DNA directed assembly of nanoparticle linear structure for nanophotonics
Baoquan Ding, Stefano Cabrini, Ronald N. Zuckermann, and Jeffrey Bokor
Citation: Journal of Vacuum Science & Technology B 27, 184 (2009); doi: 10.1116/1.3072515
View online: https://www.doczj.com/doc/8a4050426.html,/10.1116/1.3072515
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Published by the AVS: Science & Technology of Materials, Interfaces, and Processing
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DNA directed assembly of nanoparticle linear structure for nanophotonics Baoquan Ding,a?Stefano Cabrini,b?Ronald N.Zuckermann,and Jeffrey Bokor
Molecular Foundry,Lawrence Berkeley National Laboratory,1Cyclotron Rd.,Berkeley,California94720
?Received17June2008;accepted22December2008;published2February2009?
Assemblies of metal nanospheres have shown interesting properties for nanophotonics.Here the
authors describe a method to use robust DNA multicrossover molecules to organize Au
nanoparticles with different sizes to form well controlled linear chain structures with desired
distance below10nm between the particles.Au particles with only one piece of DNA attached are
puri?ed individually.Three different sizes DNA-Au conjugates then hybridize with?ve other DNA
strands to form the stiff triple crossover?TX?motif.The linkage position of Au on the TX motif is
designed so that the Au particles are organized in a well controlled linear fashion.Precise control of
the distances between the Au particles can be also achieved by design of the linkage position.This
arrangement of Au nanoparticle linear structures meets the requirement to construct an ef?cient
nanolens.?DOI:10.1116/1.3072515?
The optical properties of metallic nanoparticles and their aggregates have attracted great interest for their application to nanophotonics.Theoretical computation shows that a self-similar linear chain of several metal nanospheres with pro-gressively decreasing sizes and separations can work as an ef?cient nanolens.1
Previously,self-assembled DNA nanostructures have been produced to generate various patterns.2Using DNA to orga-nize nanoparticles was originally demonstrated by Alivisatos3and Mirkin.4Other groups used stiff DNA motifs to organize nanoparticles in well designed fashion to form one-dimensional?1D?and two-dimensional?2D?arrays,5–7 but a linear array of nanoparticles with decreasing sizes and precise control of distance under10nm has not been re-ported.Bidault et al.recently reported a plasmon-based nanolens assembled on a well de?ned DNA template.8How-ever their method still uses DNA duplex as the template,so the orientation and distance between nanoparticles is hard to control.Lithography has also been utilized to fabricate nano-photonic structures.However it is very hard to produce nanospheres by top-down methods.Here we describe a method to use a rigid DNA motif?triple crossover molecule?to organize Au nanoparticles to realize the arrangement:well controlled linear structure with desired distances.The persis-tence length of duplex DNA in solution is about50nm.9The DNA triple crossover molecule which contains three helical domains is much more rigid and has been used to con-structed2D and1D arrays10,11over relatively large length scales.Thus we reasoned that the triple crossover should work as a rigid linear template.
Here we design a nine-turn long TX motif that consists of eight DNA strands as shown in Fig.1.The DNA sequences were designed with the SEQUIN program13and listed in Table I.Three strands?labeled as2,5,and7?are thiol modi?ed at the5ft end to allow the linkage to Au particles.Au particles with only one piece of DNA attached are individually puri-?ed by agarose gel electrophoresis.Three DNA-Au conju-
gates then hybridize with?ve other DNA strands without Au
linkage to form the TX motif.We can leave a breaking point
on the DNA backbone and use thiol ended DNA at any de-
sired position.By changing the location of thiolated strands
in the TX design,we can control the position of Au attach-
ment,and thereby control the distance between Au nanopar-
ticles.One TX motif can template three different sizes of Au
nanoparticles,for example5,10,and15nm.With properly
designed sticky ends,two TX motifs can be linked together
with double cohesion to assemble six nanoparticles into a
symmetric chain.A computation study predicts that these six
particles in a centrally symmetric chain will exhibit ex-
tremely high?eld enhancement.1
First,the formation of a hybridized DNA complex with-
out Au particles is demonstrated by nondenaturing gel elec-
trophoresis.DNA strands were purchased from Integrated
DNA Technologies,Inc.14and puri?ed by denaturing poly-
acrylamide gel electrophoresis?PAGE?.The TX DNA motif
was formed by mixing an equal quantity of each strand
designed in the complex at a concentration of1?M,as es-timated by OD260,in1?TAE/Mg buffer?20mM tris,
p H7.6,2mM ethylenediaminetetraacetic acid?EDTA?,
12.5mM MgCl2?.The DNA motif was analyzed by nonde-naturing gel electrophoresis with10%nondenaturing PAGE
gel running in room temperature in1?TAE/Mg buffer.On
the gel in Fig.2,lanes A and B are the50base pair?bp?
ladder markers.Starting from lane1to8,in each lane,we
add one more DNA https://www.doczj.com/doc/8a4050426.html,ne1is strand1,lane2is the
annealed mixture of strands1and2,and lane8is the an-
nealed mixture of all8strands.There is a clear single band
in lane8and no multimerization or breakdown products
which mean these eight DNA strands are hybridized to form
a stable DNA complex.
