Label-free electronic detection of peptide post-translational modification with functional enzyme-driven assay at the physical limit

Eleonora Macchia; Kim Björkström; Amit Tewari; Ville Eskonen; Axel Luukkonen; Amir Mohammad Ghafari; Lucia Sarcina; Mariapia Caputo; Natalia Tong-Ochoa; Kari Kopra; Fredrik Pettersson; Zahra Gounani; Luisa Torsi; Harri Härmä; Ronald Österbacka

doi:10.1016/j.xcrp.2024.101874

Label-free electronic detection of peptide post-translational modification with functional enzyme-driven assay at the physical limit

Eleonora Macchia
, Kim Björkström
, Amit Tewari
, Ville Eskonen
, Axel Luukkonen
, Amir Mohammad Ghafari
, Lucia Sarcina
, Mariapia Caputo
, Natalia Tong-Ochoa
, Kari Kopra
, Fredrik Pettersson
, Zahra Gounani
, Luisa Torsi
, Harri Härmä^*
, Ronald Österbacka^*

^*Corresponding author for this work

Physics

Research output: Contribution to journal › Article › Scientific › peer-review

2 Citations (Scopus)

33 Downloads (Pure)

Abstract

High-performance, ultra-sensitive, and universal protein post-translational modification (PTM) and protein-protein interaction (PPI) technologies are eagerly pursued in the pharmaceutical industry and bioanalytical research. Novel PTM and PPI detection methods outperform traditional assays in scope and scalability, enabling the collection of information on multiple biochemical targets. Detecting peptides and proteins at the single-molecule level is done by utilizing nanosized transducing elements and assaying solutions at very high analyte concentrations, in the nanomolar range or higher. Here, a proof of principle of a biosensing platform for single-molecule PTM detection is demonstrated. This platform is based on the single molecule with a large transistor (SiMoT) technology, encompassing a millimeter-sized electrolyte-gated organic field-effect transistor, for label-free PTM detection with a zeptomolar limit of detection. Sensitivity is improved 10⁶- to 10¹²-fold compared with mass-spectrometry and luminescence-based assay methods. A functional assay for detecting enzyme-driven peptide PTMs in the zeptomolar concentration range is demonstrated using multivariate data processing, opening the way for future applications to monitor PTMs.

Original language	English
Article number	101874
Journal	Cell Reports Physical Science
Volume	5
Issue number	8
DOIs	https://doi.org/10.1016/j.xcrp.2024.101874
Publication status	Published - 21 Aug 2024
MoE publication type	A1 Journal article-refereed

