Single nucleotide discrimination with sub-two nanometer monolayer graphene pore

https://doi.org/10.1016/j.snb.2021.130792Get rights and content

Highlights

  • Sub-2-nanometered graphene pores for DNA detection are fabricated by helium ion milling.

  • The translocation of ssDNA including 20-mer, 5-mer and 3-mer can be slowed down by using RTILs.

  • Single bases are identified through the nanopores engineered in monolayer graphene for the first time.

Abstract

The effective sensing area is the key point of solid-state nanopore single-molecule detection, which is determined by the diameter of the hole and the thickness of the membrane. High spatial resolution can be achieved by using single-atom thickness two-dimensional materials, such as single-layer graphene and molybdenum disulfide. Helium ion beam microscopy provides an effective technique for manufacturing small-sized nanopores. Here, we show that the precise fabrication of sub-2 nanometer nanopores has been successfully achieved on a single-layer graphene film, which provides the best spatial resolution in room-temperature ionic liquid-assisted single-molecule detection. Many graphene nanopores of different sizes have been fabricated, with a minimum pore diameter of 1.4 nm. The application of these graphene nanopores manufactured by HIM shows that single-layer graphene nanopores can distinguish different homopolymer single-stranded DNA lengths and types, and can even identify four single nucleotides.

Introduction

A lot of effort has been invested in the research and development of DNA sequencing based on nanopore devices because they exhibit label-free, low-cost, and high-throughput characteristics [1]. Nanopore sensors work according to the Coulter counter principle, which shows that target particles passing through a biased pore can temporarily block the channel and cause the current to drop [2]. And the molecular level information of the translocation analyte is characterized by the blocking signal, that is, amplitude, residence time, and event frequency.

After the first application of biological nanopore to nucleic acid detection [3], researchers focus on achieving single-base resolution in nanopore-based DNA sequencing. In recent years, with the help of a large number of enzyme motor proteins, the high spatial resolution of biological nanopores has been verified [4], [5], [6], [7], [8], [9], [10], [11]. Although the shrinkage inside the biological nanopore can successfully distinguish individual nucleotides, the application of such pores is still limited by their insufficient mechanical stability and complex biochemical reactions. In contrast, solid-state nanopores can be used in alternative liquid environments and have geometric shapes that can be designed for specific analytes and produced by nanofabrication technology. They exhibit excellent mechanical, chemical, and thermal stability, as well as compatibility with other devices [12]. However, such long-channel nanopores cannot theoretically detect single nucleotides because their channels are much longer than the interval between two consecutive nucleobases in a single-stranded DNA strand, allowing polynucleotide blockade [13]. Two-dimensional (2D) materials such as graphene and molybdenum disulfide (MoS2) are expected to be promising alternatives for the precise detection of single nucleotides because nanopores are fabricated in ultra-thin films can provide extremely high spatial resolution.

Benefiting from the improvement of nanofabrication technology, several techniques have been applied to produce solid-state nanopores, including transmission electron microscopy (TEM) [14], [15], [16], [17], focused ion beam (FIB) [18], [19], [20], [21], dielectric breakdown (CBD) [22], [23], [24], [25], [26], electron beam sputtering [27], [28], [29], [30] and the track-etch method [31], [32], [33], [34]. However, most of these methods suffer from issues in terms of irregular shape, poor repeatability, and low throughput of nanopore fabrication. Helium ion milling (HIM) functions as an ion beam microscopy whose high-energy beam is emitted from the tungsten trimer on the top of the electrode inside of the helium gas environment, and the size of its beam spot can be extremely small--as small as 0.5 nm. The theoretical manufacture precision is expected to be about 2 nm. With exposure time in the order of seconds, the nanoscale membrane can be drilled in one step, which is different from electron beam milling that relies on the photoresist. And this technique allows multichip and structural array fabrication as well [35], [36], [37]. Our group has reported the research for HIM fabrication of graphene nanopores with a minimum diameter of 5 nm and the ssDNA translocation demonstrated the efficacy of HIM graphene nanopores in short DNA chain discrimination [38]. Hayashi et al. achieved the reproducible HIM fabrication of nanopore less than 10 nm in diameter, and a 1.5 nm graphene nanopore had been obtained [39]. Recent works of our group reported researches of the substrate influences on the defect formation of graphene membrane due to helium ion irradiation using Raman spectroscopy [40], [41].