Au colloids with diameters of5,10,and15nm?pur-
chased from Ted Pella Inc.?were stabilized by incubation
with bis?p-sulfonatophenyl?phenylphosphine dihydrate dipo-
tassium salt?Strem Chemicals Inc.?.This salt?20mg?was
added to the gold colloid solution?100ml;?1outside di-
a?Electronic mail:bding@https://www.doczj.com/doc/8a4050426.html,
b?Electronic mail:scabrini@https://www.doczj.com/doc/8a4050426.html,
184184 J.Vac.Sci.Technol.B27…1…,Jan/Feb2009
ameter at 520nm ?,and the mixture was shaken overnight at room temperature.NaCl ?solid ?was then added slowly to this mixture while stirring until the color changed to light purple.The supernatant was carefully removed with a pipette after centrifugation,and the Au nanoparticles ?NPs ?were resuspended in 1mM phosphine solution ?5ml ?.Methanol ?5ml ?was then added and the mixture was centrifuged and the supernatant was removed.The Au NPs were resuspended in 2.5mM phosphine solution ?2ml ?.The concentration of the Au NPs was estimated from the optical absorbance at 520nm.Phosphine coating gives a net negative charge on the particle surface,which stabilizes the Au NPs in high concentrations of particles.
The thiolated DNA strand was purchased from IDT and puri?ed by denaturing PAGE.The thiolated DNA strand was incubated overnight with Au NPs capped with phosphine in a ratio of 1.0:1.0in 0.5?TBE containing 50mM NaCl at room temperature.Different combinations of DNA/Au con-jugate ?particles bearing 1–4single-stranded DNAs ?ssD-NAs ??were separated by 3%agarose gel ?running buffer:0.5?TBE,loading buffer:30%glycerol,at 15V cm ?1?.Dif-ferent bands were clearly visible from the red color of the Au NPs.The band with DNA/Au in a 1:1ratio was electroeluted onto the glass ?ber ?lter and a dialysis membrane ?MWCO 10000?inserted in front of the band in the gel.The conjugate was recovered using a 0.45?m centrifugal ?lter device.The
concentration of this Au NP/DNA conjugate was estimated from the optical absorbance at 520nm.
The Au particles conjugated with the DNA strand were further stabilized by adding thiolated T5ssDNA oligomers to avoid further coagulation during annealing under high-electrolyte conditions.This stabilization was carried out by incubating a relatively high concentration ?T5/Au=100:1?of ssDNA T5relative to the concentration of DNA/Au con-jugates in 1?TBE /Na+buffer ?89mM tris,89mM boric acid,2mM EDTA,and 50mM sodium chloride,p H 8.0?.To form the Au-NP and DNA complex,three Au-NP-DNA conjugates and other ?ve DNA strands were mixed together at 80°C in an equimolar ratio and were slowly cooled to room temperature in 1?TBE buffer containing
T ABLE I.The sequences of DNA strands used in the experiment.
Strand 1:5?CAGGCA TCAGA CATAC GG ACAGC CGATGGTC CGACCGCA AGCGT GGAGT GGCAT
CGT AAGTC ACATT CAA TACGG ACAAG TAACG ACACT GCTCG TTC
Strand 2:HS-5?-TTTT TTTT CCAGC AATCG CA CC GTATG TCTGA TGCCTG
Strand 3:5?-CATCT GTTGA CC TATCC TGCTACGC CCGCTACA CCAACTGT TCACTTGG ACTCC TGCGATTGCTGG
Strand 4:5?-AATGT CACTT ACG ATGCC TGTAGCGG GCGTAGCA CCAAGTGA ACCAGTTGG ACTCC TGATGTCT
Strand 5:HS-5?TTTTTTTTACGCCAGT G GTCAT CTGGT ATCGG ACGCT TGCGGTCG GACCATCG
GCTGT GGAGT GGATA GGTCA ACAGATG
Strand 6:5?-TTGGACG ACG GTCAA GCGAC TGCAG CATAC GCGCA CCTCA CCGTA TTG
Strand 7:HS-5?TTTTTTTTACCA GCGTA CAGCC TCAGG ACTGGCGT AGACATCA CCGAT ACCAG
ATGACC TGCGA GTATG CTGCA TTGAC CGT
Strand 8:5?-GTCGCAT GAA CGAGCAACGT CGTTA CTTGT GGCTG TACGC TGGT
CGACAC
F I
G .2.Nondenaturing gel electrophoresis shows that the DNA strands hy-bridize to form the target DNA motif ?without Au particle attached ?.Lanes A and B are the 100bp ladder https://www.doczj.com/doc/8a4050426.html,nes 1–8are annealed mixtures of DNA https://www.doczj.com/doc/8a4050426.html,ne 1:strand https://www.doczj.com/doc/8a4050426.html,ne 2:strands 1and https://www.doczj.com/doc/8a4050426.html,ne 3:strands 1–https://www.doczj.com/doc/8a4050426.html,ne 4:strands 1–https://www.doczj.com/doc/8a4050426.html,ne 5:strands 1–https://www.doczj.com/doc/8a4050426.html,ne 6:strands 1–https://www.doczj.com/doc/8a4050426.html,ne 7:strands 1–https://www.doczj.com/doc/8a4050426.html,ne 8:strands
1–8.
F I
G .1.?a ?Structure of DNA triple crossover template.Strands are labeled at the 5?by numbers.Strands 2,5,and 7have thiol group at the 5?end.?b ?Three prepuri?ed Au-DNA conjugates will hybridize with other ?ve DNA strands to form the DNA motif.Thin black arrows point to the Au and DNA linkage position.
