Session: 16-01-01: Government Agency Student Poster Competition

Paper Number: 144088

144088 - Expanding the Sensing Resolution and Analyte Range for De Novo Peptide and Protein Sequencing via Ångström-Precise Nanoporation in Atomically-Thin 2d Dielectrics 

Many diseases including cancer, diabetes, and Alzheimer’s are caused by faulty transcription from DNA. Since proteins, as the ultimate product of transcription and translation, drive all cellular functions, it's crucial to monitor the abnormality of gene expression and the fluctuation of certain proteins’ level for early detection of adverse health conditions and for precision medicine. 

Traditional protein sequencing, for example, via mass spectrometry (MS), are restricted to high-abundance proteins. However, by bypassing the challenge of amplifying protein, sequencing via nanopore translocation paves the way for reduced sample size or even single-cell proteomics. This undoubtedly provides a powerful tool for identifying the proteomic signatures underlying specific pathology.

By guiding the peptide chain through a nano-scale orifice and discerning the sequential translocation events indicated by the modulation of the ionic current through the nanopore sensor, nanopore sequencing offers the advantages of real-time analysis and extensive read lengths, positioning it as a powerful next-generation sequencing approach.

1. Motivation and purpose of the research

Bio-nanopores and solid-state nanopores have thus far proven their capacity in detecting non-dehydrated amino acids, point mutations, and post-translational modifications. However, their potential for reconstructing entire amino acid sequences without a priori reference library or proteome has not been fully realized. 

Hexagonal boron nitride (hBN), an atomically-thin 2D dielectric crystal, is a highly promising material platform for achieving unprecedented resolution in nanopore sequencing. The monolayer thickness of hBN is approximately 3.3 ångström (Å), which is less than half the size of even the smallest amino acids such as alanine, glycine, and serine. By using 2D hBN, robust nanopores can be made so tiny as to accommodate only a single amino acid at a time. In this case, the ionic current level detected at each translocation event only reveals the information from that single amino acid, which greatly simplifies the signal deconvolution process towards single-molecule sequencing resolution.

2. Preliminary results and methodology

In this study, we explored nanopores made in monolayer and bilayer hBN by controlled dielectric breakdown (CBD). CBD facilitates ångström-precise control over nanopore dimensions, enabling the fabrication of a diverse range of nanopore morphologies. CBD-fabricated nanopores enable the sensing of small amino acid molecules such as glycine and whole folded proteins like bovine serum albumin (BSA). Leveraging a hybrid neural network model, our nanopore sequencing platform achieves de novo sequencing of short peptide chains down to single amino acid resolution.

3. Conclusions and contribution

This study revealed the viability of achieving ångström-precise nanopore fabrication in atomically-thin hBN using CBD. The hBN nanopore not only demonstrates the ability to detect biomolecules of diverse sizes but also accurately deciphers the sequence of short peptide chains with single amino acid resolution. In the future, hBN nanopore devices hold promise for analyzing samples with complex compositions, such as single-cell proteome and diverse protein samples like serum, saliva, or urine, containing numerous protein biomarkers indicative of various health conditions.

Presenting Author: Darley (Daiyue) Wei University of South Florida

Presenting Author Biography: Darley (Daiyue) Wei is currently pursuing her PhD in the Department of Medical Engineering and serving as a research assistant in the Department of Mechanical Engineering as well as a graduate assistant in the Nanotechnology Research & Education Center (NREC) at the University of South Florida, Tampa, FL, USA. Her research focuses primarily on two-dimensional (2D) materials processing and nanopore sequencing. She obtained her B.S. degree from China University of Petroleum, Qingdao, China, in 2017, followed by her M.S. degree from South China University of Technology, Guangzhou, China, in 2020. Prior to USF, she worked as a research assistant in the Department of Mechanical and Energy Engineering at Southern University of Science and Technology, Shenzhen, China, from 2020 to 2022.

Authors:

Darley (Daiyue) Wei University of South Florida
Zhewen Yin University of South Florida
Ossie Douglas University of South Florida
Muhammad Shahbaz Rafique University of South Florida
Ashley Valestin University of South Florida
Michael Cai Wang University of South Florida