Science advanced reports on the technology of electrodeless biomimetic nanopore chip by Chen Hongyuan and Huang Shuo of Nanjing University

In nature, the phenomenon of material transport across membrane is widely used to precisely regulate many biological processes at the molecular level by mediating the material exchange inside and outside cells Among them, the transmembrane transport of inorganic ions, glucose, nucleic acids, amino acids and other cell metabolites needs to be mediated by specific membrane channel proteins These membrane channel proteins are spontaneously assembled on the biomembrane by protein monomers or polymers to form transmembrane channels Patch clamp technology can effectively explore the biophysical properties of single membrane channel protein one by one With more and more novel membrane channel proteins being discovered, explored and understood, the gated kinetic properties and transport mechanism of these channels have been gradually interpreted The new single molecule nanopore technology is the biomimetic technology based on the patch clamp technology Its basic principle is to use electric field force to drive single molecule or ion through the picoammetric current change caused by nanopore to detect single molecule Nanopore technology is widely used in protein analysis, metal ion detection, organic small molecule recognition, nano particle characterization, single molecule chemistry, single molecule mass spectrometry and DNA sequencing However, since the first concept of nanopore sensing was presented in 1993, no matter how the instrument form of nanopore technology changes, its detection mechanism is completely from the electrical signal detection method relying on patch clamp technology Although this method has ultra-high time resolution (~ 10 μ s) and current resolution (< 0.1pA), but limited by the technical threshold and cost limitations of the existing microelectronics and microelectrode processing technology, expanding the detection flux is the main bottleneck in this field At this stage, single molecule gene sequencing is the most important application in the field of nanopore sensing At the same time, with the massive demand of data collection, with the industrialization of nanopore sequencing technology gradually unfolding, people have sequenced the ultra-high throughput nanopore The demand of 1 million channels is more and more urgent, so it is urgent to develop a new nanopore detection mode, which breaks through the limitations of the existing detection flux on the premise of not significantly improving the detection cost A series of transmembrane transport mechanisms with the simplest structure and the most efficient operation have been screened out in the billions of years of biological evolution T4 phage can specifically adsorb on the corresponding receptors on the surface of the host cell, and inject DNA from the head into the host cell through the hollow tail (2.5-3.5 nm in diameter) α - HL secreted by Staphylococcus aureus can be inserted into the cell membrane of the host cell, resulting in the loss of cell nutrients and dissolution Different from the nanopore technology driven by electrode pair and patch clamp instrument, the transmembrane transport process in nature is often spontaneous, and does not need the assistance of redundant electrodes, patch clamp and other peripheral electronic devices, so it is the most simplified material transmembrane transport molecular machine Figure 1: principle and application of electrodeless nanopore technology Top left: schematic diagram of electrodeless nanopore technology The free diffusion of calcium ions in the gel passes through the nanopores and combines with the fluorescent dye of calcium ions in the droplets to produce fluorescent signals Top right: single molecule sensing based on electrodeless nanopore technology When the molecule passes through the nanopore, it will produce specific blocking fluorescence signal, which can be used for the sensing of cyclodextrin, polyethylene glycol and double stranded DNA Bottom left: simultaneous imaging and differentiation of clya nanopore and α - HL nanopore Due to the different pore sizes (clya nanopore: D = 3.6 nm, α - HL nanopore: D = 1.4 nm), clya nanopore can produce a bright bright spot (red circle) in fluorescence mode, while α - HL nanopore shows a weak bright spot (yellow circle) Bottom right: clya nanopore micro droplet array (source: Nanjing University News Network) inspired by this, Chen Hongyuan and Huang Shuo, the National Key Laboratory of life analysis chemistry, School of chemistry and chemical engineering, Nanjing University, developed a new nanopore detection method, and named it as the electrodeless nanopore Technology (DOP, diffusioptophysics), which is also the world's first single molecule nanopore detection method without electrode The team added calcium and calcium fluorescent dyes on both sides of the nanopore Under the concentration gradient, the free diffusion of calcium ions through the nanopores and the fluorescent dye combined with the release of fluorescence signals, indirectly realized the real-time imaging of the nanohole on the bilayer of the droplet gel interface, and successfully displayed its single molecule sensing for small molecules, macromolecules and biomolecular This technology fully simplifies the traditional nanopore detection model (Nernst Planck equation, formula 1), removes the contribution of the electric migration term in the material transfer, and only describes the material transfer by diffusion, chemical reaction and fluid (formula 2): (source: Nanjing University News Network) Through further optimization of electrolyte solution and pore, its sensing performance can be comparable to the traditional electrophysiological nanopore technology, and the theoretical detection density can be as high as 104 pores / mm 2 Since DOP does not need to reserve electrode space, its measurement volume can be further reduced to ~ 30 pl the detection volume is one millionth of the existing nanopore technology, significantly reducing sample consumption, which is very suitable for the measurement of analytes with very low abundance In addition, without the use of electrodes, researchers can easily build a micro drop DOP detection array This detection mode is very suitable for multi-component, parallel high-throughput drug screening, and its theoretical detection density can reach 103 billayers / mm 2 (Figure 1) On this basis, the team has built a high-throughput disposable single molecule analysis chip, which only needs simple and nontoxic materials The cost of the existing chip is less than US $1, which is very suitable for the disposable detection needs in clinical detection and is expected to enter daily life Fig 2: detection of dsDNA with large pore diameter clya nanopore without electrode (source: Nanjing University News Network) The work is entitled "electron free nanopore sensing by diffusioptophysiology", which was recently published in science advanced (DOI: 10.1126 / sciadv.aar3309) Wang Yuqin is the first author of this paper Chen Hongyuan, academician of the school of chemistry and chemical engineering, Nanjing University, and Professor Huang Shuo are co authors of this paper This research was supported by the National Natural Science Foundation of China (Project No.: 91753108, 21327902, 21675083), the basic scientific research business expenses of the Central University (International Science and technology cooperation promotion project) (Project No.: 020514380142, 020514380174), the National Key Laboratory of life Analytical Chemistry (Project No.: 5431zzxm1804, 5431zzxm1902), and the excellence program of Nanjing University（ Project No.: zyjh004), Jiangsu Province high level entrepreneurship and innovation talent introduction plan, Nanjing University Science and technology innovation fund project and other financial support.