁高的载流子迁移率㊁长的载流子扩散长度㊁带隙可调谐等优异的光电性能,被广泛应用于光电探测器㊁太阳能电池㊁场效应晶体管㊁发光二极管等器件中㊂光电探测器是基于传统光电效应将光信号转变为电信号的器件装置,其在光通信㊁激光雷达㊁医疗诊断㊁安防监控等多个领域应用广泛㊂传统光电探测器多以无机半导体材料为主,例如Si㊁GaAs㊁GaN等材料[11]㊂近年来,随着有机-无机杂化卤化物钙钛矿半导体材料的出现,其展现出的巨大的应用潜力,有望促进光电探测器在成本和性能上取得进一步的提升和跨越㊂大量研究表明,由于较低的光吸收损耗和理想的激子扩散距离,钙钛矿单晶薄膜光电探测器[68-69,75-77]相比于单晶块体探测器,在光电探测方面已展露出明显的性能优势㊂2015年,阿卜杜拉国王科学大学Bakr教授团队首次报道利用直接生长在ITO玻璃衬底上的MAPbCl3单晶薄膜,制备一种具有金属-半导体-金属器件结构的光电导型探测器[54],并展现出出色的光电探测性能,具有较高的探测率与开关比,响应时间在ms数量级,这与当时商用的III-V族半导体光电晶体管的性能几乎相当㊂2017年,黄劲松团队利用MAPbBr3单晶薄膜制作了垂直器件结构为p-i-n型的Cu/BCP/C60/MAPbBr3/PTAA/ITO钙钛矿单晶探测器[78],如图5(a)所示,该光电探测器的探测率(D∗)高达1.5ˑ1013Jones㊂由于单晶薄膜较低的缺陷态密度,探测器对于弱光探测极为敏感,探测最低可达pW/cm2量级,同时线性动态范围高达256dB,是当时报道最高的结果㊂2018年,马仁敏教授团队系统性研究了光电探测器性能与单晶薄膜厚度之间的依赖关系[14]㊂发现随着钙钛矿单晶薄膜的厚度从10μm降低到几百nm,光电探测器的探测能力提升了2个数量级,增益提升了4个数量级㊂通过优化钙钛矿单晶薄膜的厚度以及结晶度,器件的增益可达5ˑ107,增益带宽积为70GHz㊂钙钛矿材料具有可低温㊁液相制备的特点,并可与多种柔性衬底相兼容,制备可弯折的柔性光电子器件㊂同时,钙钛矿单晶薄膜展现出较好的柔性和机械性,可用于制备柔性钙钛矿单晶薄膜光电探测器㊂为此, 2020年,马仁敏教授团队引入超薄钙钛矿单晶薄膜作为有源层,制备了高性能的柔性光电探测器[39],如图5 (b)所示,该光电探测器的单晶薄膜厚度仅为20nm,器件响应度高达5600A/W,在经过1000次循环弯折后,探测器的光电流和开关比没有出现明显的下降,展现出较好的弯折稳定性㊂高质量的钙钛矿单晶纳米线阵列有利于限制载流子在几何通道内输运,提高载流子的迁移率和扩散距离㊂2021年罗林保教授团队制备的基于MAPbI3单晶纳米线阵列的光电探测器[71],在520nm入射光照射下,随入射光功率的升高,该光电探㊀第4期张庆文等:溶液空间限域法制备有机-无机杂化卤化铅钙钛矿单晶薄膜及其器件应用研究进展579㊀测器的光电流呈线性递增,最低暗电流为0.3nA,最高光电流达350nA,总开关比高达1.2ˑ103㊂同时,该探测器的响应度为20.56A/W,探测率达到4.73ˑ1012Jones㊂由于钙钛矿单晶纳米线阵列展现出良好的偏振敏感性,该类型器件也适用于探测线偏光的偏振度㊂为了解决钙钛矿材料中铅毒性[79]和不稳定性的问题,2020年,中山大学匡代彬教授团队在ITO玻璃上原位生长不含铅元素的全无机Cs3Bi2I9单晶薄膜并制备了相应的光电探测器[80]㊂制得的Cs3Bi2I9钙钛矿单晶薄膜的陷阱态密度比多晶材料低3个数量级,载流子迁移率也高出3.8ˑ104倍㊂这些优异的性质有利于实现高性能的光电探测器,基于此材料制备的垂直结构型光电探测器的开关比高达11000㊂而且,在未封装的情况下,处在潮湿环境中1000h之后,该钙钛矿单晶薄膜光探测器的光电流仍维持初始值的91%,体现了该材料出色的环境稳定性㊂由于钙钛矿多晶薄膜内存在大量的晶界㊁空穴和缺陷态等,太阳能电池存在显著的非辐射复合能量损失,限制了钙钛矿太阳能电池PCE的进一步提升㊂而无晶界㊁低缺陷态密度的钙钛矿单晶薄膜成为解决材料内在问题及器件PCE的理想材料体系㊂2017年,中国科学院深圳先进技术研究院李江宇教授团队在FTO/TiO2衬底上直接生长MAPbI3单晶薄膜,并制造了相应的钙钛矿单晶薄膜太阳能电池,该电池器件的PCE达到了8.78%[81]㊂同年,黄劲松教授团队利用在PTAA空穴传输层上直接生长的MAPbI3单晶薄膜,构建器件结构为ITO/PTAA/MAPbI3/PCBM/C60/BCP/Cu的太阳能电池器件,如图5(c)所示[1]㊂通过优化钙钛矿单晶薄膜厚度,其电池的光谱响应范围可以扩展到820nm,比相对应的多晶薄膜材料的光谱响应要宽20nm,器件的最佳短路电流密度J sc为20.5mA/cm2,开路电压V oc为1.06V,填充因子(fill factor,FF)为74.1%,PCE可达16.1%㊂在使用MAI离子溶液对单晶薄膜表面进行钝化处理之后,有效降低了MAPbI3单晶薄膜表面的电荷陷阱,器件最佳PCE提升到17.8%㊂2019年,Bakr教授团队利用20μm厚的MAPbI3单晶薄膜制备太阳能电池,器件结构为ITO/PTAA/MAPbI3/C60/BCP/Cu[82]㊂该钙钛矿单晶薄膜电池器件的PCE达到21.09%,填充因子FF为84.3%㊂之后,该团队通过优化前驱体溶液,采用碳酸丙烯酯(propylene carbonate,PC)和γ-丁内酯(1,4-butyrolactone,GBL)的混合溶剂,90ħ下生长MAPbI3钙钛矿单晶薄膜㊂基于此单晶材料制备的钙钛矿太阳能电池的V oc明显提高,PCE达到21.9%[84]㊂2021年,该团队在之前的器件结构基础上,将钙钛矿单晶的成分改为混合阳离子FA0.6MA0.4PbI3钙钛矿单晶,如图5(d)所示,制备的钙钛矿太阳能电池对近红外响应要比纯FAPbI3器件扩展了50meV,J sc达到26mA/cm2,PCE达到22.8%[84]㊂2023年,该团队在亲水性的([2-(3,6-dimethoxy-9H-carbazol-9-yl)ethyl]phosphonic acid,MeO-2PACz)单分子层表面生长FA0.6MA0.4PbI3钙钛矿单晶薄膜,与PTAA上生长的单晶薄膜相比,MeO-2PACz有效提高了钙钛矿单晶薄膜与衬底的机械粘附力,PCE达到创纪录的23.1%[85]㊂伴随着钙钛矿单晶薄膜生长技术的更新和迭代,钙钛矿单晶薄膜太阳能电池的器件性能有望超越钙钛矿多晶太阳能电池,在太阳能电池器件领域占据一席之地[86]㊂从钙钛矿材料结构角度出发,由金属阳离子和卤化物阴离子形成的强共价或离子键相互作用结合的钙钛矿八面体骨架结构,将为材料提供高的载流子迁移率骨架模型,据理论预测的迁移率最高可达1000cm2/(V㊃s);有机阳离子可以间接扭曲无机骨架,在分子尺度上影响材料的晶体结构和电学特性㊂因此,钙钛矿材料因其展现出较高的载流子迁移率,被认为是发展新一代半导体电子技术最理想的光电材料㊂基于钙钛矿单晶薄膜材料的场效应晶体管研究起步相对较晚,2018年,阿卜杜拉国王科技大学Amassian教授团队制备了底栅顶接触的钙钛矿单晶薄膜场效应晶体管器件,器件的沟道长度为10~150μm,如图5(e)所示[87]㊂该团队设计和制备了一系列基于MAPbCl3㊁MAPbBr3㊁MAPbI3单晶薄膜的场效应晶体管器件,测量和分析器件的转移和传输特性曲线,其空穴迁移率最高分别可达2.6㊁3.1㊁2.9cm2/(V㊃s),电子迁移率分别为2.2㊁1.8㊁1.1cm2/(V㊃s),且器件开关比分别可达2.4ˑ104㊁4.8ˑ103㊁6.7ˑ103㊂该系列场效应晶体管器件展现出良好的电学输运特性,为进一步推动钙钛矿单晶薄膜材料在集成电子器件领域的应用提供了良好的研究基础㊂钙钛矿发光二极管(perovskitelight emitting diodes,PeLED)近年来也发展迅速,自2014年英国剑桥大学的Friend教授课题组首次报道室温下PeLED器件以来,PeLED以其优异的光电性能㊁较低的器件成本,以及。