Funding

The authors acknowledge Prof. Carl-Eric Wil\u00E9n and Ms. Pernilla Sund for useful discussions in this work's early stages. The following are all acknowledged for partial financial support: Academy of Finland projects 296225, 323433, 269973, 316881, 316883, 332106, 296093, and 270010; PON SISTEMA (MIUR); H2020, Electronic Smart Systems, SiMBiT (single-molecule bioelectronic smart system array for clinical testing) (grant agreement ID: 824946); Doctoral Network of Materials Research at \u00C5bo Akademi University; NoOne (a binary sensor with a single-molecule digit to discriminate biofluids enclosing zero or at least one biomarker), ERC Stg2021, GA:101040383; I primi mille clinical trial con il Centro DIGITAL ASSAY, Centro di Innovazione Regionale Digital Assay, Presidente Prof. L. Torsi; Responsabile Scientifico UNIBA E. Macchia Regione PUGLIA Delibera Regionale n 702 del 08/11/2022 (CUP B93C22000840001); PMGB \u2013 Sviluppo di piattaforme meccatroniche, genomiche e bioinformatiche per l'oncologia di precisione and ARS01_01195 \u2013 PON RICERCA E INNOVAZIONE 2014\u20132020 projects; Italian network of excellence for advanced diagnosis (INNOVA), Ministero della Salute -code PNC-E3-2022-23683266 PNC-HLS-DA, CUP: C43C22001630001; Complementary National Plan PNC-I.1 research initiatives for innovative technologies and pathways in the health and welfare sector D.D. 931 of 06/06/2022; DARE (DigitAl lifelong pRevEntion initiative), code PNC0000002, CUP: B53C22006420001; \u00C5bo Akademi University CoE bioelectronic activation of cell functions; CSGI - Center for Colloid and Surface Science; Vilho, Yrj\u00F6, and Kalle V\u00E4is\u00E4l\u00E4 Foundation; and the Drug Research Doctoral Programme (University of Turku Graduate School). Conceptualization, L.T. H.H. and R.\u00D6.; methodology, V.E. N.T.-O. K.K. L.T. H.H. and R.\u00D6.; validation, E.M. K.B. A.T. and F.P.; formal analysis, E.M. K.B. A.T. V.E. A.L. and F.P.; investigation, E.M. K.B. A.T. V.E. A.M.G. L.S. M.C. F.P. and Z.G.; resources, V.E. A.M.G. L.S. M.C. N.T.-O. K.K. and Z.G.; data curation, E.M. and A.T.; writing \u2013 original draft and review & editing, E.M. K.B. L.T. H.H. and R.\u00D6.; visualization, E.M. K.B. and A.T.; supervision, L.T. H.H. and R.\u00D6.; project administration, L.T. H.H. and R.\u00D6. The authors declare no competing interests. The authors acknowledge Prof. Carl-Eric Wil\u00E9n and Ms. Pernilla Sund for useful discussions in this work\u2019s early stages. The following are all acknowledged for partial financial support: Academy of Finland projects 296225, 323433, 269973, 316881, 316883, 332106, 296093, and 270010; PON SISTEMA ( MIUR ), H2020, Electronic Smart Systems, SiMBiT (single-molecule bioelectronic smart system array for clinical testing) (grant agreement ID: 824946 ); Doctoral Network of Materials Research at \u00C5bo Akademi University , NoOne (a binary sensor with a single-molecule digit to discriminate biofluids enclosing zero or at least one biomarker), ERC Stg2021 , GA: 101040383 ; I primi mille clinical trial con il Centro DIGITAL ASSAY, Centro di Innovazione Regionale Digital Assay ; Presidente Prof. L. Torsi; Responsabile Scientifico UNIBA E. Macchia Regione PUGLIA Delibera Regionale n 702 del 08/11/2022 ( CUP B93C22000840001 ); PMGB \u2013 Sviluppo di piattaforme meccatroniche , genomiche e bioinformatiche per l\u2019oncologia di precisione and ARS01_01195 \u2013 PON RICERCA E INNOVAZIONE 2014\u20132020 projects; Italian network of excellence for advanced diagnosis ( INNOVA ), Ministero della Salute -code PNC-E3-2022-23683266 PNC-HLS-DA , CUP: C43C22001630001 ; Complementary National Plan PNC-I.1 research initiatives for innovative technologies and pathways in the health and welfare sector D.D. 931 of 06/06/2022; DARE ( DigitAl lifelong pRevEntion initiative ), code PNC0000002 , CUP: B53C22006420001 ; \u00C5bo Akademi University CoE bioelectronic activation of cell functions , CSGI ; Vilho , Yrj\u00F6 , and Kalle V\u00E4is\u00E4l\u00E4 Foundation ; and the Drug Research Doctoral Programme (University of Turku Graduate School).

Keywords

multivariate data processing
organic bioelectronics
peptide detection
SiMoT
single-molecule assay with large transistors
single-molecule detection

Access to Document

10.1016/j.xcrp.2024.101874Licence: CC BY

1-s2.0-S2666386424001103-mainFinal published version, 3.83 MBLicence: CC BY

https://urn.fi/URN:NBN:fi-fe2024090969798

Cite this

Macchia, E., Björkström, K., Tewari, A., Eskonen, V., Luukkonen, A., Ghafari, A. M., Sarcina, L., Caputo, M., Tong-Ochoa, N., Kopra, K., Pettersson, F., Gounani, Z., Torsi, L., Härmä, H., & Österbacka, R. (2024). Label-free electronic detection of peptide post-translational modification with functional enzyme-driven assay at the physical limit. Cell Reports Physical Science, 5(8), Article 101874. https://doi.org/10.1016/j.xcrp.2024.101874

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abstract = "High-performance, ultra-sensitive, and universal protein post-translational modification (PTM) and protein-protein interaction (PPI) technologies are eagerly pursued in the pharmaceutical industry and bioanalytical research. Novel PTM and PPI detection methods outperform traditional assays in scope and scalability, enabling the collection of information on multiple biochemical targets. Detecting peptides and proteins at the single-molecule level is done by utilizing nanosized transducing elements and assaying solutions at very high analyte concentrations, in the nanomolar range or higher. Here, a proof of principle of a biosensing platform for single-molecule PTM detection is demonstrated. This platform is based on the single molecule with a large transistor (SiMoT) technology, encompassing a millimeter-sized electrolyte-gated organic field-effect transistor, for label-free PTM detection with a zeptomolar limit of detection. Sensitivity is improved 106- to 1012-fold compared with mass-spectrometry and luminescence-based assay methods. A functional assay for detecting enzyme-driven peptide PTMs in the zeptomolar concentration range is demonstrated using multivariate data processing, opening the way for future applications to monitor PTMs.",

keywords = "multivariate data processing, organic bioelectronics, peptide detection, SiMoT, single-molecule assay with large transistors, single-molecule detection",

author = "Eleonora Macchia and Kim Bj{\"o}rkstr{\"o}m and Amit Tewari and Ville Eskonen and Axel Luukkonen and Ghafari, \{Amir Mohammad\} and Lucia Sarcina and Mariapia Caputo and Natalia Tong-Ochoa and Kari Kopra and Fredrik Pettersson and Zahra Gounani and Luisa Torsi and Harri H{\"a}rm{\"a} and Ronald {\"O}sterbacka",

