Commentary and Perspective
Accelerating biophysical studies and applications by label-free nanopore sensing
2023 年 20 巻 1 号 論文ID: e200010
詳細
2023 年 20 巻 1 号 論文ID: e200010
Label-free single-molecule sensing technologies are attractive tools for investigating the properties of biological molecules via the understanding of molecular functionality. Among these technologies, nanopore sensing has become one of the growing technologies [1,2]. Nanopore sensing operates in the principle of resistive pulse sensing, where sensing molecules, such as DNA, RNA, and protein, pass through a pore under the electrical field, resulting in a blockade current due to the molecular occupation in a pore. The physical properties of sensing entities were obtained by analyzing blockade current, which can provide a fingerprint of sensing molecules (Figure 1) [3].
The fundamental working principle of nanopore sensing
In this commentary article, we review the eight presentations at the symposium “Innovative label-free nanopore sensing toward biophysical studies and applications” of the 60th Annual Meeting of the Biophysical Society of Japan held in September 2022 and introduce how this sensing technology can be used as a tool to open new biophysical science or applications other than DNA sequencing.
Kyle Briggs at Ottawa University/Northern Nanopore Instruments talked about an automated method of electrical-based nanopore fabrication, which is one of the gold standard fabrication methods in the lab, and introduced how to accelerate solid-state nanopore research using this method [4,5]. He also presented the automated muti-pore fabrication tools having multi-channels fluidic flow cells with multi-membrane chips. Finally, he showed the nanopore trace analysis software, Nanolyzer, which has multiple functions such as multi-level blockade current fitting, overlay translocation events, kernel density estimation, etc.
Kan Shoji at Nagaoka University of Technology presented a probe-type planer bilayer lipid membrane (pBLM) system [6,7] and its application for scanning ion conductance microscopy (SICM) [8]. In this system, pBLMs can be repeatedly formed at the tip of probes by inserting probes into a layered bath solution of an oil/lipid mixture and electrolyte. He mounted the probe into a SICM setup and demonstrated spatially-resolved chemical sensing by manipulating the probe. Additionally, he introduced an efficient current measurement system for synthetic DNA nanopores. Although DNA nanopore structures are expected to be applied for nanopore sensing, it is challenging to efficiently insert DNA nanopores into pBLMs. He prepared DNA nanopore-tethered gold electrodes and formed pBLMs on the surface of electrodes by inserting electrodes into the bath solution. Resultantly, efficient insertions of DNA nanopores were observed, and this method potentially accelerates applications of DNA nanopores for nanopore sensing.
Hiroki Ueda at the University of Tokyo introduced a new self-developed nanopore sequencing software, nanoTune and nanoDoc [9]. nanoTune is a cloud-based software for the sequence-to-signal assignment, which uses the probability of basecaller output to realign the signal to the reference. nanoDoc is designed to detect DNA/RNA sequences using deep neural networks. This software analyzes the deviation of current signal caused by DNA/RNA modification using Deep One-Class Classification. He also showed that the genetic DNA/RNA modification can be identified without a training dataset using nanoTune and nanoDoc.
Ping Liu and Ryuji Kawano at Tokyo University of Agriculture and Technology talked about the determination of DNA methylation and demethylation intermediates by stochastic nanopore sensing. The determination of 5-methylcytosine (5mC) and its oxidized derivatives is crucial to understand how genes regulate cell function and development [10]. To determine the position of 5mC and demethylation intermediates, they utilized the epigenetic modulation of cytosine dynamics in dsDNA, which destabilizes the Watson-Crick base pair. Because modified cytosine interacts with the amino acids in the α-hemolysin nanopore, the duration time and blocking ratio associate with the position and type of modified cytosine [11]. They determined the position of 5mC and demethylation intermediates at the single-nucleotide level by analyzing blocking signals with the bootstrap method [12], and this method offers a simple and powerful tool for the detection of DNA methylation and demethylation intermediates.
Hiromu Akai and Kan Shoji at Nagaoka University of Technology introduced an ATP-detectable DNA nanopore that can repeatedly open and close the aperture of nanopores in response to ATP. They designed the DNA nanopore structure with an ATP-binding DNA aptamer as a molecular recognition domain and investigated the relationship between the open-close ratio of the DNA nanopore and ATP concentration by measuring ion currents through the nanopore. Resultantly, the open-close ratio was linearly changed in the range of ATP concentrations from 300 μM to 3 mM, suggesting the potential for application as ATP sensors.
Akihide Arima at Nagoya University presents a nanopore-based method that can identify virus species by analyzing blockade current traces using machine-learning [13,14]. To apply machine-learning for nanopore sensing, his team detected various virions to obtain training datasets of characteristic trace features and used them for the classification in a high-dimensional feature space. This machine-learning approach can identify five different virus species with an accuracy higher than 99%. The demonstration of this approach shows the combination of nanopore sensing and machine-learning can be an ideal way for future novel diagnostic systems with rapid screening.
Qing Zhao and Rui Hu at Peking University showed how mechanically stable solid-state nanopore sensors can be used to study a ubiquitinated histone on mononucleosomes [15]. First, the demonstration of voltage dependence of nucleosome showed that nucleosome can be ruptured and passes through a pore when the voltage reaches above the threshold voltage (rupture threshold voltage) as similar to previous study [16]. Moreover, by comparing two different ubiquitinated histones (ubH2A and ubH2B), the rupture threshold voltage of ubH2A showed higher than ubH2B, revealing ubH2A forms a much more stable structure. This study pointed out that a solid-state nanopore sensor can be developed as a portable device for rapidly screening post-translational modifications.
Hirohito Yamazaki at the University of Tokyo presents how optical technologies can be used for nanopore sensing [17,18]. Since light absorption of solid-material results in various phenomena, e.g., heating and chemical reactions, it can enhance the modality of nanopore sensing. The photothermal heating of silicon nitride (SiN) pore causes the increase of current trace, which can be a temperature indicator of a pore (nanopore thermometer). Using this thermometer, the thermal kinetics of biomolecules can be probed by passing them into photothermally heated pores [19]. Another effect of light absorption of SiN in electrolyte solution was photochemical etching. Using this etching with controlled dielectric breakdown, a pore in a few nm diameter and thickness can be fabricated using a custom-made feedback system [20]. The molecular sensing using this type of pore shows the potential for a wide range of applications, such as biomarker detection.
In conclusion, the symposium provided the opportunity to share and discuss the potential for new tools to work on biophysical research and upgrade to new biotechnological applications. We believe the symposium could motivate researchers in other fields to use nanopore sensing for accelerating their research.
We would like to appreciate the committee members at the 60th Annual Meeting of the Biophysical Society of Japan to support holding our nanopore symposium. Also, we would like to thank to Dr. Briggs, Prof. Ueda, Prof. Zhao, Dr. Hu, Prof. Arima, Prof. Kawano, Ms. Liu and Mr. Akai for their presentations and supports for this commentary preparation. We acknowledge Exploratory Research for Advanced Technology (ERATO; JPMJER2002) from JST.