JVST B -Microelectronics and Nanometer Structures
50mM NaCl.This assembled complex is then analyzed by agarose gel electrophoresis:3%agarose gel with running buffer 0.5?TBE ?89mM tris,89mM boric acid,2mM EDTA,p H 8.0?,loading buffer 30%glycerol,at 15V cm ?1.Figure 3shows the agarose https://www.doczj.com/doc/8a4050426.html,ne 1contains the an-nealed mixture of three Au-DNA conjugates with other ?ve DNA https://www.doczj.com/doc/8a4050426.html,ne 2contains prepuri?ed 5nm Au with single DNA https://www.doczj.com/doc/8a4050426.html,ne 3contains prepuri?ed 10nm Au nanoparticle with a DNA https://www.doczj.com/doc/8a4050426.html,ne 4contains prepuri-?ed 15nm Au nanoparticle with a DNA attached.Judging from the position of single Au-DNA conjugates in lanes 2–4,we expect that band A is the target band that contains all three particles;band B is the partial hybridization product that contains 10and 15nm Au particles;and band C is the mixture of extra 15nm Au particles and other small partial hybridization products.B and D is extra 10nm Au particles.The total yield of the target band with chain structure ?band A ?is less than 15%,but optimization of the stoichiometry among the three Au particles linked DNA and the other ?ve DNA strands will increase the yield.Corresponding
bands
F I
G .3.Agarose gel electrophoresis of hybridization product of DNA motif with Au https://www.doczj.com/doc/8a4050426.html,ne 1:Annealed mixture of three Au-DNA conju-gates with other ?ve DNA https://www.doczj.com/doc/8a4050426.html,ne 2:Prepuri?ed 5nm Au with single DNA https://www.doczj.com/doc/8a4050426.html,ne 3:Prepuri?ed 10nm Au with single DNA https://www.doczj.com/doc/8a4050426.html,ne 4:Prepuri?ed 15nm Au with single DNA attached.Band A :target band that contains all three particles.Band B :partial hybridization product that contains 10and 15nm Au particles.Band C :Mixture of extra 15nm Au particle and other small partial hybridization product.Band D :Extra 10nm Au
particle.
F I
G .4.?a ?SEM images of band B sample which correspond to the partial hybridization product that contains 10and 15nm Au particles.?b ?Zoom-in image with particle sizes
labeled.
F I
G .5.?a ?SEM images of band A sample that contains the target linear structure.5,10,and 15nm Au particles form straight linear structure.The size of particles is labeled on the images.?b ?Measured distances between particles are consistent with designed structure.The average center to center distance of 15nm particle and 5nm particles is about 25.4nm and the gaps between particles is about 2.5nm.
J.Vac.Sci.Technol.B,Vol.27,No.1,Jan/Feb 2009
are extracted from the gel.Samples of Au-NP-DNA complex were deposited on PolyLysine treated Si surface.Images were taken by Zeiss Gemini Ultra-55analytical scanning electron microscope.The SEM images are consistent with our expectation.Figure4?a?is the SEM image from the band B sample.We can see clearly that there is only a partial hybridization product that contains10and15nm Au par-ticles.Figure4?b?is the zoom-in image that shows the sizes of the particles.Figure5?a?is the scanning electron micros-copy?SEM?image from target band A.The linear structure of the three different size Au nanoparticles is clearly demon-strated.The sizes of particles are labeled on the images.The measurement of distances between the three particles is shown in Fig.5?b?.The average center to center distance of 15nm particle and5nm particles is about25.4nm,and the gaps between particles is about2.5nm;which are consistent with our design.
In conclusion,linear chains of different size Au particles with controllable distance were successfully fabricated.This structure is expected to work as an ef?cient nanolens.The next step will be the optical characterization of these struc-tures.The six particle centrally symmetric linear chain is predicted to show extremely high?eld enhancement.This linear structure can be produced by connecting two stiff TX motifs through robust double cohesion.Our method could apply to other stiff DNA template motifs to organize other sizes of nanoparticles.For example,DNA origami12can be use to organize more and larger different sizes particles.Here we have demonstrated that we can use biological material as the template for the nanoscale fabrication of inorganic object in a controllable fashion.The same method could be applied to the nanofabrication of other materials.
Work at the Molecular Foundry was supported by the Of-?ce of Science,Of?ce of Basic Energy Sciences,of the U.S. Department of Energy under Contract No.DE-AC02-05CH11231.
This paper was presented at the52nd International Con-ference on Electron,Ion,and Photon Beam Technology and Nanofabrication held in Portland,Oregon on May27–30, 2008.
