基于热还原氧化石墨烯的单分子表面诱导荧光衰逝技术

樊秦凯; 杨晨光; 胡书新; 徐春华; 李明; 陆颖

doi:10.7498/aps.72.20230450

摘要

单分子表面诱导荧光衰逝(single molecule surface-induced fluorescence attenuation, smSIFA)技术是一种基于二维材料受体、用于研究生物大分子法向运动的精密测量方法, 该方法不受二维平面运动的干扰. 作为受体的二维材料, 其特征淬灭距离决定法向上探测的距离和精度. 近年来以氧化石墨烯(graphene oxide, GO)和石墨烯作为介质受体的SIFA技术在生物大分子的研究中发挥了重要作用, 但石墨烯和GO具有固定的特征淬灭距离, 探测范围有限. 调整探测范围需要更换介质材料, 面临材料选择与制备的困难, 亟需开发用于技术的可调控材料. 本文改良了以GO为介质受体的单分子SIFA技术, 利用热还原的方法对GO进行还原, 通过控制还原温度, 制备出了还原程度不同的还原氧化石墨烯(reduced graphene oxide, rGO), 调控特征淬灭距离, 利用荧光标记的DNA测量rGO的特征淬灭距离. 将rGO用于单分子SIFA技术, 对Holliday junction构象变化的观察, 论证了rGO的探测范围.

关键词:

Abstract

Single-molecular surface-induced fluorescence attenuation (smSIFA) is a precise method of studying the vertical movement of biological macromolecules based on two-dimensional material receptors. This method is not affected by two-dimensional planar motion of membrane or proteins. However, the detection range and accuracy of vertical movement are determined by the properties of two-dimensional materials as receptors. In recent years, surface induced fluorescence attenuation based on graphene oxide and graphene has played an important role in studying biomacromolecules. However, the detection range of graphene and graphene oxide are limited owing to the fixed and limited characteristic quenching distance. Adjusting the detection range requires replacing the medium material, which poses difficulties in selecting and preparing materials. Therefore, it is urgently needed to develop controllable materials for single-molecular SIFA. In this study, the single-molecule SIFA with graphene oxide as the medium acceptor is improved by reducing graphene oxide through thermal reduction. By controlling the reduction temperature, reduced graphene oxides to different reduction degrees are prepared and the characteristic quenching distances are adjusted. The characteristic quenching distance is measured by fluorescent labeled DNA. Single-molecule SIFA based on reduced graphene oxide is used to observe the conformational changes of Holliday junction, and the detection range of reduced graphene oxide is demonstrated.

Keywords:

reduced graphene oxide /
surface-induced fluorescence attenuation /
characteristic quenching distance /
fluorescence resonance energy transfer

作者及机构信息

1.
中国科学院物理研究所, 北京凝聚态物理国家研究中心, 软物质物理重点实验室, 北京　100190

2.
中国科学院大学, 北京　100049

通信作者: 陆颖, yinglu@iphy.ac.cn

基金项目: 国家重点研发项目 (批准号: 2019YFA0709304)、国家自然科学基金(批准号: T2221001, 12090051, 11974411, 91753104, U1930402, 12090050, 12022409, 32000871)、中国科学院战略性先导科技专项 (批准号: XDB37000000, XDB0480000)、中国科学院前沿科学重点研究计划(批准号: ZDBS-LY-SLH015)和中国科学院青年创新促进会(批准号: Y2021003)资助的课题.

Authors and contacts

1.
Key Laboratory of Soft Matter Physics, Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China

2.
University of Chinese Academy of Sciences, Beijing 100049, China

Corresponding author: Lu Ying, yinglu@iphy.ac.cn

Funds: Project supported by the National Key Research and Development Program of China (Grant No. 2019YFA0709304), the National Natural Science Foundation of China (Grant Nos. T2221001, 12090051, 11974411, 91753104, U1930402, 12090050, 12022409, 32000871), the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant Nos. XDB37000000, XDB0480000), the Key Research Program of Frontier Sciences, Chinese Academy of Sciences (Grant No. ZDBS-LY-SLH015), and the Youth Innovation Promotion Association of Chinese Academy of Sciences (Grant No. Y2021003).