转玉米C4型PEPC基因油菜叶片特异性表达的可行性探讨

转玉米C4型PEPC基因油菜叶片特异性表达的可行性探讨摘要：C4型PEPC基因导入C3植物可提高C3植物的光合能力，抑制油菜C3型PEPC基因的表达能提高油菜含油量，因此要进一步提高油菜的光合生产力和促进油脂积累，有必要在油菜叶片中特异性地表达玉米的C4型PEPC基因。

通过克隆玉米、水稻和油菜的rbcS叶片特异性启动子，构建rbcS调控的玉米C4型PEPC基因叶片特异性表达载体，转化油菜获得叶片特异性高效表达的转PEPC基因植株，与原种和转玉米C4型PEPC基因油菜相比较，研究各株系PEPC 酶活性和光合生理表现；根、茎、叶与种子器官玉米C4型PEPC基因的表达差异；分析各株系油菜油脂和蛋白质含量、单株产量等农艺性状。

通过在油菜叶片中特异性地表达玉米的C4型PEPC基因，提高油菜单叶的光合速率，促进油菜油脂积累，为油菜的高产育种提供新途径。

关键词：油菜；PEPC基因；rbcS启动子；光合速率1 玉米C4型PEPC基因导入油菜的意义油菜（Rape or Rapeseed or Canola）是以菜子榨油为种植目的的一年生或越年生草本植物，已成为四大油料作物（大豆、油菜、向日葵和花生）之一。

与世界上油菜单产水平高的国家相比，中国的差距还很大，但这也说明中国油菜生产在栽培和育种技术方面还大有潜力可挖。

在适宜的环境条件下，田间的光能利用率可达5%，而现有光能利用率只有1.11%左右[1]，可见改善光合效率的潜力还很大。

但分析现有品种的增产潜力，在叶面积指数达到9以上时，扩大叶面积指数，会引起群体内光能分布的恶化，因此在适宜的光合面积上提高单叶的光合速率，是进一步增加油菜生产能力的一条有效途径。