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JVST B-Microelectronics and Nanometer Structures
People don’t always need advice. Sometimes all they need is a hand to hold, an ear to listen, and a heart to understand. 人们并不总是需要建议。有时候，人们只是需要有人来 执手支持，有人来侧耳倾听，有人来读懂心声 Me? Weird? Please, I'm limited edition. 我？很怪异？拜托，我可是限量版 Never expect, never assume, and never demand. Just let it be, because if it's meant to be, it will happen the way you want it to... 永不期待，永不假设，永不强求。顺其自然，若是注定 发生，必会如你所愿 How much you put into a relationship determines how much it will hurt when it ends. 一段 感情里，你付出了多少，在结束的时候，就决定了你有多伤 Sometimes she'll wait and see if you will text her first just to see if you really care. 有时， 女生会等着，看你会不会先给她发短信，来判断你是否真的在乎 You can love someone easily. But it's not easy to love someone unconditionally. 爱上一个人 很容易，但无条件的爱则很难 I don’t ever need anyone to promise me that they will never hurt me because sooner or later... it will happen. 不需要任何人承诺说，不会伤我。因为迟早，这都会发生 The best gift you can give to someone is your time, because you're giving them something you can never get back. 你能给别人的最好礼物就是你的时间，因为你给了他们你再也得不到 的东西 There are many words that I want to say to you, but I don’t know how…有很多话我想跟你 说，却不知从何说起 Take a risk. If the outcome isn’t what you expected, at least you can say you tried. 冒险一 试。就算结果不是你所期望的，至少你可以说你已经试过了 What makes you different makes you beautiful. 你的与众不同让你美丽动人。 It’s the rule of life that everything that you have always wanted comes at the very second you stop looking for it. 你一直渴望得到的东西，却在你即将放弃的那一刻不期而至，这就是生 活的法则 Don’t expect someone to stay sweet forever because even the sweetest candy has an expiration date. 不要期待有人会一直甜美可爱，因为就算是最甜的糖果也有保质期 A real woman avoids drama, because she knows her time is precious and she's not wasting it on unimportant things. 一个真正的女人会避免小题大做，因为她知道时间宝贵，不会任之浪 费在无谓的事情上 When you’re up, your friends know who you really are. When you’re down, you know who your friends are. 当你春风得意之时，你的朋友都知道你是谁；当你落魄潦倒之时，你就知道 谁是你的朋友了 Don’t need a perfect boyfriend; I just want someone who won’t give up on me. 不需要完美 的男朋友，我只想要一个不会放弃我的人 Love turns the smart to foolish, the evil to kind, and the weak to strong. 爱，让智者变愚钝， 让恶人变善良，让懦夫变坚强
关于伙伴选择的英语作文 Your Ideal Partner personality Appearance Intelligence Job/Future Person A * * * * * * * * * * * * Person B * * * * * * * * * * * * * Person C * * * * * * * * * * * * Directions: The chart indicates some of the characteristics of three people of approximately your age. Which of them would you prefer as a partner in life, as husband, wife, or closest friend? Explain your reason for your choice. If I were to choose one person from the three people in the chart, to be my partner in life, I would choose A. This is because of his characteristics which I think will suit my characteristics. His personality attracted me most when I was considering this question. Since I myself am of an amiable character, I find that I always enjoy the company of friendly persons. A‘s pleasant appearance also attracts me which is a fact I can‘t deny. I would
漂亮的英文翻译 1.你可知我百年的孤寂只为你一人守侯，千夜的恋歌只为你一人而唱。 You know my loneliness is only kept for you, my sweet songs are only sung for you. 2.如果活着，是上帝赋予我最大的使命，那么活者有你，将会是上帝赋予我使命的恩赐…… If living on the earth is a mission from the lord… living with you is the award of the lord… 3.你知道思念一个人的滋味吗，就像喝了一大杯冰水，然后用很长很长的时间流成热泪。 Do you understand the feeling of missing someone? It is just like that you will spend a long hard time to turn the ice-cold water you have drunk into tears.
4.在这充满温馨的季节里，给你我真挚的祝福及深深的思念。 In such a soft and warm season, please accept my sincere blessing and deep concern for you. 5.一份不渝的友谊，执着千万个祝福，给我想念的朋友，温馨的问候。 For our ever-lasting friendship, send sincere blessings and warm greetings to my friends whom I miss so much. 6.想你，是一种美丽的忧伤的甜蜜的惆怅，心里面，却是一种用任何语言也无法表达的温馨。 It is graceful grief and sweet sadness to think of you, but in my heart, there is a kind of soft warmth that can’t be expressed with any choice of words. 7.不同的时间，不同的地点，不同的人群，相同的只有你和我；时间在变，空间在变，不变的只有对你无限的思念！ You and I remains the same in different time, at different places,among different people; time is changing, space is changing and everything is changing except my miss to you! 8.没有杯子……咖啡是寂寞的……没有你……我是孤独的…… Coffee is lonely without cups. I am lonely without you. 9.每一天都为你心跳，每一刻都被你感动，每一秒都为你担心。有你的感觉真好。