文章全文 : translate this paragraph

参考文献

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施引文献

图 1 调控d₀的SIFA方法　(a) SIFA方法示意图; (b) 荧光供体光强衰减和表面距离的关系; (c) SIFA探测灵敏度和荧光供体距表面距离的关系

Fig. 1. SIFA method of adjustable d₀: (a) Schematic representation of SIFA method; (b) relationship between degree of attenuation of a fluorescent donor and donor-surface distance; (c) relationship between detection sensitivity of SIFA and donor-surface distance.

下载: 全尺寸图片幻灯片

图 2 初始GO以及rGO薄膜的XPS谱　(a) 初始GO薄膜C 1s的XPS谱(左)以及XPS全谱(右); (b) 300 ℃-2 h-rGO薄膜C 1s的XPS谱(左)以及XPS全谱(右); (c) 400 ℃-2 h-rGO薄膜C 1s的XPS谱(左)以及XPS全谱

Fig. 2. XPS spectra of original GO and rGO thin films: (a) C 1s XPS spectra (left) and XPS survey spectra (right) for original GO thin films; (b) C 1s XPS spectra (left) and XPS survey spectra (right) for 300 ℃-2 h-rGO thin films; (c) C 1s XPS spectra (left) and XPS survey spectra (right) for the 400 ℃-2 h-rGO thin films.

下载: 全尺寸图片幻灯片

图 3 荧光标记DNA测量rGO的d₀　(a) DNA成像实验示意图; (b) 在300 ℃-2 h-rGO (上)以及400 ℃-2 h-rGO (下)样品腔内观察DNA; Cy3标记在1 bp (c), 9 bp (d)和21 bp (e) 处的DNA在玻璃以及rGO上成像时的单分子光强

Fig. 3. Determination of d₀ of rGO by fluorescence labeled DNA: (a) Schematic representation of DNA imaging; (b) DNA imaging on 300 ℃-2 h-rGO (upper) and 400 ℃-2 h-rGO (lower); intensities of Cy3 labeled at 1 bp (c), 9 bp (d) and 21 bp (e) of DNA on glass and rGO.

下载: 全尺寸图片幻灯片

图 4 SIFA观察Holliday junction的构象变换, 在玻璃 (a), GO (b)和400 ℃-2 h-rGO (c)上观察Cy3标记的Holliday junction, 左列为单个Cy3的光强时间曲线, 中间为Cy3的光强统计图, 右列为Holliday junction构象变换导致Cy3光强变化的示意图

Fig. 4. Observing conformational transformation of Holliday junction by SIFA, observing the Cy3 labeled Holliday junction on glass (a), GO (b) and 400 ℃-2 h-rGO (c), left columns show intensity-time curves of a single Cy3, middle columns show distribution of intensities of Cy3, right columns show schematic representation of the change of Cy3 light intensity caused by the conformational transformation of Holiday junction.

下载: 全尺寸图片幻灯片

表 2 DNA Holliday junction的核苷酸序列

Table 2. Nucleotide sequence of DNA Holliday junction.

名称	核苷酸序列
X	CCC AGT TGA GAG CTT GAT AGG G
B	CCC TAT CAA GCC GCT GTT ACG G
R	CCC ACC GCT CTT CTC AAC TGG G
H	biotin-CCG TAA CAG CGA GAG CGG TGG G(Cy3)

下载: 导出CSV

表 1 荧光标记DNA测量rGO的d₀

Table 1. Determination of d₀ of rGO by fluorescence labeled DNA.

样品	1 bp (7.5 nm)	9 bp (8.9 nm)	21 bp (10.9 nm)
300 ℃-2h-rGO	I = (0.66 ± 0.08)I₀ d₀ = (6.4 ± 0.7) nm	I = (0.79 ± 0.07)I₀ d₀ = (6.2 ± 0.6) nm	I≈ I₀
400 ℃-2h-rGO	I = (0.45 ± 0.09)I₀ d₀ = (7.9 ± 0.7) nm	I = (0.60 ± 0.10)I₀ d₀ = (8.1± 0.8) nm	I = (0.80 ± 0.09)I₀ d₀ = (7.8 ± 0.9) nm