note = "Publisher Copyright: {\textcopyright} 2024 The Author(s)",

year = "2024",

month = aug,

day = "21",

doi = "10.1016/j.xcrp.2024.101874",

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Macchia, E, Björkström, K, Tewari, A, Eskonen, V, Luukkonen, A, Ghafari, AM, Sarcina, L, Caputo, M, Tong-Ochoa, N, Kopra, K, Pettersson, F, Gounani, Z, Torsi, L, Härmä, H & Österbacka, R 2024, 'Label-free electronic detection of peptide post-translational modification with functional enzyme-driven assay at the physical limit', Cell Reports Physical Science, vol. 5, no. 8, 101874. https://doi.org/10.1016/j.xcrp.2024.101874

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AU - Macchia, Eleonora

AU - Björkström, Kim

AU - Tewari, Amit

AU - Eskonen, Ville

AU - Luukkonen, Axel

AU - Ghafari, Amir Mohammad

AU - Sarcina, Lucia

AU - Caputo, Mariapia

AU - Tong-Ochoa, Natalia

AU - Kopra, Kari

AU - Pettersson, Fredrik

AU - Gounani, Zahra

AU - Torsi, Luisa

AU - Härmä, Harri

AU - Österbacka, Ronald

PY - 2024/8/21

Y1 - 2024/8/21

N2 - High-performance, ultra-sensitive, and universal protein post-translational modification (PTM) and protein-protein interaction (PPI) technologies are eagerly pursued in the pharmaceutical industry and bioanalytical research. Novel PTM and PPI detection methods outperform traditional assays in scope and scalability, enabling the collection of information on multiple biochemical targets. Detecting peptides and proteins at the single-molecule level is done by utilizing nanosized transducing elements and assaying solutions at very high analyte concentrations, in the nanomolar range or higher. Here, a proof of principle of a biosensing platform for single-molecule PTM detection is demonstrated. This platform is based on the single molecule with a large transistor (SiMoT) technology, encompassing a millimeter-sized electrolyte-gated organic field-effect transistor, for label-free PTM detection with a zeptomolar limit of detection. Sensitivity is improved 106- to 1012-fold compared with mass-spectrometry and luminescence-based assay methods. A functional assay for detecting enzyme-driven peptide PTMs in the zeptomolar concentration range is demonstrated using multivariate data processing, opening the way for future applications to monitor PTMs.

AB - High-performance, ultra-sensitive, and universal protein post-translational modification (PTM) and protein-protein interaction (PPI) technologies are eagerly pursued in the pharmaceutical industry and bioanalytical research. Novel PTM and PPI detection methods outperform traditional assays in scope and scalability, enabling the collection of information on multiple biochemical targets. Detecting peptides and proteins at the single-molecule level is done by utilizing nanosized transducing elements and assaying solutions at very high analyte concentrations, in the nanomolar range or higher. Here, a proof of principle of a biosensing platform for single-molecule PTM detection is demonstrated. This platform is based on the single molecule with a large transistor (SiMoT) technology, encompassing a millimeter-sized electrolyte-gated organic field-effect transistor, for label-free PTM detection with a zeptomolar limit of detection. Sensitivity is improved 106- to 1012-fold compared with mass-spectrometry and luminescence-based assay methods. A functional assay for detecting enzyme-driven peptide PTMs in the zeptomolar concentration range is demonstrated using multivariate data processing, opening the way for future applications to monitor PTMs.

KW - multivariate data processing

KW - organic bioelectronics

KW - peptide detection

KW - SiMoT

KW - single-molecule assay with large transistors

KW - single-molecule detection

UR - https://www.scopus.com/pages/publications/85189680655

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DO - 10.1016/j.xcrp.2024.101874

M3 - Article

AN - SCOPUS:85189680655

SN - 2666-3864

VL - 5

JO - Cell Reports Physical Science

JF - Cell Reports Physical Science

IS - 8

M1 - 101874

ER -

Label-free electronic detection of peptide post-translational modification with functional enzyme-driven assay at the physical limit

Abstract

Funding

Keywords

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Åbo Akademi Functional Printing Center

BACE: Center of Excellence in Bioelectronic Activation of Cell Functions

SIMBIT: Single molecule bio-electronic smart system array for clinical testing

Cite this

Label-free electronic detection of peptide post-translational modification with functional enzyme-driven assay at the physical limit

Abstract

Funding

Keywords

Access to Document

Other files and links

Fingerprint

Equipment

Åbo Akademi Functional Printing Center

Projects

BACE: Center of Excellence in Bioelectronic Activation of Cell Functions

SIMBIT: Single molecule bio-electronic smart system array for clinical testing

Cite this