Nanopore technology is a biomimetic single-channel approach adapted from the patch clamp electrophysiology. It has been widely used for protein analysis, metal ion detection, small molecule recognition, single-molecule chemistry, and DNA sequencing. However, all reported nanopore sensing applications result from the same measurement configuration that relies on the patch clamp and Ag/AgCl electrodes. It makes the parallel nanopore recording limited in cost and throughput due to the introduced complexities from electronic integration. With the industrialization of single-molecule nanopore sequencing, the demand for high-throughput nanopore array (greater than 1 million channels) is growing. Such an urgent need thus stimulates us to reconsider a simplified strategy for high-throughput nanopore recordings.
Biological evolution has screened out the most streamlined transmembrane transport mechanisms. Bacterial phage T4 injects its genomic DNA through channel proteins when infecting host cells. Staphylococcus aureus α-hemolysin (α-HL) leads to hemolysis of target cells due to passive leakage of nutrients through inserted channels. These spontaneous molecular transport processes, remind us that external electronics are not indispensable formolecular transport.
Figure 1: Principle and applications of the electrode-free nanopore technology. Top left: Schematic diagram of the electrode-free nanopore technology. The calcium ions in the gel diffuse freely through the nanopores and bind with the fluorescent dye in the pore mouth, producing the fluorescence signal around the pore. Top right: Electrode-free nanopore sensing. The molecule translocation through a single nanopore gives rise to a reduced fluorescence signal, which can be employed for single-molecule detection of cyclodextrin, polyethylene glycol and dsDNA. Bottom left: simultaneous imaging of α-HL and ClyA nanopores. Due to the difference in the pore size, ClyA-RR nanopore appears as a huge and dazzling fluorescence spot(red circle), whereas an α-HL nanopore appears small in size with a dim intensity (yellow circle). Bottom right: Micro-droplet nanopore array.
Inspired by transmembrane transports and recent advances in calcium fluorescence imaging, Hong-Yuan Chen and Shuo Huang’s team at State Key Laboratory of Analytical Chemistry for Life Sciences, have recently developed the first electrode-free nanopore technology dubbed DiffusiOptoPhysiology (DOP). By optically monitoring the diffused calcium flux through the nanopore, direct sensing of small molecules, polymers, and biomacromolecules was subsequently demonstrated from fluorescence readout. A finite element method (FEM) simulation was also established by removing the electromigration term in molecular transfer (Formula 1), which was a simplified form of the traditional electrophysiological nanopores detection model (Nernst-Planck equation, Formula 2).
Formula 1 (DOP model):
Formula 2(Electrophysiological model):
Upon optimizations from electrolytes and pore sizes, the sensing performance of DOP could be comparable to that of traditional electrophysiological nanopores technology, while the advantages of low cost (<1$ for disposable devices for each measurement) and high throughput (~104 pores/mm2) were retained. By omitting the need for an electrode arrangement, the measurement volume of DOP was reduced to ~30 pl, which can pronouncedly reduce the sample consumption and may be suitable for measurements of analytes with an extremely low abundance. As a further extension, a fingertip-sized nanopore chip was developed for multiplex single- channel recordings from an array of microdroplets at a theoretical density of 103 bilayers/ mm2. It suggests a new concept of clinical diagnosis using disposable nanopore chips.
Figure 2：Real time detection of dsDNA using a ClyA nanopore.
The related paper entitled Electrode-free Nanopore Sensing by DiffusiOptoPhysiology has been published on Science Advances on September 6, 2019 (DOI: 10.1126/sciadv.aar3309，paper link：https://advances.sciencemag.org/content/5/9/eaar3309) . Yuqin Wang，a phD student is the first author of the paper. Academician Hong-Yuan Chen and Professor Shuo Huang are co-corresponding authors of the paper. This work is supported by the National Natural Science Foundation of China (grant nos. 21327902, 21675083, and 91753108), the Fundamental Research Funds for the Central Universities (grant nos. 020514380142 and 020514380174), the State Key Laboratory of Analytical Chemistry for Life Science (grant nos. 5431ZZXM1804 and 5431ZZXM1902), the Excellent Research Program of Nanjing University (grant no. ZYJH004), the 1000 Plan Youth Talent Program of China, the Program for High-Level Entrepreneurial and Innovative Talents Introduction of Jiangsu Province, and the Technology Innovation Fund Program of Nanjing University.