Nanotechnologies / Materials
Medical & healthcare, Smart device

Single-molecule measurements for single-cell multiomics
analyses
Department of Bio-Nanotechnology, SANKEN (The Institute of Scientific and Industrial Science)
Associate Professor Makusu Tsutsui
https://researchmap.jp/makusu

Nanotechnologies / Materials

Abstract
Rapid advance in single-molecule techniques allowed a sensor
platform that enables to address biological information at unprecedented resolution and throughput. This research aims at creating
nanosensors that can count the individual proteins and genomes
within a cell. The sensor structure is comprised of two nanopore
membranes three-dimensionally stacked on a silicon wafer. It canperform cell electrolysis, intracellular molecule extraction, and single molecule detections on-chip by ionic current measurements.
The mechanism was tested for E. coli for the proof of concept,
where it was observed that single-molecule protein and DNA in thebacterial cell were shown to be detectable. This ongoing projectcurrently focuses on identifying the cellular biomolecules by themachine learning-assisted ionic current signal pattern analyses.

▍ Background & Results

ionic current measurements. The mechanism was tested for E. coli
for the proof of concept, where it was observed that single-molecule protein and DNA in the bacterial cell were shown to be detectable. This ongoing project currently focuses on identifying the cellular biomolecules by the machine learning-assisted ionic current
signal pattern analyses.

▍ Significance of the research and Future perspective
This technology offers a way for quantitative analyses of protein
expression in single cells by simple electrical measurements without special pretreatments, which may contribute to innovation in life
sciences and personalized medicine. In addition, its application is
not limited to cells; when applied to viruses, for example, it can bea tool for surveillance of infectious strains that can detect a small
mutation at a single virion level.

Nanotechnology-based single-molecule measurement techniques have made dramatic progress in the last ten years andstarted to be applied in omics analysis. The nanopore and theSMRT sequencing technologies are typical examples that canperform DNA and RNA sequence readout at the single-molecule
level in real time. Notably, their high throughput and ultralong read
length characteristics are already widely practiced in the fields ofgenomics and transcriptomics. And nowadays there is growinginterest beyond the genome sequencing: significant efforts havebeen devoted in recent years to novel technologies that can countthe numerous proteins in a cell by measuring their structure oramino acid sequence at a single-molecule level for the whole proteoform analysis.
This project aims to develop such technology by integrated
nanopore sensors. A nanopore is a nanoscale hole formed in amembrane. When a voltage is applied in the electrolyte liquid,ions move inside the nanopore, whose process can be observed
via ionic current measurements. When an object such as protein
passes through this small hole, the ionic current changes rapidly
due to the temporal blockage of the ion transport by its volume
thereby allowing a single-molecule sensor of simple mechanism aspracticed in the nanopore sequencers. However, it is not straightforward to apply the nanopore approach to proteins since there isno simple method like PCR amplification to amplify the molecules,
which makes it difficult to detect rare molecules. Or more simply,
if a biological sample contains large particles or aggregates suchas amyloid, the nanopore will be easily clogged by them. In orderto solve these problems, we devised a structure in which two nanopore membranes are stacked on a silicon chip. A cell can betrapped on nanopores in the upper membrane, and then exposedto the local electric field and Joule heat there for the electrolysis.
Subsequently, molecules smaller than the nanopores move toward
the second membrane. Here, the distance between the two membranes is made short so that the extracted molecules will swiftly
pass through the nanopore in the lower layer for the detection byP a t e n t Japanese Patent Application No. 2022-160261
Tsutsui, Makusu; Takaai, Takayuki; Yokota, Kazumichi et al. Deep Learning-Enhanced Nanopore Sensing of Single-Nanoparticle Translocation Dynamics,
Small Methods, 2021, 5, 2100191, doi: 10.1002/smtd.202100191
Tsutsui, Makusu; Yokota, Kazumichi; Akihide, Arima et al. Detecting Single Molecule Deoxyribonucleic Acid in a Cell Using a Three-Dimensionally
Tr e a t i s e
Integrated Nanopore, Small Methods, 2021, 5, 2100542, doi: 10.1002/smtd.202100542
Tsutsui, Makusu; Arima, Akihide; Yokota, Kazumichi et al. Ionic heat dissipation in solid-state pores, 2022, 8, eabl7002. Science Advances,doi: 10.1126/sciadv.abl7002
URL
K e y w o r d nanopore, single molecule measurements, single cell analysis, machine learning