下载: 导出CSV

[1]	Lerner E, Barth A, Hendrix J, et al. 2021 Elife 10 e60416 Google Scholar
[2]	Keller A M, DeVore M S, Stich D G, Vu D M, Causgrove T, Werner J H 2018 Anal. Chem. 90 6109 Google Scholar
[3]	Ishikawa-Ankerhold H C, Ankerhold R, Drummen G P 2012 Molecules 17 4047 Google Scholar
[4]	贾棋, 樊秦凯, 侯文清, 杨晨光, 王利邦, 王浩, 徐春华, 李明, 陆颍 2021 物理学报 70 158701 Google Scholar Jia Q, Fan Q K, Hou W Q, Yang C G, Wang L B, Wang H, Xu C H, Li M, Lu Y 2021 Acta Phys. Sin. 70 158701 Google Scholar
[5]	Almen M S, Nordstrom K J V, Fredriksson R, Schioth H B 2009 Bmc. Biology. 7 50 Google Scholar
[6]	White S H, Wimley W C 1999 Annu. Rev. Bioph. Biom. 28 319 Google Scholar
[7]	马东飞, 侯文清, 徐春华, 赵春雨, 马建兵, 黄星榞, 贾棋, 马璐, 刘聪, 李明, 陆颖 2020 物理学报 69 038701 Google Scholar Ma D F, Hou W Q, Xu C H, Zhao C Y, Ma J B, Huang X Y, Jia Q, Ma L, Liu C, Li M, Lu Y 2020 Acta Phys. Sin. 69 038701 Google Scholar
[8]	Ponmalar, II, Cheerla R, Ayappa K G, Basu J K 2019 Proc. Natl. Acad. Sci. USA 116 12839 Google Scholar
[9]	King C, Raicu V, Hristova K 2017 J. Biol. Chem. 292 5291 Google Scholar
[10]	King C, Sarabipour S, Byrne P, Leahy D J, Hristova K 2014 Biophys. J. 106 1309 Google Scholar
[11]	Li Y, Qian Z, Ma L, Hu S, Nong D, Xu C, Ye F, Lu Y, Wei G, Li M 2016 Nat. Commun. 7 12906 Google Scholar
[12]	Ma L, Li Y, Ma J B, Hu S X, Li M 2018 Biochemistry 57 4735 Google Scholar
[13]	Jiang X, Yang C G, Qiu J, Ma D F, Xu C, Hu S X, Han W J, Yuan B, Lu Y 2022 Nanoscale 14 17654 Google Scholar
[14]	Ma L, Hu S X, He X L, Yang N, Chen L C, Yang C G, Ye F F, Wei T T, Li M 2019 Nano. Lett. 19 6937 Google Scholar
[15]	Kaminska I, Bohlen J, Yaadav R, Schuler P, Raab M, Schroder T, Zahringer J, Zielonka K, Krause S, Tinnefeld P 2021 Adv. Mater. 33 e2101099 Google Scholar
[16]	Kaminska I, Bohlen J, Rocchetti S, Selbach F, Acuna G P, Tinnefeld P 2019 Nano. Lett. 19 4257 Google Scholar
[17]	Federspiel F, Froehlicher G, Nasilowski M, Pedetti S, Mahmood A, Doudin B, Park S, Lee J O, Halley D, Dubertret B, Gilliot P, Berciaud S 2015 Nano. Lett. 15 1252 Google Scholar
[18]	Gaudreau L, Tielrooij K J, Prawiroatmodjo G E D K, Osmond J, de Abajo F J G, Koppens F H L 2013 Nano. Lett. 13 2030 Google Scholar
[19]	Li W, Wojcik M, Xu K 2019 Nano. Lett. 19 983 Google Scholar
[20]	Eda G, Fanchini G, Chhowalla M 2008 Nat. Nanotechnol. 3 270 Google Scholar
[21]	Pei S F, Cheng H M 2012 Carbon 50 3210 Google Scholar
[22]	Stankovich S, Dikin D A, Piner R D, Kohlhaas K A, Kleinhammes A, Jia Y, Wu Y, Nguyen S T, Ruoff R S 2007 Carbon 45 1558 Google Scholar
[23]	Sulowska K, Wiwatowski K, Szustakiewicz P, Grzelak J, Lewandowski W, Mackowski S 2018 Materials (Basel) 11 1567 Google Scholar
[24]	Kim J, Cote L J, Kim F, Huang J X 2010 J. Am. Chem. Soc. 132 260 Google Scholar
