论文设计基于银纳米粒子构建荧光传感平台用于核酸检测24917

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1、基于银纳米粒子构建荧光传感平台用于核酸检测张瑛洧1 李海龙1,2 孙旭平1* 1（中国科学院长春应用化学研究所，长春 130012）2（中国科学院研究生院，北京 100039）摘要我们首次报道了基于银纳米粒子构建的荧光传感平台，并成功地应用于核酸检测。这种荧光传感平台的核酸检测通常是基于以下的设计策略：首先，荧光团标记的单链DNA探针被吸附到银纳米粒子的表面，荧光团由于和银纳米粒子近距离接触而发生荧光淬灭，接下来加入同探针DNA序列互补的目标DNA，两者杂交形成双链DNA后从银纳米粒子的表面脱离下来，从而荧光得到恢复。这种银纳米粒子构建的荧光传感平台对于完全互补和碱基错配的DNA序列具有良好的

2、区分能力。关键词核酸检测；荧光传感平台；银纳米粒子1 引 言随着对基因结构及功能研究的不断深入，人们已经越来越多地发现，人类的许多遗传疾病都与DNA 分子中碱基序列的突变有关，因此，发展迅速、有效、灵敏和低成本的核酸检测方法对于研究基因的表达、临床疾病的诊断和治疗都有着重要的意义1。随着纳米结构的日益发展，其在生物技术体系的诊断应用引起了人们广泛的关注，并且已有一些成功应用的例子2,3。据此，研究人员将纳米结构引入到荧光核酸检测中并做了许多工作，其机理主要是基于荧光共振能量转移或淬灭机理4。已证实纳米结构在这种检测体系中扮演的是“纳米淬灭剂”的角色，并且一种纳米结构可同时淬灭不同发射波长的多种

3、染料。应用纳米结构做淬灭剂，消除了以前检测方法中要挑选荧光团-淬灭剂对的麻烦。目前，已报道的被成功地应用于荧光核酸检测的纳米结构有以下这些，比如：金纳米粒子、单壁碳纳米管、多壁碳纳米管、碳纳米粒子、介孔碳微粒、氧化石墨、聚对苯二胺纳米带、配位聚合物胶体球等4-28。尽管如此，继续发展新材料的纳米结构来构建荧光检测平台以提高荧光核酸检测的选择性和灵敏度等仍然很有必要。 在本文中，经实验证实，银纳米粒子也可以作为淬灭剂来构建一个新的荧光传感平台用于核酸检测，其展示了很高的选择性，可实现对完全互补和单碱基错配DNA序列的区分，其中单碱基、两个碱基错配的目标所引起的荧光恢复是完全互补目标引起荧光恢复的

4、83%和60%。此外该平台的检测灵敏度很高，检测限可低至5 nM。如图1所示，检测的设计策略如下：荧光团标记的单链DNA探针被吸附到银纳米粒子的表面引起荧光淬灭，之后与目标DNA杂交得到双链DNA，其从银纳米粒子的表面脱离使得荧光得到恢复。这种设计主要是基于单双链DNA在银纳米粒子表面有不同的吸附能力7。图 1 使用银纳米粒子构建荧光传感平台进行荧光增强核酸检测的原理图。Fig. 1. A schematic diagram (not to scale) to illustrate the fluorescence-enhanced nucleic acid detection using A

5、g nanoparticle as a sensing platform.2实验部分2.1仪器与试剂透射电子显微镜照片在H-8100型透射电子显微镜（日本Hitachi公司）上获得，加速电压为200 kV。荧光光谱测定在RF-5301PC（日本Shimadzu公司）荧光分光光度计上完成。实验所用的寡核苷酸，都是上海生工生物工程技术服务有限公司（中国上海）合成，通过测量260 nm的紫外吸收值来标定所用DNA的浓度。实验所用的其它化学试剂都是从阿拉丁有限公司（中国上海）购买，直接使用未再纯化。实验用水经由Milli-Q高纯水处理系统纯化。本实验所采用的DNA 序列如下（错配位置由下划线标出）：P

6、HIV (荧光素FAM染料标记的作为探针的单链DNA):5-FAM-AGT CAG TGT GGA AAA TCT CTA GC-3T1 (互补的目标):5-GCT AGA GAT TTT CCA CAC TGA CT-3T2 (单碱基错配的目标):5-GCT AGA GAT TGT CCA CAC TGA CT-3 T3 (两个碱基错配的目标):5-GCT AGA GAT TGT ACA CAC TGA CT-32.2 实验方法银纳米粒子的制备过程如下：45 mg AgNO3用500 mL水溶解，加入10 mL（1%）的柠檬酸钠水溶液，在90 加热半小时，得到黄色的胶体溶液8，待进一步的表征