1.Please don't see me off.The journey I'm walking on alone is lonely and dangerous. 请不要为我送行。我即将独自踏上的旅途是孤独且布满荆棘的。 2.I will always keep my eyes wide open so that I can know everything in your heart. 我会一直睁大眼睛这样的话我就能读出你心底的一切了。
3.I love the way of rain drops falling on the leaves because that is the way you loved me . 我喜欢雨滴落在树叶上的方式因为你也曾经这样爱过我。
4.I miss you. I miss you. I miss you. Even if let me say this one thousand times,I will never get tired of it. 我想你我想你我想你呀即使让我说一千遍我也永远不会厌倦。
5.Look at the stars in the sky,that's all my wishes especiallly for you. 你看到夜空中的星星了么那都是专属于你我的祝福啊。 6.Yep.I'm wondering if you will give all yourself to me when I need you. 是的。我在想我需要你的时候你会不会把全部的你托付给我。
世界真的很小，好像一转身，就不知道会遇见谁…世界真的很大，好像一转身，就不知道谁会消失…The world is really small, when you turn around, you don’t know who you’re gonna encounter with…The world is really big, when you turn around, you don’t know who’s already gone. Don’t look back——you’re not going that way! 别回头——那不是你要走的方向！ Your presence is a present to the world. 你的存在是献给世界的一份礼物。 Every story has an ending.but in life, every end is a new beginning.每段故事都有一个结局。但是在人的一生中，每一个终点同时也是一个新的起点。 I love you not because of who you are,but because of who I am when I am with you.我爱你，不是因为你是一个什么样的人，而是因为我喜欢与你在一起时的感觉。 You’re unique and one of a kind. 你是唯一的，无可替代。 Whenever you say “I love you”, please say it honestly. 无论何时说“我爱你”，请真心实意。 In fact, just intoxicate wine does not drink it remembered that unbearable past.其实酒不醉人，只是在喝的时候想起了那不堪的过去。 For in all adversity of fortune the worst sort of misery is to have been happy. 在所有不幸中，最不幸的事是曾经幸福过。 It takes a minute to have a crush on someone, an hour to like someone and a day to love someone ，but it takes a lifetime to forget someone.一分钟心动，一小时喜欢，一天爱上。忘记他，却是一辈子。
漂亮的英文怎么写 【漂亮：pretty; beautiful; good-looking; handsome】 漂亮[piào liang] (好看;美观) handsome; good-looking; pretty; beautiful: be prettily dressed; 衣服穿得漂亮 trim oneself up; 把自己打扮得漂漂亮亮 This photograph flatters you. 这照片比你本人漂亮。 (出色) smart; remarkable; brilliant; splendid; beautiful: well done; 干得漂亮 speak prettily; 说得漂亮 You write a beautiful hand. 这字写得真漂亮。 pretty例句: 1.Today was a pretty project based intensive day. 今天是项目相当密集的一天。
2.She is a pretty girl. 她是个漂亮的女孩。 3.Morocco's king seems pretty safe. 摩洛哥国王看上去似乎很安全。 4.Life can be pretty complicated. 生活是非常复杂的。 5.Balancing national security concerns against moral responsibilities is never pretty. 平衡国家安全问题和道德责任从来不是一件容易的事。 beautiful例句: 1.But paris is more beautiful than tokyo. 但巴黎是比东京更美丽。 2.You have a beautiful house. 你有个漂亮的房子。 3.There is nothing more beautiful than a wedding. 没有什么比婚礼更美的了。 https://www.doczj.com/doc/8a4050426.html, 4.South africa is a beautiful country. 南非是一个美丽的国度。 5.Then she began a beautiful love song. 接着她开始演唱一首优美的爱情歌曲。
I don’t understand why fate brings two people who can’t stay together forever to each other. 我不明白，为什么命运要让两个不可能在一起的人相遇。 I’m proud of my heart. It’s been played, burned, and broken, but it still works. 我为自己的心感到骄傲。它曾受玩弄，曾经心焦，曾遭破碎，却依然鲜活跳动。 If you don’t understand my silence, you will never understand my words.—如果你不懂我的沉默，你也永远不会明白我说的话语。 When life gives you a hundred reasons to cry, show life that you have a thousand reasons to smile.—当生活给你100个伤心的原因，你就还它1000个微笑的理由。 Learn to use the understanding of the vision to see and appreciate each other, in order to opinionated care to pipe each other.—学会用理解的，欣赏的眼光去看对方，而不是以自以为是的关心去管对方。 Whatever with the past has gone, the best is always yet to come.—无论过去发生过什么，你要相信，最好的尚未到来。 If we can only encounter each other rather than stay with each other, then I wish we had never encountered.—如果只是遇见，不能停留，不如不遇见。 There will be a tear that lets you grow in a twinkling.总会有一次流泪，让我们瞬间长大。 You are so lucky, because you can choose to love me or not, but myself only have to choose from loving you or loving you more.—你是幸运的，因为你可以选择爱我或不爱我，而我只能选择爱你还是更爱你。 