SpyDirect: A Novel Biofunctionalization Method for High Stability and Longevity of Electronic Biosensors

Keying Guo, Raik Grünberg, Yuxiang Ren, Tianrui Chang, Shofarul Wustoni, Ondrej Strnad, Anil Koklu, Escarlet Díaz-Galicia, Jessica Parrado Agudelo, Victor Druet, Tania Cecilia Hidalgo Castillo, Maximilian Moser, David Ohayon, Adel Hama, Ashraf Dada, Iain McCulloch, Ivan Viola, Stefan T. Arold*, Sahika Inal*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Electronic immunosensors are indispensable tools for diagnostics, particularly in scenarios demanding immediate results. Conventionally, these sensors rely on the chemical immobilization of antibodies onto electrodes. However, globular proteins tend to adsorb and unfold on these surfaces. Therefore, self-assembled monolayers (SAMs) of thiolated alkyl molecules are commonly used for indirect gold–antibody coupling. Here, a limitation associated with SAMs is revealed, wherein they curtail the longevity of protein sensors, particularly when integrated into the state-of-the-art transducer of organic bioelectronics—the organic electrochemical transistor. The SpyDirect method is introduced, generating an ultrahigh-density array of oriented nanobody receptors stably linked to the gold electrode without any SAMs. It is accomplished by directly coupling cysteine-terminated and orientation-optimized spyTag peptides, onto which nanobody-spyCatcher fusion proteins are autocatalytically attached, yielding a dense and uniform biorecognition layer. The structure-guided design optimizes the conformation and packing of flexibly tethered nanobodies. This biolayer enhances shelf-life and reduces background noise in various complex media. SpyDirect functionalization is faster and easier than SAM-based methods and does not necessitate organic solvents, rendering the sensors eco-friendly, accessible, and amenable to scalability. SpyDirect represents a broadly applicable biofunctionalization method for enhancing the cost-effectiveness, sustainability, and longevity of electronic biosensors, all without compromising sensitivity.

Original languageEnglish (US)
JournalAdvanced Science
DOIs
StateAccepted/In press - 2023

Bibliographical note

Publisher Copyright:
© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.

Keywords

  • cysteine-peptide linker
  • nanobody
  • organic bioelectronics
  • organic electrochemical transistor
  • protein sensor
  • self-assembled monolayer

ASJC Scopus subject areas

  • Medicine (miscellaneous)
  • General Chemical Engineering
  • General Materials Science
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)
  • General Engineering
  • General Physics and Astronomy

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