提高植物的光合效率可能有多种途径，其中将C4光合基因PEPC导入C3植物以提高C3植物的C4光合特性，取得了显著的成绩，Ku等[2]通过农杆菌介导系统，首次成功地将玉米C4光合途径的关键酶PEPC的基因导入C3植物水稻中，获得了高表达的转基因植株。

具有烷基磺酸侧链的凝胶型聚苯并咪唑质子交换膜的制备与表征

化工进展Chemical Industry and Engineering Progress2024 年第 43 卷第 4 期具有烷基磺酸侧链的凝胶型聚苯并咪唑质子交换膜的制备与表征朱泰忠1，张良1，黄泽权1，罗伶萍1，黄菲1，薛立新1，2（1 浙江工业大学化工学院膜分离与水科学技术中心，浙江杭州 310014；2温州大学化学与材料工程学院，浙江温州 325035）摘要：磷酸（PA ）掺杂聚苯并咪唑（PBI ）以其优异的热化学稳定性和高玻璃化转变温度成为高温质子交换膜燃料电池（HT-PEMFCs ）的首选材料。

然而，由于低温下磷酸较弱的解离度和传递速率，导致膜的质子传导性能不佳，电池冷启动困难。

因此，研发可在宽温湿度范围内高效运行的高温质子交换膜成为当前挑战。

特别是拓宽其低温运行窗口、实现冷启动对这类质子交换膜燃料电池在新能源汽车领域的实际应用具有重要意义。

本文通过多聚磷酸溶胶凝胶工艺与内酯开环反应设计并合成了一系列磷酸掺杂的具有柔性烷基磺酸侧链的凝胶型聚苯并咪唑质子交换膜。

重点探究了烷基磺酸的引入以及侧链长度对磷酸掺杂水平、不同温湿度下的质子传导率及稳定性的影响规律。

研究结果表明，所制备的质子交换膜具有凝胶型自组装片层堆叠的多孔结构，有利于吸收大量磷酸并提供质子快速传输通道。

其中，PA/PS-PBI 展现出了在宽温域范围内均优于目前所报道的其他工作的质子传导性能。

特别是常温下，其质子传导率从原膜的0.0286S/cm 提升至0.0694S/cm 。

80℃下，其质子传导率从原膜的0.1117S/cm 提升至0.1619S/cm 。

200℃下，其质子传导率从原膜的0.2609S/cm 提升至0.3578S/cm 。

此外，该膜在80℃和0%相对湿度（RH ）条件下仍可具有与Nafion 膜在100%RH 时相当的质子传导率，为打破质子交换膜经典定义、实现宽温域（25~240℃）运行提供新的方案。

普鲁兰多糖为骨架的新型阳离子非病毒基因载体

的ＰＰＩ —Ｅ是一种新的有潜在应用价值的非病毒基因载体．关键词普鲁兰多糖；乙烯亚胺；聚绿色荧光蛋白表达质粒ｐＦ；ＧＰ非病毒基因载体
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光蛋白表达质粒（ＧＰ及荧光素酶表达质粒（Ｇ３转染实验结果表明，载体ＰＰＩＮＰ高达１．ｐＦ）ｐＬ） —Ｅ在／２５时对细胞ＭＣ一，ｅａＣＳ７的毒性低于ＰＩＦ７ＨＬ和Ｏ－Ｅ；当ＮＰ为６２／．５时能有效将ｐＦＧＰ和ｐＬＧ３带入Ｈｌｅａ细胞并表达，最佳转染效率及荧光素酶活分别为［３．７０２）和（８７ ± ．９ × ０Ｌ／ｇ蛋白，ＲＵ为光强（６６ ± ．５％２．３７１）１ＲＵｍＬ度］，比Ｌｐ００（９１＋．１％，５．７７６）１Ｌ／蛋白）略低．因此以Ｐｌｌｉ２０［４．３０６）ｏ＿（８４ ± ．２ × ０ＲＵｍｇｕｌａｕｎ为骨架材料
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普鲁兰多糖（ｕｌｌ，子量为１００山东中清生物科技有限公司）琥珀酸酐（Ｐｌａ分ｕｎ０００，；国药集团化学试剂有限公司）；高分支度聚乙烯亚胺（Ｅ，＝１０武汉强龙化工新材料有限责任公司）一ＰＩＭ００，；１乙基．（．甲基氨基丙基）酰二亚胺盐酸盐［ＤＨＣ，吉尔生化（３二碳ＥＣ・１上海）限公司］Ｎ一有；羟基琥珀酰亚胺（ＨＳＮ，上海蓝源生物科技有限公司）４二甲氨基吡啶（ＭＰ，；一ＤＡ阿拉丁产品）；ＭＣ－，ＬＦ７Ｈｅａ和ＣＳ７Ｏ