[25]	Kovtyukhova N I, Ollivier P J, Martin B R, Mallouk T E, Chizhik S A, Buzaneva E V, Gorchinskiy A D 1999 Chem. Mater. 11 771 Google Scholar
[26]	Hummers W S, Offeman R E 1958 J. Am. Chem. Soc. 80 1339 Google Scholar
[27]	Chen X, Meng D, Wang B, Li B W, Li W, Bielawski C W, Ruoff R S 2016 Carbon 101 71 Google Scholar
[28]	Lazauskas A, Baltrusaitis J, Grigaliūnas V, Guobienė A, Prosyčevas I, Narmontas P, Abakevičienė B, Tamulevičius S 2014 Superlattices Microstruct. 75 461 Google Scholar
[29]	Li J, Ma J, Kumar V, Fu H, Xu C, Wang S, Jia Q, Fan Q, Xi X, Li M, Liu H, Lu Y 2022 Nucleic. Acids. Res. 50 7002 Google Scholar
[30]	Ma J B, Chen Z, Xu C H, Huang X Y, Jia Q, Zou Z Y, Mi C Y, Ma D F, Lu Y, Zhang H D, Li M 2020 Nucleic. Acids. Res. 48 3156 Google Scholar
[31]	陈泽, 马建兵, 黄星榞, 贾棋, 徐春华, 张慧东, 陆颖 2018 物理学报 67 118201 Google Scholar Chen Z, Ma J B, Huang X Y, Jia Q, Xu C H, Zhang H D, Lu Y 2018 Acta Phys. Sin. 67 118201 Google Scholar
[32]	Wei A, Wang J X, Long Q, Liu X M, Li X G, Dong X C, Huang W 2011 Mater. Res. Bull. 46 2131 Google Scholar
[33]	Luo D, Zhang G, Liu J, Sun X 2011 J. Phys. Chem. C. 115 11327 Google Scholar
[34]	Xu S T, Liu J K, Xue Y, Wu T Y, Zhang Z F 2017 Fuller. Nanotub. Car. N. 25 40 Google Scholar
[35]	Zhen X J, Huang Y F, Yang S S, Feng Z Z, Wang Y, Li C H, Miao Y J, Yin H 2020 Mater. Lett. 260 126880
[36]	Dessinges M N, Maier B, Zhang Y, Peliti M, Bensimon D, Croquette V 2002 Phys. Rev. Lett. 89 248102 Google Scholar
[37]	Baumann C G, Smith S B, Bloomfield V A, Bustamante C 1997 Proc. Natl. Acad. Sci. U. S. A. 94 6185 Google Scholar
[38]	Son S, Takatori S C, Belardi B, Podolski M, Bakalar M H, Fletcher D A 2020 Proc. Natl. Acad. Sci. U. S. A. 117 14209 Google Scholar
[39]	Demirel G B, Caykara T 2009 Appl. Surf. Sci. 255 6571 Google Scholar
[40]	Lu J R, Su T J, Thomas R K 1999 J. Colloid. Interf. Sci. 213 426 Google Scholar
[41]	P. C. Weber J J W, f M. W. Pantoliano, and F. R. Salemme 1992 J. Am. Chem. Soc. 114 3197 Google Scholar
[42]	Liu Y L, West S C 2004 Nat. Rev. Mol. Cell. Bio. 5 937 Google Scholar
[43]	Clegg R M, Murchie A I, Lilley D M 1994 Biophysical. J. 66 99 Google Scholar
[44]	McKinney S A, Tan E, Wilson T J, Nahas M K, Declais A C, Clegg R M, Lilley D M J, Ha T 2004 Biochem. Soc. T 32 41 Google Scholar
[45]	McKinney S A, Declais A C, Lilley D M J, Ha T 2003 Nat. Struct. Biol. 10 93 Google Scholar
[46]	Hohng S, Joo C, Ha T 2004 Biophys. J. 87 1328 Google Scholar
[47]	Lee J, Lee S, Ragunathan K, Joo C, Ha T, Hohng S 2010 Angew. Chem. Int. Ed. Engl. 49 9922 Google Scholar
[48]	Uphoff S, Holden S J, Le Reste L, Periz J, van de Linde S, Heilemann M, Kapanidis A N 2010 Nat. Methods. 7 831 Google Scholar

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