Sometimes, you just have to pretend that you are happy just to stop everyone from asking you what the hell happened—有时候，你不得不假装很快乐，只是为了不让别人问“你怎么了？”Try to hold the right hand with your left hand, and gave yourself most simple warmth. We should learn to get it by ourselves instead of craving for warmth from others.试着用左手握住右手，给自己最简单的温暖，不再奢求别人的给予，开始学着自己给自己。 Among those people that appear in our life, some are to teach us, some to comfort us, some to share and some to love. 在我们生命中出现的人，一些给我们上课，一些让我们痊愈，有的用来分担分享，有的用来真爱。 等翻译：我喜欢你。是一句藏在心里很久的话。你可以不用回复我，但是，我却必须把它告诉你。 I’m proud of my heart. It’s been played, burned, and broken, but it still works. 我为自己的心感到骄傲。它曾受玩弄，曾经心焦，曾遭破碎，却依然鲜活跳动。 I don’t think that when people grow up.Conversely, I think it’s a selecting process, knowing what’s the most important and what’s the least. And then be a simple man.—人的心智成熟是一个逐渐剔除的过程，知道自己最重要的是什么，知道不重要的东西是什么。而后，做一个纯简的人。 Forget all the reason why it won’t work and believe the one reason why it will. ------ 忘掉所有那些“不可能”的借口，去坚持那一个“可能”。 Best way to not get your heart broken, is pretend you don’t have one.—不想伤心最好的办法就是假装自己没心没肺。 Memory is a wonderful thing if you don’t have to deal with the past。回忆本来是非常美好的，只要你能让过去的都过去 I‘d rather love someone I can‘t have than have someone I can‘t Love 。我宁愿爱上一个我不能拥有的人，也不想拥有一个我无法爱上的人。 There is still a long way to go. You may cry, but you have to keep on moving and never stop.前面
接下来帖一篇各国公司的缩写，这些缩写对我们在国际贸易准确的辨别国别信息有很大帮助。同时也可利用利用搜索引擎搜索客户资料。与列位共享。。 新加坡法律规定私人企业名称中必须出现Pte.字样，而其他国家则绝少有此现象。由于有的缩略语为某个国家独有，有的则是同一语种国家和地区所共有。 参考其他类似缩写词： 交易国别是我国国际收支统计记录中一个重要的申报要素。在对交易对方和交易国别的核查过程中，我们发现很多国家和地区的收付款人（公司）名称中存在各式各样的表示企业类型的缩略语，比如：Co.Ltd、Corp.、S.A.、GmbH和BHD SDN等。经过调查，我们发现上述现象存在的主要原因主要是：（1）各国语言文字不同，同一词汇在不同的国家具有不同的称谓，比如，“公司”在英语里一般简称Co.、Inc.或Corp.，在瑞典语里的简称则是AB；（2）各国法律制度和使用习惯存在差异，导致一些国家和地区的企业名称比较独特，比如，新加坡法律规定私人企业名称中必须出现Pte.字样，而其他国家则绝少有此现象。由于有的缩略语为某个国家独有，有的则是同一语种国家和地区所共有。因此，我们能通过交易对方中的缩略语来排除掉或推定出某一笔国际收支交易的国别。 有鉴于此，我们以近两年全国部分地区的近20万条国际收支统计交易记录为索引，对我国主要贸易伙伴国的《公司法》、《投资法》进行了查阅和研究，梳理出许多在国际收支统计信息中出现频率较高、可用于国际收支交易国别判断的交易对方缩略语，现列举和剖译如下： 一、SDN BHD与马来西亚的关系 SDN系马来西亚语Sendirian的缩写，意即“私人”。BHD系Berhad的缩写，意为“公司”。SDN BHD是指“私人有限公司”，单BHD一般指“公众有限公司”。 在马来西亚，企业一般注册为个人企业、合伙人企业或私人有限公司，其中以SDN BHD私人有限公司最为常见。如： CSP CORPORATION M ALAYSIABHD UNITED MS ELECTRICAL MFG(M) S DN BHD 除新加坡、文莱（马来语是两国正式语言）企业名称偶尔出现SDN BHD外，其他国家企业名称基本上没有S DN BHD字样，因此，如果交易对方中出现S DN BHD，而交易国别不是马来西亚，则基本上可认定交易国别有误。 二、GmbH是德语区国家除德国外还有奥地利、列支敦士登、瑞士、比利时和卢森堡 GmbH系德文Gesellschaft Mit Beschrankter Haftung的缩写，等于英文中的Limited liability company，即“有限责任公司”。 有限责任公司为介于大型股份公司与小型合伙企业之间的企业形态，为目前德国采用最为广泛的企业形式。如： B.TEAM EDV.EDITION B.BREIDENSTEIN GMBH（德国） MESSE FRANKFURT MEDJEN UND SERVICE GMBH（德国） 此类公司形式主要是德语区存在，除德国外，将德语作为母语之一的还有奥地利、列支敦士登、瑞士、比利时和卢森堡，上述国家企业名称中都有可能出现GmbH。如：Hutchison 3G Austria GmbH即为地址在维也纳的奥地利企业。 因此，绝不可将交易对方中出现GMBH的交易记录一概认为是德国的。
1 我的世界不允许你的消失,不管结局是否完美. No matter the ending is perfect or not, you cannot disappear from my world. 2 爱情是一个精心设计的谎言 Love is a carefully designed lie. 3 承诺常常很像蝴蝶,美丽的飞盘旋然后不见 Promises are often like the butterfly, which disappear after beautiful hover. 4 凋谢是真实的盛开只是一种过去 Fading is true while flowering is past 5 为什么幸福总是擦肩而过,偶尔想你的时候….就让….回忆来陪我. Why I have never catched the happiness? Whenever I want you ,I will be accompanyed by the memory of... 6 如果你为着错过夕阳而哭泣，那么你就要错群星了 If you weeped for the missing sunset,you would miss all the shining stars 7 如果只是遇见，不能停留，不如不遇见 If we can only encounter each other rather than stay with each other,then I wish we had never encountered . 8 宁愿笑著流泪,也不哭著说后悔心碎了,还需再补吗？ I would like weeping with the smile rather than repenting with the cry,when my heart is broken ,is it needed to fix? 9 天空没有翅膀的痕迹，而鸟儿已飞过 There are no trails of the wings in the sky, while the birds has flied away. 10 当香烟爱上火柴时,就注定受到伤害 When a cigarette falls in love with a match,it is destined to be hurt 11 人活着总是要得罪一些人的就要看那些人是否值得得罪 When alive ,we may probably offend some people.However, we must think about whether they are deserved offended. 12 命里有时终需有命里无时莫强求 You will have it if it belongs to you,whereas you don't kveth for it if it doesn't appear in your life.