7、和应用。3 结果与讨论3.1 电镜表征图2给出的是银产物的透射电镜照片。低倍照片显示产物完全是大量的纳米粒子，高倍照片显示粒子尺寸在50-100 nm的范围。图2产物的（）低倍 和（）高倍的透射电镜照片。Fig. 2 (a) Low and (b) high magnification TEM images of the products thus formed.3.2 荧光检测应用所制备的银纳米粒子（AgNPs）构建荧光传感平台进行核酸检测，并采用与人类免疫缺陷病毒(HIV)相关联的DNA序列构建模型体系。图3显示的是荧光团FAM标记的单链DNA探针（PHIV）在不同条件下的荧光发射谱。如曲

8、线a所示，当体系中没有银纳米粒子存在时，由于荧光素染料的修饰，探针DNA展示了很强的荧光发射。然而当加入银纳米粒子进入检测体系超过分钟时，根据曲线c，48%的荧光淬灭发生，这表明银纳米粒子可以有效的吸附单链DNA并淬灭其荧光。相反地，当加入同探针DNA互补的目标DNA (T1)时，探针DNA同银纳米粒子的复合物展示了明显的荧光增强，根据曲线d, 荧光恢复达到88%。需要说明的是，如曲线b所示，当仅有目标T1和探针DNA共存而无纳米粒子存在时，探针DNA的荧光强度几乎没有变化。如图4所示，当向检测体系中加入5 nM T1时，可观察到明显的荧光恢复，因此我们认为该检测平台的检测限低至5 nM。 通

9、过采集随时间变化的荧光发射谱，可以研究检测过程中的动力学情况。图5a 给出了银纳米粒子存在时探针DNA（PHIV）随时间变化的荧光淬灭情况，在最初的1分钟，荧光强度迅速降低，在随后的29分钟缓慢降低直至达到平衡。图5b 给出了加入目标T1后荧光的恢复过程，在最初的1分钟，荧光强度迅速增强，随后趋于平缓直至15分钟后达到平衡。图3 探针DNA(PHIV) (50 nM)在不同条件下的荧光发射谱：(a) PHIV; (b) PHIV + 300 nM T1; (c) PHIV + AgNPs; (d) PHIV + AgNPs + 300 nM T1。 所有测量均在Tris-HCl (pH: 7.

10、4，15 mM Mg2+)缓冲液中完成。Fig.3 Fluorescence emission spectra of PHIV (50 nM) at different conditions: (a) PHIV; (b) PHIV + 300 nM T1; (c) PHIV + AgNPs; (d) PHIV + AgNPs + 300 nM T1. Excitation was at 480 nm. All measurements were done in Tris-HCl buffer in the presence of 15 mM Mg2+ (pH: 7.4).图4 (a) 探针DN

11、A(PHIV) (50 nM)的荧光被银纳米粒子淬灭后的荧光发射曲线， (b) 加入5 nM的检测目标T1后的荧光恢复曲线。 激发波长在480 nm，所有测量均在Tris-HCl (pH: 7.4，15 mM Mg2+)缓冲液中完成。Fig. 4 (a) Fluorescence quenching of PHIV (50 nM) by AgNPs and (b) fluorescence recovery of PHIV-AgNPs by T1 (5 nM). Excitation was at 480 nm. All measurements were done in Tris-HCl b

12、uffer in the presence of 15 mM Mg2+ (pH: 7.4).图5 (a) 探针DNA(PHIV)的荧光被银纳米粒子淬灭随时间变化的过程， (b) 加入300 nM的检测目标T1后，荧光强度随时间恢复的过程。激发波长在480 nm，监控518 nm处的发射峰强度，所有测量均在Tris-HCl (pH: 7.4，15 mM Mg2+)缓冲液中完成。Fig. 5 (a) Fluorescence quenching of PHIV (50nM) by AgNPs and (b)fluorescence recovery of PHIV-AgNPs by T1 (300