L-脯氨酸-4-羟化酶的异源表达和合成反式-4-羟基-L-脯氨酸的条件优化

L-脯氨酸-4-羟化酶的异源表达和合成反式-4-羟基-L-脯氨酸的条件优化周海岩;李会帅;王培;柳志强【摘要】L-脯氨酸-4-羟化酶(L-Proline-4-hydroxylase,P4H)是依赖α-酮戊二酸(α-KG)和Fe2+的双加氧酶成员之一,在反式4-羟基-L-脯氨酸(trans-4-hydroxy-L-proline,t-4Hyp)等重要手性化合物的生物合成中发挥关键作用.本研究构建了来源于Bradyrhizobium japonicum USDA 6的P4H重组大肠杆菌Escherichia coli BL21(DE3)/pET-28b-p4hBJ,SDS-PAGE和酶活检测结果表明,该菌株具有表达可溶性P4H和催化合成t-4Hyp的能力.通过优化,确定了该重组菌全细胞催化合成t-4Hyp较优的反应体系和条件:10 mLpH 6.5 80 mmol/LMES缓冲液、9 mmol/L L-Pro,6 mmol/L L-抗坏血酸,6 mmol/L α-KG,0.8 mmol/LFeS04·7H2O,反应温度为35℃;在20 g/L湿细胞的催化反应中,t-4Hyp的合成量达到34.86 mg/L,比优化前(17.53 mg/L)提高了98.86％.该工作为进一步利用P4H生物催化法合成t-Hyp奠定了一定的技术基础.%L-Proline-4-hydroxylase (P4H) is a member of Fe (Ⅱ)/c-ketoglutarate (cα-KG)-dependent dioxygenase superfamily,playing an important role in biosynthesis of trans-4-hydroxy-L-proline (t-4Hyp) and other important chiral building-block chemicals.In this work,recombinant Escherichia coli BL21 (DE3)/pET-28b-p4hBJ harboring P4H gene p4HBJ,originated from Bradyrhizobium japonicum USDA 6 was constructed.The results of SDS-PAGE and enzyme activity solubly analysis showed that P4H was expressed solubly and actively in E.coli BL21 (DE3)/pET-28b-p4hBJ.Furthermore,the whole cell catalysis conditions with E.coli BL21 (DE3)/pET-28b-p4hBJ was investigatedand optimized for t-4Hyp biosynthesis.The optimum reaction system and conditions were as follows:10 mL pH 6.5 MES buffer,9 mmol/L L-Pro,6 mmol/L L-ascorbic acid,6 mmol/L α-KG,0.8 mmol/L FeSO4 · 7H2O;the reaction temperature was 35 ℃.Under the optimized conditions,a t-4Hyp yield of 34.86 mg/L was achieved via whole cell catalysis reaction with 20 g/L wet cells,which was 98.86％ higher than that of pre-optimization.The work provided an important theoretical foundation for the further research of t-4Hyp biosynthesis with P4H as biocatalyst.【期刊名称】《工业微生物》【年(卷),期】2017(047)001【总页数】9页(P1-9)【关键词】L-脯氨酸-4-羟化酶;反式-4-羟基-L-脯氨酸;双加氧酶;生物合成;生物催化;优化【作者】周海岩;李会帅;王培;柳志强【作者单位】浙江省生物有机合成技术研究重点实验室,生物工程学院,浙江工业大学,浙江杭州310014;生物转化与生物净化教育部工程研究中心,浙江杭州310014;浙江省生物有机合成技术研究重点实验室,生物工程学院,浙江工业大学,浙江杭州310014;生物转化与生物净化教育部工程研究中心,浙江杭州310014;浙江省生物有机合成技术研究重点实验室,生物工程学院,浙江工业大学,浙江杭州310014;生物转化与生物净化教育部工程研究中心,浙江杭州310014;浙江省生物有机合成技术研究重点实验室,生物工程学院,浙江工业大学,浙江杭州310014;生物转化与生物净化教育部工程研究中心,浙江杭州310014【正文语种】中文反式-4-羟基-L-脯氨酸(trans-4-hydroxy-L-proline，t-4Hyp)是一种重要的L-羟脯氨酸(L-Hydroxyproline，简称Hyp)同分异构体，在生物体的一些生理和病理过程中发挥关键的作用，广泛应用于食品、化妆品、医药和化工等领域[1]。

蛋白质折叠的热力学和动力学

蛋白质折叠的热力学和动力学药学院 10489629 苟宝迪蛋白质是一种生物大分子，基本上是由20种氨基酸以肽键连接成肽链。

肽链在空间卷曲折叠成为特定的三维空间结构。

有的蛋白质由多条肽链组成，每条肽链称为亚基，亚基之间又有特定的空间关系，称为蛋白质的四级结构。

所以蛋白质分子有非常特定的复杂的空间结构。

诺贝尔奖得主Anfinsen认为每一种蛋白质分子都有自己特有的氨基酸的组成和排列顺序，由这种氨基酸排列顺序决定它的特定的空间结构。

具有完整一级结构的多肽或蛋白质, 只有当其折叠形成正确的三维空间结构才可能具有正常的生物学功能. 如果这些生物大分子的折叠在体内发生了故障, 形成错误的空间结构, 不但将丧失其生物学功能, 甚至会引起疾病.蛋白质异常的三维空间结构可以引发疾病，疯牛病、老年性痴呆症、囊性纤维病变、家族性高胆固醇症、家族性淀粉样蛋白症、某些肿瘤、白内障等等都是“折叠病”。

蛋白质折叠的研究（图1[1]），是生命科学领域的前沿课题之一。

不仅具有重大的科学意义，而且在医学和在生物工程领域具有极大的应用价值。

图1蛋白质折叠的热力学研究蛋白质折叠的研究，比较狭义的定义就是研究蛋白质特定三维空间结构形成的规律、稳定性和与其生物活性的关系。

这里最根本的科学问题就是多肽链的一级结构到底如何决定它的空间结构？X-射线晶体衍射是至今为止研究蛋白质结构最有效的方法, 所能达到的精度是其它任何方法所不能比拟的. 但是, 蛋白质分离纯化技术要求高, 蛋白质晶体难以培养,晶体结构测定的周期较长, 从而制约了蛋白质工程的进展. 随着近代物理学、数学和分子生物学的发展, 特别是计算机技术的进步, 人们开始用理论计算的方法, 利用计算机来预测蛋白质的结构. 同源模建方法是最常用、最有效的蛋白质结构预测方法. 但是, 利用同源模建方法预测蛋白质结构时, 需用同源蛋白质的已知结构作为模板. 当缺乏这种模板结构时, 预测则很难奏效. 这是该方法的天生缺陷. 是否能从蛋白质序列出发, 直接预测蛋白质的结构?从理论上最直接地去解决蛋白质的折叠问题，就是根据测得的蛋白质的一级序列预测由Anfinsen原理决定的特定的空间结构。