Nevertheless, DNA sequencing with a 2D nanopore has been limited by its fast translocation speed [42]. Many efforts have been devoted to regulating the translocation velocity of DNA to realize nucleotides sequencing ultimately, such as increasing aqueous viscosity [43], [44], lowering the temperature [45], lowering the bias voltage [46], decreasing nanopore diameter [47], and increasing the potential barrier of DNA translocation [48], [49], [50], [51], [52]. Particularly, Feng et al. [53] presented research based on the high spatial-resolution of MoS2 nanopore and the slowing down effect of high viscous room temperature ionic liquids (RTILs) 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF6]), in which they successfully identified four nucleotide types. However, Shankla et al.[54] reported all-atom molecular dynamics research of MoS2 nanopore, which demonstrated the interface of KCl aqueous and RTILs produced junction potential and the free energy difference between two-phase might cause the retardation of DNA translocation. While Lee et al. [55] proposed a method of adding 3% water into [bmim][PF6] to improve the solubility and the conductivity of ssDNA. Although different nucleotides could not be identified, the ssDNA detection results using 2D h-BN nanopores showed higher nanopore sensitivity. In this work, we demonstrated a size-adjustable manufacturing technology that uses a helium ion beam microscope to produce single-layer graphene nanopores to achieve precise control of size and shape with high manufacturing efficiency. The smallest geometry of graphene nanopores with a diameter of 1.4 nm has been achieved, which is theoretically close to the manufacturing limit of HIM technology. Combined with RTILs water, we conducted a series of translocation experiments using the prepared graphene nanopores to detect DNA molecules of different lengths and successfully distinguished four types of single nucleotides.

Section snippets

Fabrication of silicon nitride chips

The silicon nitride (SiNx) chips were fabricated by the method of the combination of the photolithography technique and wet etching on silicon-based low-pressure chemical vapor deposition (LPCVD) SiNx wafer (Fig. SI1). Pretreatment of SiNx chips using 10% HCl and acetone ensured that the alkaline and organic residues left over by chips etching were eliminated. Then SiNx films were punctured with 240 nm holes using gallium FIB (Ga-FIB, Orion NanoFab, Carl Zeiss). The Ga-FIB acceleration was set

Nanopore fabrication using Helium ion beam microscopy

Fig. 1a is a schematic diagram of the HIM instrument. Since different trimers will produce an inconstant helium ion beam current, we could adjust the beam current to the range of 0.7–0.9 pA by controlling the helium flow (0.3–1.0 e−6 Torr). HIM allows fabrication in scan mode or spot mode. In this work, the spot mode has been applied to drill nanopores, which means that the target area of the graphene sheet is exposed to a specific beam about 0.8 pA for a period of time, and the size of the

Conclusion

In summary, we have developed an effective method to directly drill graphene membrane into sub-2 nanometer pore devices. Using the fabricated graphene nanopores, DNA translocation experiments have been performed. With the aid of BMIMCl aqueous solution, the ssDNA of poly-dN20, poly-dN5, poly-dN3, and dNTP has been successfully identified. The detection signal confirmed the ability of BMIMCl aqueous solution to slow down DNA translocation. According to the data of the translocation experiment,

