Introduction:
Traditional protein sequencing methods, such as mass spectrometry, struggle with novel protein variants, isoforms, and the precise locations of post-translational modifications (PTMs). We propose a graphene-based sensing platform for single-point amino acid and PTM analysis, utilizing graphene field-effect transistors (GFETs) to capture the distinct electrochemical fingerprints of amino acids and their modifications.
Methods:
We modeled the electrochemical dynamics of amino acids during pH titration [1], leveraging their amphoteric nature to generate unique charge and capacitance profiles measured using GFETs. To validate our model, we started with optimizing a functionalization protocol for directional amino acid attachment to graphene, monitored via surface plasmon resonance (SPR). We developed three platforms to enable the process in GFETs: one for sensor preconditioning, another for functionalization with amino acids and peptides, and a third for pH titration-based amino acid fingerprint analysis using GFETs.
Results:
The coupling reactions with the graphene linker and directional amino acid attachment were monitored with single-layer precision. SPR results showed the regulation of the number of molecules, ensuring reproducible surface density control critical for the analysis of surface potential measurements. The functionalization chemistry also enables in situ peptide synthesis on graphene. Additionally, our platforms function effectively in both aqueous and organic solvents, and the Dirac point of graphene has been tracked in different conditions.
Conclusion:
This work presents a promising graphene-based framework for single-point protein sequencing, enabling detailed amino acid characterization. By optimizing protocols and developing platforms for consistent GFET measurements, we controlled molecular coverage for electrochemical profiling. The building blocks for this technology have been optimized for our platforms and can offer an alternative to traditional methods, with applications in proteomics, biomarker discovery, and diagnostics. Furthermore, our platforms and chemistry offer versatility to probe chemical affinity to peptides.
