生物标志物的高灵敏度、高选择性、快速检测，对于疾病的临床诊断和流行疾病的预防十分重要。其中，荧光检测技术由于灵敏度高、选择性好，已成为生物分子检测的优选手段之一。单层二维纳米片对染料标记的单链DNA（ssDNA）具有选择吸附和荧光猝灭能力，能够实现DNA与小分子的有效检测。

　　氧化石墨烯（）是最早报道的具有荧光淬灭能力的二维材料，石墨烯与核酸之间的π-π堆积作用，使得染料标记的ssDNA能够强烈吸附于表面，与染料分子之间的荧光共振能量转移使荧光染料淬灭。但双链DNA（dsDNA）由于带负电荷的磷酸骨架对核酸碱基的屏蔽效应，降低了dsDNA与分子的相互作用，不能发生荧光淬灭。

　　石墨炔（GD）是一种新的二维碳材料，计算结果表明，它应当具有比石墨烯更强的吸附染料分子和捕获电子的能力，因而有望产生更有效的荧光淬灭，提高检测灵敏度。中科院过程工程研究所王丹研究员及其合作者课题组采用锂插层法对石墨炔进行剥离，获得了少层的石墨炔纳米片（图1），其具有高的荧光猝灭能力，以及对ssDNA与dsDNA不同的亲和力，实现了对多种DNA的实时检测，检测限低至25×10-12 M（图2）。而且，与和MoS2纳米片相比，该少层石墨炔纳米片具有更高的灵敏度和更短的检测时间。该检测方法可在均相溶液中进行，完全适用于原位检测，可携带多种荧光指示剂，能够实现对多种生物分子的实时检测。该研究开拓了基于GD纳米探针的简便、快速、高效的生物分子荧光检测技术，为二维纳米生物传感材料的研发及其在生物分析上的应用提供了全新的思路。

　　相关研究结果发表在《先进材料》（Adv. Mater 2017, 29, 1606755）上。该研究得到了国家自然科学基金杰出青年基金（21031005），国家自然科学基金（21590795, 51672276, 21671016, 51372245, 51541206），中国科学院创新交叉团队，生化工程国家重点实验室等支持。

图1. a) 少层石墨炔纳米片的TEM照片（插图：剥离前的石墨炔纳米片的TEM照片）; b) 少层石墨炔纳米片的AFM照片（插图显示厚度约为1.1nm）; c) MoS2纳米片的TEM照片; d) 氧化石墨烯的TEM 照片.

图2. a-c) 不同靶DNA浓度下（T1, T2, T3: 0-5×10-9 M）染料标记的ssDNA的荧光光谱（P1: H1N1-FAM，P2: H5N1-Texas Red，P3: M13-FAM）;  d-f) 靶DNA检测的校准曲线

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