Single-molecule sequencing based on solid-state nanopores can greatly expand the application of nanopore technology for DNA, proteins, and saccharides. Solid-state nanopores in ultrathin two-dimensional materials have the potential to offer higher spatial resolution in single-molecule sequencing. However, the conventional solid-state nanopores, which are created by drilling substrate materials with focused ion/electron beam, often suffer from low reproducibility at the atomic scale. Hence, solid-state nanopores have not yet been available for single-molecule DNA sequencing. 2D covalent organic framework (COF) is a class of covalently bonded crystalline polymers, which naturally has atomically consistent nanopore arrays in their reticular structure, providing an opportunity for single-molecule DNA sequencing.
Recently, COF nanopore devices (pore size≈1.1 nm) based on quartz nanopipette were designed for single-molecule DNA sensing by Prof. Kang Wang, Prof. Kai Xi and Assoc. Prof. Li-Na Ji at Nanjing University. The COF nanopore device could roughly distinguish dAMP, dCMP, dGMP, and dTMP. Furthermore, a certain percentage of the current blockades originating from 150 nucleotides model DNA molecules show distinct DNA sequence specific concave and convex resistive current patterns. This study is a significant step toward single-molecule DNA sequencing by solid-state nanopores.
Firstly, two different COF nanopore devices were obtained by covering the ultrathin 2D COF nanosheets on the tip of nanopipette with small (7 ± 3 nm) and big orifice (20 ± 5 nm), respectively (Figure 1). During the electrophoresis, one and only one obvious current drop occurred in both current traces, suggesting that both nanopipettes could only be covered by one piece of COF nanosheet. The I-V curves show that the current passing through the COF nanopores within the small substrate orifice is always smaller than that passing through nanopores within the big one, indicating that the decrease in the nanopipette orifice can lower the background current for subsequent detection.
Figure 1. The preparation and characterization of COF nanopore device
To explore the effect of substrate orifice size on the performance of COF nanopores for single-molecule detection, we compared the translocation behaviors of mononucleotides dAMP and dCMP using two COF nanopore devices (Figure 2). In contrast, dAMP and dCMP can be roughly discriminated using COF nanopores within the small substrate orifice. The greatly decreased nanopipette orifice encircles fewer COF nanopores and improves the signal-to-noise ratio.Furthermore, four types of mononucleotides (dAMP, dCMP, dGMP, and dTMP) also can be distinguished using another COF nanopore device with a small substrate orifice.
Figure 2. Identification of dAMP and dCMP
Then, two DNA oligonucleotides (dA50dC50dA50 and dC50dA50dC50) were allowed to pass through the COF nanopores within a small substrate orifice. A portion of current blockades shows distinct DNA sequence specific concave and convex resistive current pattern (Figure 3), suggesting that 2D framework material nanopores have great potential in single-molecule DNA sequencing.
Figure 3. DNA sequence-specific identification through COF nanopores
Three types of current blockades (TypeⅠ, Type Ⅱ and Type Ⅲ) were observed when DNA pass through the COF nanopores within a small substrate orifice (Figure 4). The variation in current blockades can be attributed to the specific location of each COF nanopore with respect to the wall of the nanopipette. The quartz nanopipette is negatively charged under the experimental conditions. Therefore, the cation (K+) is the dominant charge carrier in the COF-covered nanopore system and the concentration of K+ within the electrical double layer (EDL) is much higher than that along the central axis. When a DNA molecule passes through a COF nanopore within EDL, an obvious current drop will occur due to the partially blocked K+ translocation, and then concave and convex resistive patterns can be observed. Meanwhile, the simulation results show a clear trend that the appearance of current steps greatly depends on the transporting location of DNA strands.
Figure 4. Exploration of the single-molecule DNA translocation behavior
This work demonstrates the promising application of 2D porous framework materials in single-molecule sequencing.
The related paper has been published in ACS Nano (2024, doi: org/10.1021/acsnano.4c09848). Prof. Kang Wang, Prof. Kai Xi and Assoc. Prof. Li-Na Ji are the corresponding author of the paper. Dr. Linru Guo is the first author. This work is supported by the National Natural Science Foundation of China (Grant Numbers 22327802 and 22174060) and the State Key Laboratory of Analytical Chemistry for Life Science (Grant Number 5431ZZXM1907).