少層石墨炔納米片用于DNA的多重實時熒光檢測取得重要進展
生物標志物的高靈敏度、高選擇性、快速檢測，對于疾病的臨床診斷和流行疾病的預防十分重要。其中，熒光檢測技術由于靈敏度高、選擇性好，已成為生物分子檢測的優選手段之一。單層二維納米片對染料標記的單鏈DNA（ssDNA）具有選擇吸附和熒光猝滅能力，能夠實現DNA與小分子的有效檢測。
氧化石墨烯（）是最早報道的具有熒光淬滅能力的二維材料，石墨烯與核酸之間的π-π堆積作用，使得染料標記的ssDNA能夠強烈吸附于表面，與染料分子之間的熒光共振能量轉移使熒光染料淬滅。但雙鏈DNA（dsDNA）由于帶負電荷的磷酸骨架對核酸堿基的屏蔽效應，降低了dsDNA與分子的相互作用，不能發生熒光淬滅。
石墨炔（GD）是一種新的二維碳材料，計算結果表明，它應當具有比石墨烯更強的吸附染料分子和捕獲電子的能力，因而有望產生更有效的熒光淬滅，提高檢測靈敏度。中科院過程工程研究所王丹研究員及其合作者課題組采用鋰插層法對石墨炔進行剝離，獲得了少層的石墨炔納米片（圖1），其具有高的熒光猝滅能力，以及對ssDNA與dsDNA不同的親和力，實現了對多種DNA的實時檢測，檢測限低至25×10-12 M（圖2）。而且，與和MoS2納米片相比，該少層石墨炔納米片具有更高的靈敏度和更短的檢測時間。該檢測方法可在均相溶液中進行，完全適用于原位檢測，可攜帶多種熒光指示劑，能夠實現對多種生物分子的實時檢測。該研究開拓了基于GD納米探針的簡便、快速、高效的生物分子熒光檢測技術，為二維納米生物傳感材料的研發及其在生物分析上的應用提供了全新的思路。
相關研究結果發表在《先進材料》（Adv. Mater 2017, 29, 1606755）上。該研究得到了國家自然科學基金杰出青年基金（21031005），國家自然科學基金（21590795, 51672276, 21671016, 51372245, 51541206），中國科學院創新交叉團隊，生化工程國家重點實驗室等支持。
圖1. a) 少層石墨炔納米片的TEM照片（插圖：剝離前的石墨炔納米片的TEM照片）; b) 少層石墨炔納米片的AFM照片（插圖顯示厚度約為1.1nm）; c) MoS2納米片的TEM照片; d) 氧化石墨烯的TEM 照片.
圖2. a-c) 不同靶DNA濃度下（T1, T2, T3: 0-5×10-9 M）染料標記的ssDNA的熒光光譜（P1: H1N1-FAM，P2: H5N1-Texas Red，P3: M13-FAM）; d-f) 靶DNA檢測的校準曲線
Researchers Find Ways for Multiplexed Real-Time DNA Detection using Few-Layer Graphdiyne Nanosheets
There are increased demands for high sensitivity, selectivity, and rapidity sensing devices to meet the needs of in-clinical diagnostics and epidemic prevention. Homogeneous assays for target molecules with fluorogenic probes have attracted intense attentions due to their operational convenience and ease of automation. Single-layer 2D nanosheets (NSs) have demonstrated to be a class of efficient sensing platform for detecting deoxyribonucleic acid (DNA) and small molecules, attributing to their selective adsorption and fluorescence-quenching abilities toward dyelabeled single-stranded DNA (ssDNA).
Graphene oxide (), a water-soluble derivative of graphene (GR), was the earliest reported 2D materials to possess fluorescence quenching properties. Due to the π-π stacking interactions between graphene and nucleic acid, ssDNAs can be strongly adsorbed on , resulting in a substantial fluorescence quench of the organic dyes labeled on ssDNAs through the fluorescence resonance energy transfer between the hexanal cells of graphene and the dye molecular. However, double-stranded DNAs (dsDNA) cannot quench the fluorescence because of their negatively charged phosphate backbone will shield the nucleobases to reduce the interactive force between the dsDNA and .
Being a new 2D carbon materials, the unique structural characteristics of graphdiyne (GD) makes it very promising for the biosensing applications. Recently, researchers’ density functional theory calculation results suggest that the dye molecular absorption on GD is stronger than that on GR. Moreover, the GD NSs exhibits a superior electrons capturing ability than that of GR. The electrochemical lithium-intercalation method was applied to prepare the few-layered GD NSs. Fig 1 shows the TEM images of the GD NSs with and without lithium-intercalation treatment. The AFM data reveal that the thickness of the as-prepared GD is about 1.1 nm, and such a thickness is comparable to those common 2D MoS2 and . For the first time, few-layer GD NSs have been demonstrated to possess high fluorescence quenching abilities and different affinities toward the ssDNA versus dsDNA. Such superior properties of GD NSs can be advantageously used to develop new biosensing principles for multiplexed real-time fluorescent detection of DNA in a highly sensitive manner with a limit of detection as low as 25 × 10-12 M (Fig 2). Importantly, comparing with and MoS2 nanomaterials-based sensors, the GD NSs-based biosensor exhibits high sensitivity and short detection time for detection of multiplexed DNA. In addition, the assay can be carried out in homogeneous liquid phase, making it perfectly suits the in situ detection applications. The efficient GD nanoquenchers can be readily synthesized in large-area which can be loaded with different dye-labeled ssDNA make the material have the advantages for analysis of multiplexed DNA. We expect that the GD NSs nanoprobes demonstrated in this work for facile, rapid, and cost effective multiplexed detection of biological molecules would pave a way for widespread biological analysis using 2D nanomaterials-based biosensing systems.
Fig 1. a) TEM image of a typical GD nanosheet after lithium-intercalation treatment (inset: GD
nanosheet without lithium-intercalation treatment); b) AFM image of GD nanosheets, deposited on Si/SiO2 substrate (inset: Height profile of the dotted line, showing thickness of ~1.1 nm); c) TEM image of MoS2; d) TEM image of .
Fig 2. a-c) Fluorescence spectra of the dye-labeled ssDNA (P1:H1N1-FAM, P2: H5N1-Texas Red, P3:M13-FAM) in the presence of various concentrations of target DNA (T1, T2, and T3, 0-5 × 10?9 M); d–f) Calibration curve for target DNA detection. GD was 20.0 μg mL-1. With the excitation/emission wavelengths of a,c) 494 nm/516 nm and b) 595 nm/612 nm, respectively.
The research team includes a group led by Prof. WANG Dan from the Institute of Process Engineering (IPE) of the Chinese Academy of Sciences (CAS), a group led by YU Ranbo from University of Science & Technology Beijing, a group led by HUANG Ling from Nanjing Tech University, a group led by WANG Lianhui from Nanjing University of Posts and Telecommunications and a group led by ZHAO Huijun from Griffith University. The research was supported by the National Science Fund for Distinguished Young Scholars (No. 21325105), National Natural Science Foundation of China (Nos. 21590795, 51672276, 21671016, 51372245, 51541206), CAS Interdisciplinary Innovation Team, the Foundation for State Key Laboratory of Biochemical Engineering, and et al.
Their work entitled “Few-layer graphdiyne nanosheets applied for multiplexed real-time DNA detection” has been published in Adv. Mater (2017, 29, 1606755).
http://onlinelibrary.wiley.com/doi/10.1002/adma.201606755/full
Contact:
Prof. Dan Wang
State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, P. R. China.
E-mail: danwang@ipe.ac.cn