婴配乳粉中2′-岩藻糖基乳糖和乳糖-N-新四糖含量离子色谱法检测

第41卷2024 年 3 月应用化学CHINESE JOURNAL OF APPLIED CHEMISTRY第3期405⁃414婴配乳粉中2′-岩藻糖基乳糖和乳糖-N -新四糖含量离子色谱法检测詹胜群葛城* 周荣杰周钧（澳优乳业（中国）有限公司，长沙 410200）摘要研究了离子色谱法同时测定婴配乳粉中2′-岩藻糖基乳糖和乳糖-N -新四糖的含量及影响因素。

采用离子色谱柱分离，脉冲安培检测器检测和外标法定量分析等方法。

通过对不同净化方式、酶解处理和洗脱条件等过程进行考察和优化，比较确定色谱条件和前处理条件对分离的影响，并就线性范围进行探讨。

结果表明，优化后的方法线性关系良好， R 2＞0.999；方法精密度好，相对标准偏差（n =7）＜2.7%；方法准确度好，2′-岩藻糖基乳糖回收率为95.97%~103.38%，乳糖-N -新四糖回收率为100.09%~104.01%。

该方法能对婴配乳粉中的2′-岩藻糖基乳糖和乳糖-N -新四糖同时测定，操作简单，具有良好的准确度和精密度，可为婴配乳粉质量监控方法提供参考。

关键词母乳低聚糖；离子色谱；2′-岩藻糖基乳糖；乳糖-N -新四糖；婴配乳粉中图分类号：O657.7 文献标识码：A 文章编号：1000-0518（2024）03-0405-10母乳低聚糖（HMOs ）是一种多功能聚糖，是人乳中的第三大成分［1］，其结构主要由5种基本单糖组成：葡萄糖（Glucose ， Glc ）、半乳糖（Galactose ， Gal ）、N -乙酰葡糖氨（Nacetylglucosamine ， GlcNAc ）、岩藻糖（Fucose ， Fuc ）和N -乙酰神经氨酸（N -Acetylneuraminic acid ， NeuAc ），通常由半乳糖和葡萄糖与其它一种或多种单糖聚合形成多种结构［2］。

HMOs 对于婴幼儿的生长健康有着重要的意义，除了可预防感染、过敏以外，还能促进肠胃发育、增强自身免疫、预防糖尿病和心血管疾病［3-5］等。

利用RAPD技术构建四倍体苜蓿遗传连锁图谱

用ＡＦＰ（ｍｐｉｅａｍｅｔｅｇｈｐｌｍｏｐｉ扩增片段长度多态性）ＲＤ（ａｄｍｍｐｉｅｏｙｒＬａｌｄｆｇｎｎｔｏｙｒｈｓｉｆｒｌｍ，和ＡＰｒｎｏａｌｉｐｌｍｏ — ｆｄ
ｐｉＤｈｃＮＡ，随机扩增多态性）记构建了四倍体苜蓿遗传图谱。Ｅｊｙ等于２０标ｕａｌ０５年利用来自紫花苜蓿（Ｍ．
进行ＲＤＥ分子标记的遗传作图研究。旨在优化苜蓿分子遗传学实验体系，ＡＰ。构建苜蓿遗传连锁框架图，为进一
步构建饱和的苜蓿遗传连锁图谱及开展苜蓿分子育种研究奠定基础。
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生物学中的蛋白质折叠研究

生物学中的蛋白质折叠研究蛋白质折叠是生物学中的一个重要问题，也是一个极其复杂的问题。

蛋白质（protein）是生命体中最重要的大分子，它的性质和功能取决于它的三维结构，也就是所谓的蛋白质结构。

而蛋白质的结构是由蛋白质分子中的氨基酸（amino acid）所组成，这些氨基酸通过互相连接形成了一个线性的多肽链（peptide chain），然后在一定的条件下，整个多肽链会经历一种特殊的自组织现象，最终形成了一个稳定的、具有空间结构的蛋白质分子。

这个自组织现象就被称为蛋白质折叠（protein folding），它同时也是一种能量最低化的过程。

化学上所谓的“能量最低化”，其实是指在一定的条件下，多肽链中的每一个氨基酸都会倾向于采取最佳的构象（conformation），在这个构象中，每一个氨基酸的电子云都能够保持最稳定的状态，同时也能同时满足蛋白质分子内部的相互作用和外部环境的影响。

最终形成的蛋白质结构，就是由无数个氨基酸所构成的“折叠图谱”（folding landscape）所决定的。

美国生命科学研究所的Gregory S. Patience教授曾经将蛋白质折叠比喻为一个“生命大拼图”，他说：“如果我们把一个完全由氨基酸所构成的蛋白质比作一个亿级别的拼图，那么其中有数以亿计的互动环节，甚至比宇宙中的星系还要多！”所以，迄今为止，几乎所有的科学家都认为，蛋白质折叠是生命科学中最重要、同时也是最难的问题之一。