1.I love three things in this world. Sun, moon and you. Sun for morning, moon for night , and you forever.予独爱世间三物。昼之日，夜之月，汝之永恒。 2.Life has taught us that love does not consist in gazing at each other, but in looking outward together in the same direction. 生活教会我们，爱并不在于长久地凝视，而在于眺望远方同一方向的希望。 3.Life isn't about waiting for the storm to pass, it's about learning to dance in the rain.生活不是等待暴风雨过境，而是学会在雨中跳出最美的舞姿。 4.You know my loneliness is only kept for you, my sweet songs are only sung for you.你可知我百年的孤寂只为你一人守侯，千夜的恋歌只为你一人而唱。 5.If living on the earth is a mission from the lord…living with you is the award of the lord…如果活着，是上帝赋予我最大的使命，那么活者有你，将会是上帝赋予我使命的恩赐…… 6.Do you understand the feeling of missing someone? It is just like that you will spend a long hard time to turn the ice-cold water you have drunk into tears.你知道思念一个人的滋味吗，就像喝了一大杯冰水，然后用很长很长的时间流成热泪。 7.In such a soft and warm season, please accept my sincere blessing and deep concern for you.在这充满温馨的季节里，给你我真挚的祝福及深深的思念。 8.For our ever-lasting friendship, send sincere blessings and warm greetings to my friends whom I miss so much.一份不渝的友谊，执着千万个祝福，给我想念的朋友，温馨的问候。 9.It is graceful grief and sweet sadness to think of you, but in my heart, there is a kind of soft warmth that can’t be expressed with any choice of words.想你，是一种美丽的忧伤的甜蜜的惆怅，心里面，却是一种用任何语言也无法表达的温馨。 10.You and I remains the same in different time, at different places,among different people; time is changing, space is changing and everything is changing except my miss to you!不同的时间，不同的地点，不同的人群，相同的只有你和我；时间在变，空间在变，不变的只有对你无限的思念！ 11.Coffee is lonely without cups just as I am lonely without you.没有杯子……咖啡是寂寞的……没有你……我是孤独的…… 12.My heart beats for you every day. I am inspired by you every minute, and I worry about you every second. It is wonderful to have you in my life.每一天都为你心跳，每一刻都被你感动，每一秒都为你担心。有你的感觉真好。 13.No matter the ending is perfect or not, you cannot disappear from my world.我的世界不允许你的消失,不管结局是否完美. 14.Love is a carefully designed lie.爱情是一个精心设计的谎言. 15.Promises are often like the butterfly, which disappear after beautiful hover.承诺常常很像蝴蝶,美丽的飞盘旋然后不见 16.Fading is true while flowering is past凋谢是真实的,盛开只是一种过去 17.Why I have never catched the happiness? Whenever I want you ,I will be accompanyed by the memory of...为什么幸福总是擦肩而过,偶尔想你的时候….就让….回忆来陪我. 18.Love ,promised between the fingers.Finger rift,twisted in the love爱情…在指缝间承诺指缝….在爱情下交缠. 19.If you weeped for the missing sunset,you would miss all the shining stars如果你为着错过夕阳而哭泣，那么你就要错群星了 20.To feel the flame of dreaming and to feel the moment of dancing,when all the romance is far away,the eternity is always there.感受梦的火焰，感觉飞舞瞬间，当一切浪漫遥远，永恒依然
时尚潮流英文句子 1、Fashion,isakindofaestheticview.Brotherisapunk,yousatisfied 时尚，就是一种审美观。哥就朋克，你不服吗? 2、Wheatcolorskintoahealthysenseofvitality,wearingNikeacomple tesetofpurewhitepinkedgesportswear,thetinycurlybrownhairti edinarelaxedandlivelybraids,alwaystheconfidenceofcuteexpres sions. 小麦色的皮肤给人一种健康活力的感觉，穿着耐克的一整套的纯白带粉色边运动服，微卷的褐色头发扎成一个轻松活泼的辫子，总是那自信可爱的表情。 3、Devilkillerbody,alargewavygoldenhairshine,slenderlegswearin gayellowgooseminiskirts,showfigureoftheperfect. 魔鬼般惹火的身材，一头大波浪形金黄卷发发出耀眼的光芒，修长的大腿穿着一条鹅黄色的超短迷你裙，显出身材的完美绝伦。 4、Whitecapsetheruplonghairandhalfofhisfaceisobscured,butfelts hemustbeverybeautiful,breathtakingbeauty!