13、nM) as a function of incubation time. Excitation was at 480 nm, and the emission was monitored at 518 nm. All measurements were done in Tris-HCl buffer in the presence of 15 mM Mg2+ (pH: 7.4).3.3 对碱基错配的区分 这种荧光传感平台对于完全互补和碱基错配的DNA序列具有很好的区分能力。如图6所示，当分别加入完全互补的目标T1、单碱基错配的目标T2和两个碱基错配的目标T3 到检测体系中时，我们观察到了不同

14、的荧光恢复情况。用荧光强度的比值F/F0（F0 和F 分别是没有检测目标和已经加入检测目标时的荧光强度）来具体分析荧光恢复的差异。其中，单碱基错配的目标T2所引起的荧光恢复是T1的83%，而两个碱基错配的目标T3 所引起的荧光恢复仅为T1的60%，图6的插图给出了对应的柱状图和误差棒。以上实验结果表明该荧光传感平台在进行单碱基错配区分方面有很好的应用前景。图6探针DNA(PHIV) (50 nM)在不同条件下的荧光发射谱：(a) PHIV; (b) PHIV + 300 nM T1; (c) PHIV + AgNPs; (d) PHIV + AgNPs + 300 nM T1。插图：对应的荧光

15、强度的柱状图及误差棒。激发波长在480 nm，监控518 nm处的发射峰强度，所有测量均在Tris-HCl (pH: 7.4，15 mM Mg2+)缓冲液中完成。Fig. 6 Fluorescence emission spectra of PHIV (50 nM) at different conditions: (a) PHIV-AgNPs complex; (b) PHIV-AgNPs complex + 300 nM T1; (c) PHIV-AgNPs complex + 300 nM T2; (d) PHIV-AgNPs complex + 300 nM T3. Inset: fl

16、uorescence intensity histograms with error bar. Excitation was at 480 nm, and the emission was monitored at 518 nm. All measurements were done in Tris-HCl buffer in the presence of 15 mM Mg2+ (pH: 7.4). 另外，银纳米粒子的用量对于检测过程中荧光的淬灭和恢复都有很大的影响。图7给出了银纳米粒子不同用量（0, 50, 100, 150, 200, 250, 300-L）时荧光强度的柱状图，如图可见，

17、银纳米粒子用量的增加有利于荧光淬灭效率的增大，但不利于荧光的恢复。我们认为其原因如下：大量的银纳米粒子有利于更多的单链DNA在其粒子表面的吸附，因而有利于荧光的淬灭；另一方面，过量的银纳米粒子引入到检测体系中，将有过多的表面空位吸附目标DNA分子从而阻止其同探针DNA的杂交，因而不利于荧光的恢复。基于以上的实验结果，选择200-L作为最优化的银纳米粒子用量。图7 当分别加入0, 50, 100, 150, 200, 250, 和 300-L银纳米粒子时，发生荧光淬灭（PHIV + AgNPs）和荧光恢复（PHIV + AgNPs + T1）所对应的荧光强度的柱状图，(PHIV=50 nM; T

18、1=300 nM; ex=480 nm)。Fig.7 Fluorescence intensity histogram of PHIV + AgNPs and PHIV + AgNPs + T1 with the using of 0, 50, 100, 150, 200, 250, and 300-L AgNPs sample in this system (PHIV=50 nM; T1=300 nM; ex=480 nm).4结论本文成功地证明了银纳米粒子可以构建一个有效的荧光传感平台进行荧光增强的核酸检测，且这种传感平台可以很好地区分完全互补和错配的DNA序列。本文的研究工作扩展了银纳米

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27、Key Lab of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022)2 (Graduate School of the Chinese Academy of Sciences, Beijing 100039)Abstract In this study, we report on the use of Ag nanoparticles (AgNPs) as an effective fluorescent s

28、ensing platform for nucleic acid detection for the first time. The general concept used in this approach is based on adsorption of the fluorescently labeled single-stranded DNA (ssDNA) probe by AgNP, which is accompanied by substantial fluorescence quenching due to their close proximity, followed by

29、 specific hybridization with its target to form a double-stranded DNA (dsDNA). The use of this sensing platform to differentiate complementary and mismatched sequences is also demonstrated. The fluorescence recovery values of single-base mismatched target and two-base mismatched target are only abou

30、t 83% and 60% respectively, compared with the fluorescence recovery of complementary target. Moreover, this sensing platform also presents high sensitivity, and the detection limit as low as 5 nM is achieved.Keywords Nucleic acid detection; Fluorescent sensing platform; Ag nanoparticleEditors note:

