Plasmonic two-nanopore technology for single-molecule protein sequencing (TwoPoreProSeq)


Single-molecule DNA sequencers are difficult to sequence proteins as they can detect only 3 of the 20 kinds of amino acids (AAs) that are building blocks of proteins. The nanogap sensor in our TwoPoreProSeq project has enhanced the AA Raman signals by 100-million folds to achieve label-free single-molecule detection of all 20 AAs and reading single AA residues within single peptide molecules.
The plasmonic nanogap biosensor that detected single residues in single vassopresin peptide.
The plasmonic nanogap biosensor that detected single residues in single vassopresin peptide.

Project information

Project duration


Funded by

Academy of Finland - Academy Research Fellow

Funding amount

847 421 EUR

Project coordinator

University of Oulu

Contact information

Project leader

Project description

While portable single-molecule DNA sequencers have been brought to African and Brazil villages for sequencing Ebola and Zika virus, no corresponding technologies exist for protein sequencing. The big gap hampers not only mechanistic studies but also clinical applications. The latter includes digital health and personalized medicine that aims to provide customized treatment to individual patients based on dynamic personal health data collected by Point-of-care diagnostic devices.

The TwoPoreProSeq project aims to develop a novel plasmonic two-nanopore device able to unfold and sequence single protein molecules by the nanogap-enhanced Raman spectroscopy [1]. Our recent results have demonstrated label-free Raman spectroscopic reading single amino-acid residues within single peptide molecules [2] and single-molecule Raman detection of all 20 amino acids [3]. The project can provide a single-molecule protein sequencing technology to Point-of-Care devices, allowing peoples in both developed and underdeveloped countries to acquire data of their health and diseases at home for health management.

Related works:

  1. J.A. Huang et al., SERS Discrimination of Single DNA Bases in Single Oligonucleotides by Electro-plasmonic Trapping, Nature Communications 10, 5321 (2019)

  2. J.A. Huang*et al., Multiplex characterization of single amino acid residue in polypeptide by single SERS hot spot, Angewandte Chemie International Edition, 59, 11423-11431 (2020)

  3. Y. Zhao, et al., Label-Free Optical Analysis of Biomolecules in Solid-State Nanopores: Toward Single-Molecule Protein Sequencing, ACS Photonics 9 (3), 730–742, 2022.

Project results

01.06.2023: Yingqi´s work of single-molecule manipulation and SERS detection by a plasmonic bowl-shaped nanopore is published on Nano Letters.

21.07.2022: Results on single-molecule Raman analysis of 20 proteogenic amnio acids and peptides can be found in my invited talk video (15 min) at the META 2022 conference:

Partners and Collaborators

The TwoPoreProSeq project is so multi-disciplinary that we are receiving help and support on biochemistry, nanophotonics, electronics, bioinformatics, nanofluidics and machine leanring from the following collaborators:

  1. Dr. Francesco De Angelis and Dr. Aliaksandr Hubarevich in Italian Institute of Technology (Italy) for nanofabrication, plasmon nanotechnologies and multiphysics simulation;
  2. Dr. Cuifeng Ying in Nottingham Trent University (UK) for nanopore fabrication and resistive pulse sensing of single-molecule DNA and proteins;
  3. Prof. Xuejin Zhang in Nanjing University (China) for nanophotonics measurements and simulation;
  4. Prof. Caglar Elbuken in University of Oulu for microfluidic sensor integration;
  5. Prof. Simo Saarakkala in University of Oulu for machine learning data analysis;
  6. Dr. Ilkka Nissinen in University of Oulu for digital electronic control automation;
  7. Dr. Valerio Izzi in University of Oulu for bioinformatic reconstruction of protein sequence;
  8. Prof. Lloyd Ruddock, Prof. Johanna Myllyharju, Prof. Aki Manninen, Prof. Lari Lehtiö in University of Oulu for biochemistry of pepetide, protein and their post-translational modifications.