在许多领域，如生物医学、生物工程和生物计算等，蛋白质折叠都具有举足轻重的地位。

然而，正因为其复杂性和重要性，蛋白质折叠问题难以被完全理解和解决。

在现代生物学和计算机科学的帮助下，科学家们利用各种技术手段，通过对蛋白质折叠过程的不断研究，逐渐揭示了折叠过程中的一些规律和机理。

其中，使用凝胶过滤、光散射、表面等离子共振、荧光共振能量转移、小角X射线散射等手段测量蛋白质分子稳定性、热力学和动力学性质等方法属于常用技术。

单细胞基因组测序技术新进展及其在生物医学中的应用

Hereditas (Beijing) 2021年2月, 43(2): 108―117 收稿日期: 2020-12-01; 修回日期: 2021-01-03基金项目:中国博士后科学基金面上项目(编号：2019M651377)和上海市“超级博士后”激励计划项目(2018-2020)资助[Supported by ChinaPostdoctoral Science Foundation Grant (No. 2019M651377), and Shanghai “Super Postdoctoral Fellow” Program (2018-2020)]作者简介: 王卓，博士，研究方向：循环肿瘤细胞鉴定与单细胞测序。

E-mail:*********************.cn 通讯作者:施奇惠，博士，研究员，研究方向：液态活检与单细胞分析。

E-mail:******************.cn DOI: 10.16288/j.yczz.20-363 网络出版时间: 2021/1/22 10:34:26URI: https:///kcms/detail/11.1913.R.20210122.0900.002.html综述单细胞基因组测序技术新进展及其在生物医学中的应用王卓，申笑涵，施奇惠复旦大学生物医学研究院, 上海 201100摘要: 随着单细胞基因组测序技术的建立与发展，对细胞基因组特征的分析进入了单细胞水平。

单细胞的基因组分辨率不但使研究人员能够在单细胞尺度上分析肿瘤细胞的异质性，也使得传统上难以检测的稀有细胞的基因组研究成为可能。

这些稀有细胞往往具有重要的生物学意义或临床价值，如癌症患者血液中循环肿瘤细胞(circulating tumor cell, CTC)的基因组检测或三代试管婴儿植入前胚胎细胞的遗传缺陷诊断与筛查(preim-plantation genetic diagnosis/screening, PGD/PGS)。

P4HA2通过激活PI3KAKTmTOR信号通路促进肝癌的发生和发展

肝细胞癌（HCC ）近几十年来发病率上升，虽然在临床和实验性癌症治疗方面取得了很大进展，但由于术后肿瘤复发和转移率高，HCC 患者的总体预后较差［1-3］。

肝癌的发生发展可能是一个多因素、多步骤的过程［3］，但目前关于其具体的分子机制尚不清楚。

因此，更好地了解HCC 发生发展的分子机制对肝癌靶向治疗具有重要意义。

细胞外基质（ECM ）由多种大分子组成，包括胶原蛋白、纤维连接蛋白、弹性蛋白、层粘连蛋白、透明质酸和蛋白多糖［4］。

ECM 作为肿瘤微环境中含量最丰富的成分，可以调控肿瘤细胞行为和肿瘤进展，胶原蛋白是ECM 的主要成分，具有促进肿瘤发展的作用，例如IP4HA2promotes occurrence and progression of liver cancer by regulating the PI3K/Akt/mTOR signaling pathwaySHANG Ling 1,JIANG Wendi 1,ZHANG Junli 1,WU Wenjuan 1,21Key Laboratory of Cancer Research and Clinical Laboratory Diagnosis,2Department of Biochemistry and Molecular Biology,School of Laboratory Medicine,Bengbu Medical College,Bengbu 233030,China摘要：目的探讨脯氨酸4-羟化酶II （P4HA2）在肝癌细胞发生发展中的作用及相关机制。

方法利用GEPIA 、Human Protein Atlas 数据库预测P4HA2在肝癌中的表达情况，利用K-M plotter 在线数据库分析P4HA2的表达情况与肝癌预后的关系，采用qRT-PCR 和Western blot 检测肝癌细胞和正常肝细胞中P4HA2的表达。

仿生体系的富电子折叠体与四价离子环番的自组装

ｎｐｔｙｏ，ｉｘｐｅｙ）ｇｏｐｉｃｒｏａｅｏｄｍｅｓａｄＣＱＴｎＣＨＣ１／ｅｗｅｎｓｍｅ１５ｄｏｏａｈｈｌ（ｒｌ４ｄｏｏｈｎ１ｒｕｎｏｐｒｔｄｆｌａｒｎＢＰｉ３＇ＤＭＳＯ（１：１ｉａｙｓｌｅｔｓｓｅｄｅｔｈｒｅｔａｓｅｒｍｈｏｏｓｔ）ｂｎｒｏｖｎｙｔｍｕＯｃａｇｒｎｆｒｆｏｔｅｄｎｒＯＣＢＰＱＴ．Ｔｈｏｏｃｅｔｒｉｔｒｃｉｎｅｄｎｒａｃｐｏｎｅａｔｏｒｌｔｓｔｈｕｅａｅｏｔｅｎｍｂｒｏ１５ｄ。ｏａｈｈｌｇｏｐｏ４ｄｏｏｈｎｌｇｏｐｉｈｏｄｍｅｓｅｆ，－ｉｘｎｐｔｙｒｕｒ１，ｉｘｐｅｙｒｕｎｔｅｆｌａｒ．ＣＢＱＴｈｗｅＰｓｏｄ
ＩＩＳｉｏｇ・ＨＥｎ —ｉｇＷＡＮＧｅｌｎＨＥＮＧｅ— ｉＵｈｎｙＢｉｇｊｎＸｕｉ，ＺａＷｉｘｎ，ＺＨＯＵ —ｈｎ，ＩＱｉｏｇｚＪＡＮＧａｊａｇ（ｐｒ— ＨＨ —ｉｎＤｅａｔｍｅｔ／Ｃｅｓｒｉｏｒｈｍｉｔｙ，ＴａｚｏｉｅｓｔｉｈｕＵｎｖｒｉｙ，Ｌｉｈｉ３７０ｎａ１００，ＺｈｊａｇＰｒｖｎｅ，Ｃｈｎ）ｅｉｎｏｉｃｉａＳｌａｓｍｂｙｏｂｏｍｅｉｌｃｒｎｒｃｏｄｍｅｓａｄｅｒｃｔｎｃｃｃｏｈｎ．ＪｕｎｌｆｈｊｎＵｎｖｒｉｙｅｆｓｅｌｆｉｍｉｔｅｅｔｏｉｈｆｌａｒｎｔｔａａｉｉｙｉｐａｅｏｒａ－ｃｏｏＺｅｉｇａｉｅｓｔ