31、Judson Jones is a meteorologist, journalist and photographer. He has freelanced with CNN for four years, covering severe weather from tornadoes to typhoons. Follow him on Twitter: jnjonesjr (CNN) - I will always wonder what it was like to huddle around a shortwave radio and through the crackling sta

32、tic from space hear the faint beeps of the worlds first satellite - Sputnik. I also missed watching Neil Armstrong step foot on the moon and the first space shuttle take off for the stars. Those events were way before my time.As a kid, I was fascinated with what goes on in the sky, and when NASA pul

33、led the plug on the shuttle program I was heartbroken. Yet the privatized space race has renewed my childhood dreams to reach for the stars.As a meteorologist, Ive still seen many important weather and space events, but right now, if you were sitting next to me, youd hear my foot tapping rapidly und

34、er my desk. Im anxious for the next one: a space capsule hanging from a crane in the New Mexico desert.Its like the set for a George Lucas movie floating to the edge of space.You and I will have the chance to watch a man take a leap into an unimaginable free fall from the edge of space - live.The (l

35、ack of) air up there Watch man jump from 96,000 feet Tuesday, I sat at work glued to the live stream of the Red Bull Stratos Mission. I watched the balloons positioned at different altitudes in the sky to test the winds, knowing that if they would just line up in a vertical straight line we would be

36、 go for launch.I feel this mission was created for me because I am also a journalist and a photographer, but above all I live for taking a leap of faith - the feeling of pushing the envelope into uncharted territory.The guy who is going to do this, Felix Baumgartner, must have that same feeling, at

37、a level I will never reach. However, it did not stop me from feeling his pain when a gust of swirling wind kicked up and twisted the partially filled balloon that would take him to the upper end of our atmosphere. As soon as the 40-acre balloon, with skin no thicker than a dry cleaning bag, scraped

38、the ground I knew it was over.How claustrophobia almost grounded supersonic skydiverWith each twist, you could see the wrinkles of disappointment on the face of the current record holder and capcom (capsule communications), Col. Joe Kittinger. He hung his head low in mission control as he told Baumg

39、artner the disappointing news: Mission aborted.The supersonic descent could happen as early as Sunday.The weather plays an important role in this mission. Starting at the ground, conditions have to be very calm - winds less than 2 mph, with no precipitation or humidity and limited cloud cover. The b

40、alloon, with capsule attached, will move through the lower level of the atmosphere (the troposphere) where our day-to-day weather lives. It will climb higher than the tip of Mount Everest (5.5 miles/8.85 kilometers), drifting even higher than the cruising altitude of commercial airliners (5.6 miles/

41、9.17 kilometers) and into the stratosphere. As he crosses the boundary layer (called the tropopause), he can expect a lot of turbulence.The balloon will slowly drift to the edge of space at 120,000 feet (22.7 miles/36.53 kilometers). Here, Fearless Felix will unclip. He will roll back the door.Then,

42、 I would assume, he will slowly step out onto something resembling an Olympic diving platform.Below, the Earth becomes the concrete bottom of a swimming pool that he wants to land on, but not too hard. Still, hell be traveling fast, so despite the distance, it will not be like diving into the deep e

43、nd of a pool. It will be like he is diving into the shallow end.Skydiver preps for the big jumpWhen he jumps, he is expected to reach the speed of sound - 690 mph (1,110 kph) - in less than 40 seconds. Like hitting the top of the water, he will begin to slow as he approaches the more dense air close

44、r to Earth. But this will not be enough to stop him completely.If he goes too fast or spins out of control, he has a stabilization parachute that can be deployed to slow him down. His team hopes its not needed. Instead, he plans to deploy his 270-square-foot (25-square-meter) main chute at an altitu

45、de of around 5,000 feet (1,524 meters).In order to deploy this chute successfully, he will have to slow to 172 mph (277 kph). He will have a reserve parachute that will open automatically if he loses consciousness at mach speeds.Even if everything goes as planned, it wont. Baumgartner still will fre

46、e fall at a speed that would cause you and me to pass out, and no parachute is guaranteed to work higher than 25,000 feet (7,620 meters).It might not be the moon, but Kittinger free fell from 102,800 feet in 1960 - at the dawn of an infamous space race that captured the hearts of many. Baumgartner will attempt to break that record, a feat that boggles the mind. This is one of those monumental moments I will always remember, because there is no way Id miss this.