白色的鸭舌帽把她那盘起的长发和半张脸都给遮住了，但能感觉出她一定很漂亮，惊人的漂亮! 5、Fashionisnotonlyakindofappearance,oraninner,popularmaynot besuitableforyou,butaccordingtotheirowncharacteristicstodre ssupyourself,youbelongtothatkind,mature,lady,orsimpleandna tural,orpure,ormovement,afactthatcanallbefashionable. 时尚不光是一种外表，还是一种内在，时下流行的不一定都适合你，而是要根据自身的特点来打扮自己，看你属于那一种类型，成熟的，淑女的，还是简单自然的，还是清纯的，还是运动的，其实那一种都可以是时尚的。 6、Hip-hop,cowboywind,andthewindwindwind,occupation,fur,all-match,hippie,ladiesfashion,Korean,Japanese,whatisitFashionist heurbanspeciallogo,isacityinthevastcityofspecialpsychological needs. 嘻哈风、牛仔风、欧美风、职业风、皮草风、百搭、嘻皮、淑女、韩流、哈日，（next88）时尚到底是什么?时尚其实是都市特殊的标志，是都市人在纷繁芜杂的城市中特殊的心理需要。 7、Wearingablueskirt,microstripwheatcolorskinlookssohealthy,bl ackhairlikeawaterfallverticallyovertheshoulders,withareddishfa
1.你可知我百年的孤寂只为你一人守侯，千夜的恋歌只为你一人而唱。 You know my loneliness is only kept for you, my sweet songs are only sung for you. 2.如果活着，是上帝赋予我最大的使命，那么活者有你，将会是上帝赋予我使命的恩赐…… If living on the earth is a mission from the lord… living with you is the award of the lord… 3.你知道思念一个人的滋味吗，就像喝了一大杯冰水，然后用很长很长的时间流成热泪。 Do you understand the feeling of missing someone? It is just like that you will spend a long hard time to turn the ice-cold water you have drunk into tears. 4.在这充满温馨的季节里，给你我真挚的祝福及深深的思念。 In such a soft and warm season, please accept my sincere blessing and deep concern for you. 5.一份不渝的友谊，执着千万个祝福，给我想念的朋友，温馨的问候。 For our ever-lasting friendship, send sincere blessings and warm greetings to my friends whom I miss so much. 6.想你，是一种美丽的忧伤的甜蜜的惆怅，心里面，却是一种用任何语言也无法表达的温馨。 It is graceful grief and sweet sadness to think of you, but in my heart, there is a kind of soft warmth that can’t be expressed with any choice of words.
I missed you but I missing you. I missing you but I missed you .I see you but I seeing you . I seeing you but I see you.------明明已经错过你，但我却还在想念你。当我想念你的时候，但我 不能再拥有你。明明已经别离，却又再次相遇。当我们再次相遇时，却不得不说再见 ?
Some disappointment is inevitable, but most of the disappointed, because of you overestimate themselves. 有些失望是不可避免的，但大部分的失望，都是因为你高估了自己。 ?
Can I don't have a boyfriend, and don't have money, but I can't do without you 我可以没有男朋 友，没有钱，可是我不能没有你 ?
Sometimes I wish I could just fast forward time just to see if in the end it’s all worth it.---有时 候，我真希望我能快进时间，这样我就能看看，最终的结果是不是值得。 ?
I love you not for who you are, but for who I am before you. 我爱你不是因为你是谁，而是我在 你面前可以是谁。 ?
Guys use the word “friendship” to start a relationship. Girls use the word “friendship” to end it.男 人喜欢用“友情”开始一段爱情，女人喜欢用“友情”终结一段爱情。 ?
If one really cares for you,he is able to squeeze time for you.No excuses,no lies,no undeliverable promises. 如果一个人真的足够在乎你，那么他总能挤出时间来陪你，没有借口、谎言，没
?Never say you're not good enough, if that person can't see you for who you are, they are the ones who aren't good enough. 永远别说自己不够好，如果那个人无法看清真实的你，那他们也不怎么样。 ?Sometimes that mountain you've been climbing is just a grain of sand. What you've been out there searching for forever is in your hands. 有时候，你攀登的山峰也许只是一粒沙子。而你一直找寻的永远，不经意已经在你的手上。 ?People say, we'd rather have something than nothing at all. Truth is, to have something halfway is harder than having nothing at all. 人们总说，有总比没有好。事实是，不能彻底的拥有，比一无所有更难受。 ?I used to believe in forever, but forever is too good to be true. 我曾相信永远，但永远太美好，而不真实。 ?The hardest part of moving on is not looking back at the memories you once shared with someone. 分手之后继续前行中最艰难的部分就是不去回首那些你曾与某人共同享有的回忆。 ?Happiness depends upon ourselves. –Aristotle幸福，由我们自己掌控。 ?Never flaunt what you have - there's always someone who has more than you do. As soon as you start to become arrogant, losing everything could be right around the corner. 永远不要炫耀自己所拥有的，总有人比你富足。一旦你开始狂妄，离失去这一切就不远了。 ?No matter how much life sucks, if you have good friends, they can make you laugh. 无论生活多么操蛋，要是你有帮好朋友，他们也会逗你开心。 ?You don't have to be a size zero to be pretty. Beauty should be judged by the size of your heart. 身材纤细不一定美丽。美丽应该由你心胸气度决定。 ?You can't lose what you never had, you c an't keep what’s not yours and you can't hold on to something that does not want to stay. 从未拥有过，你就无法失去。本不属于你，你就无法拥有。本不想为你停留，你就无法坚持挽留。 ?I really love the people never will I go, no matter how much trouble.真心爱我的人永远不会我走，不管遇到多大的困境。 ?t’s a great and terrible life. Don’t take it too seriously because none of us get out alive. 生活既美好又糟糕。别太认真，因为没有人能活着离开这个世界。 ?Share a Smile, pass it along and brighten another person's day! 分享笑容，传递笑容，照亮他人的一天。