端基寡肽对不同分子量聚异戊二烯性能的影响

分子量及其分布用美国Ｗａｔｅｒｓ公司生产的凝胶渗透色谱仪测定试样的数均分子量及多分散指数，以聚苯乙烯作标样，ＴＨＦ为流动相。
傅里叶变换红外光谱（ＦＴＩＲ）用美国Ｎｉｃｏ— ｌｅｔ公司生产的ＮｉｃｏｌｅｔｉＳ１０型ＦＴＩＲ仪分析试样。
ＣＯＯＨ均在１７０６ｅｍ处出现了羧基吸收峰，表明了羧基的存在；其进一步与四肽进行缩合反应之后，１７０６ｅｌＴｌ处的吸收峰消失，证明羧基参与了反应。由图１（ｂ）可见，通过对ＮＭＲ谱图中化
· ２６８·
合成橡胶工业
第４１卷
学位移分别为５．１２和４．５６处的聚异戊二烯和四肽的特征峰进行积分后，计算二者比值可得试样１１—４Ａ的封端率为６７．４％，３０ —４Ａ为６３．４％；同时化学位移在５．１２和４．７２处分别为顺式和反式聚异戊二烯结构的特征峰，通过对二者分别计算可得，试样１１—４Ａ的顺式结构摩尔分数为９６．５％，３０—４Ａ为９９．５％，这表明由稀土催化体系所制备试样为高顺式聚异戊二烯橡胶。
通入足量二氧化碳过夜，制得ｉ１一ＣＯＯＨ试样。四肽封端低分子量聚异戊二烯（１１—４Ａ）
将１．７４ｇ的１１一ＣＯＯＨ胶样于１５０ｍＬ的ＴＨＦ中搅拌溶解后迅速加入７．９５ｇ的ＰｙＢＯＰ（溶解于８０ｍＬ的ＤＣＭ中）和３ｍＬ的ＤＩＥＡ，充分反应后加入２７ｍｇ的４Ａ、２ｍＬ的ＤＭＳＯ和１．６ｍＬ的ＤＩＥＡ混合物，反应过夜后制得１１—４Ａ试样，其反应式如下：

生物物理学中的蛋白质折叠：探索蛋白质折叠的动力学过程与折叠机理

生物物理学中的蛋白质折叠：探索蛋白质折叠的动力学过程与折叠机理摘要蛋白质折叠是生物物理学中的核心问题之一，对于理解蛋白质的功能和生命活动的本质至关重要。

本文深入探讨蛋白质折叠的动力学过程和折叠机理，回顾了经典理论和实验技术，介绍了最新的研究进展，并展望了未来的研究方向。

蛋白质折叠是一个复杂的多尺度过程，涉及到氨基酸序列、分子间相互作用、溶剂效应等多个因素。

通过综合运用实验和计算模拟手段，我们逐步揭示了蛋白质折叠的奥秘，为蛋白质工程、药物设计和生物技术的发展提供了理论基础。

引言蛋白质是生命活动的主要执行者，其功能与其独特的三维结构密切相关。

蛋白质折叠是指多肽链从无序状态自发地折叠成具有生物活性的特定三维结构的过程。

蛋白质折叠问题是生物物理学中的一个重要挑战，其研究不仅有助于我们理解蛋白质的结构与功能关系，还对蛋白质工程、药物设计和生物技术的发展具有重要意义。

蛋白质折叠的经典理论1. 安芬森法则（Anfinsen's dogma）：安芬森法则认为蛋白质的氨基酸序列完全决定其天然结构。

这一法则为蛋白质折叠研究奠定了基础，但对于复杂的蛋白质体系，折叠过程并不总是简单的自发过程。

2. 能量景观理论（Energy landscape theory）：能量景观理论将蛋白质折叠过程描述为在高维能量景观上的搜索过程。

蛋白质从高能量的非折叠态逐渐向低能量的折叠态转变，最终达到能量最低的天然态。

3. 漏斗模型（Funnel model）：漏斗模型是能量景观理论的一种简化形式，认为蛋白质折叠是一个从宽到窄的漏斗状过程。

蛋白质在折叠过程中会遇到多个中间态，但最终都会收敛到天然态。

蛋白质折叠的实验技术1. X射线晶体学（X-ray crystallography）：通过分析蛋白质晶体的X射线衍射图谱，可以获得蛋白质的高分辨率三维结构信息。

2. 核磁共振（NMR）：核磁共振可以提供蛋白质在溶液中的结构信息，有助于研究蛋白质的动态过程。

含RGD四肽的合成与功能

含 RG D 四肽的合成与功能赵明彭师奇 3 迪丽努尔 3 3周琴璐 3 3 3 赵文忠 3 3 3王银叶 (北京医科大学药学院 ,北京 100083 , 3 3 3 北京市体育科学研究所 ,北京 100050)摘要在血小板依赖的血栓形成中 ,纤维蛋白与血小板表面 G P I I b / I I I a 受体结合是重要步骤。