CRediT authorship contribution statement

Z.Z, H.C. and D.W. conceived and planned the study. D.P. performed the CVD-graphene synthesis; Z.Z. performed nanopore detection assays and analyzed the data; Z.Z, H.C. and D.W. wrote the article. All authors discussed the results and contributed to the final manuscript.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by research funding from the Natural Science Foundation of Chongqing, China (Grant No. cstc2018jcyjAX0310, cstc2017jcyjB0105, cstc2018jcyjAX0304), the National Natural Science Foundation of China (Grant No. 61701474, 31800711), the Instrument development program of the Chinese Academy of Sciences (Grant No. YZ201568), the Pioneer Hundred Talents Program of the Chinese Academy of Sciences (Liang Wang) and the Youth Innovation Promotion Association of the Chinese Academy

Zi-Yin Zhang, obtained her Ph.D. degree in 2020 from the College of Instrumentation and Electrical Engineering, Jilin University, Changchun. Currently she is a research assistant and post doctor at Chongqing Institute of Green and Intelligent Technology (CIGIT), Chinese Academy of Sciences. Recently, she is devoted to the fabrication technology of nanopore devices using focused ion beam microscopy for biomolecules detection and DNA sequencing, as well as the application of 2D materials in the

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    Zi-Yin Zhang, obtained her Ph.D. degree in 2020 from the College of Instrumentation and Electrical Engineering, Jilin University, Changchun. Currently she is a research assistant and post doctor at Chongqing Institute of Green and Intelligent Technology (CIGIT), Chinese Academy of Sciences. Recently, she is devoted to the fabrication technology of nanopore devices using focused ion beam microscopy for biomolecules detection and DNA sequencing, as well as the application of 2D materials in the development of DNA sequencer.

    Hong-Liang Cui, professor of Jilin University, distinguished researcher of Chongqing Institute of Green Intelligence, Chinese Academy of Sciences (CIGIT). In 1981, he was admitted to the first Li Zhengdao physics doctoral program in the United States, and received his Ph.D. degree in theoretical physics from Stevens Institute of Technology in 1987. He has served as Assistant Professor, Associate Professor and Professor in the Department of Physics and Engineering Physics at Stevens Institute of Technology. Since 2009, he has been a professor of Applied Physics at New York University Institute of Technology and became a professor at Jilin University in 2011. He has been engaged in research in optoelectronics, optical fiber communication and sensing, solid-state electronics, semiconductor physics and devices, terahertz technology and other fields for many years. He has supervised more than 30 doctoral graduates, published more than 200 papers, published two monographs, and won a number of invention patents in the United States and China.

    De-Ping Huang, graduated from Shandong University with a master degree of science in 2012 and then worked in Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences. In recent years, mainly engaged in the growth of the graphene films, modification, transfer and other aspects of graphene research. She has accumulated rich experience in the preparation of high-quality graphene films, and obtained 8 authorized invention patents which formed one of the key technologies in the "large-scale single-layer graphene films growth method and preparation technology system " and won the first prize of Technological Invention in Chongqing. Currently, this technology has been applied in the graphene film production line of Chongqing Moxi Technology Co., LTD. At present, her research projects related to graphene and two-dimensional materials have been funded by the National Natural Science Foundation of China and the Pioneering Science Foundation of Chongqing Natural Science Foundation.

    De-Qiang Wang, SPIE member, the director of Chongqing Key Lab of Multi-scale Manufacturing Technology and the director of the single molecule diagnostic technology research center. He received the B.S. in the Department of Electrical Engineering from University of Jilin, Changchun in 2001 and the Ph.D. degree from the Institute of Microelectronics, Chinese Academy of Sciences, in 2006. Currently, he is a principal investigator at Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences. Previously, he worked on DNA sequencing projects based on nanopore at UIUC and IBM T.J Watson Research Center, NY. His main research interests are single molecule nanopore optoelectronic detection and nanopore-based DNA sequencing. His research activities also include single molecule detection, Solid-state Nanopore, Novel lithography, micro and nano fabrication. He coauthored over 60 peer reviewed journal papers and conference presentations. He has about 18 granted US and 17 CN patents.

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