在受体识别中 , R G D (Ar g 2G ly 2Asp ,精〃甘〃天冬) 是关键序列。

含 R G D 多肽的竞争抑制作用 ,表现为抗血栓作用。

本文合成了 R G DS , R G DV 和 R G D F 3 种四肽 ;测定了它们的舒血管活性和抗血栓活性 ;讨论了溶液中的构象对生物活性的影响。

在大鼠血栓模型上 ,用 510 μmol 〃kg - 1 剂量 , R G DS 和 R G DV 无明显的抗血栓作用。

用 215 μmol 〃kg - 1 剂量 , R G D F 的抗血栓作用有明显的统计意义。

该结果与它们在溶液中的构象相关。

3 种四肽对主动脉条的舒血管作用体现另一种趋势 , R G DS 和 R G DV 显示明确的舒血管活性。

小剂量时 R G D F 的舒血管作用与对照组相比 ,无统计学意义。

关键词 R G D 序列 ;生物活性受到重视。

文献 2 ,4 ～6曾用固相法合成 R G DS 及类似物 ,研究它们的抗血小板聚集和抗血栓作用。

作者用液相法合成过 R G D S ,发现除抗血小板聚集作用外 ,它还可舒张血管7 。

为了更恰当地使用先导化合物 ,本文用液相法合成 R G DS 、R G DV 和 R G D F 3 种四肽 ,比较它们的抗血栓作用和舒血管作用 ,并讨论溶液中构象对活性的影响。

采用 B oc 保护氨端 ,侧链用适宜的保护基保护 L 2氨基酸 , 按图 1 所示的路线 , 液相法逐步接肽 ,制得对应的带保护基的中间体 (1～3) 。

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3 (GCN4-P1) has been shown to form a cooperatively folded helical dimer. [12,13,18-22]. This peptide is an excellent system for studying protein folding because it is quite simple, and yet contains a well-packed helix/helix interface, as found in globular proteins. It has been shown to exist in a two-state equilibrium between unstructured monomers and fully alpha-helical dimers12. The alpha-helical secondary structure and the double-helical folded structure apparently form concomitantly.[13,17-20,23] To simplify the folding reaction, Kim and coworkers have introduced a covalent disulfide tether between the two-peptide chains. The crosslink stabilizes the proteins towards thermal denaturation, although the peptide continues to fold in an apparent, two-state equilibrium. [24] One purpose of this study is to investigate the microscopic features of a macroscopically observed kinetic model. GCN4-P1 exhibits two-state folding kinetics when in bulk solution [17,20], although a folding intermediate has occasionally also been observed for some coiled coils [13,19,23]. It has been shown, however, that macroscopic averaging can create effective two-state kinetics in the bulk even when there are multiple unfolded states of the protein.[10] Single molecule experiments are sensitive to mechanistic heterogeneity. Since single molecule experiments are immune to ensemble averaging, it is possible, in principle, to distinguish between a pathway-based folding mechanism and a landscape multi-path mechanism. If a protein has a single folding pathway, then the distribution of observed kinetic rates should be narrow. Conversely if there are multiple pathways there would, in general, be multiple rates associated with folding and an appropriate distribution of rates would be observed in a single molecule experiment.
1
Department of Chemistry and 2Department of Biophysics and Biochemistry University of Pennsylvania, Philadelphia, PA 19104 Abstract We have prepared a bichromophoric crosslinked variant of GCN4-P1 for single molecule fluorescence energy transfer experiments (GCN4-Pf). The folding and unfolding fluctuations of single GCN4-Pf molecules are measured in a two channel confocal microscope with which donor and acceptor fluorescence trajectories are measured simultaneously. The energy transfer efficiency is thereby determined and its probability distributions as a function of added denaturant [urea] are calculated. The distributions indicate that single molecule GCN4-Pf is in dynamic folding equilibrium with the position of the equilibrium being altered by the concentration of urea.

Folding Dynamics of Single GCN-4 Peptides by Fluorescence Resonant Energy Transfer Confocal Microscopy
Yiwei Jia1, David S. Talaga1, Wai Leung Lau2, Helen S. M. Lu2, William F. DeGrado2, Robin M. Hochstrasser1
2 Introduction Single molecule measurements are now moving from the realm of technological demonstrations to making contributions to new understanding of chemical systems particularly those that are microscopically inhomogeneous and exhibit dynamics over many time scales.[1-9] Using single molecule detection, it is now possible to follow the evolution in time of individually selected members of an equilibrium ensemble. This trajectory can be used to evaluate rates, rate constants, and distributions of other properties. On the other hand, bulk measurements usually give only ensemble averaged value of the molecular property in question. Proteins and other biological assemblies exhibit microscopic structural heterogeneity and are therefore of particular interest for single molecule studies. The structural fluctuations of proteins can result in folding and unfolding of the primary sequence of amino acids between a well defined three-dimensional native structure and a broadly distributed set of denatured structures. [4,10-14] In one example, fluorescence spectral fluctuations were attributed to dynamics amongst protein substrates in single molecule experiments.[6] In another study, protein conformational dynamics was cited as the origin of fluorescence intensity and polarization fluctuations of single Staphylococcal nuclease.[4] This paper concerns the study of a peptide derived from the yeast transcription factor, GCN4. The DNA binding domain of this protein includes a sequence that forms a short segment of a two-stranded coiled coil [15,16], as shown in figure 1. Since their discovery, coiled coils have provided very simple model systems for the study of the folding of water-soluble proteins.[12,13,17] A peptide spanning the coiled coil of GCN4