Functional Nanochannels for Sensing Tyrosine Phosphorylation.

Area of Science:

• Biotechnology and Nanotechnology
• Molecular Biology and Biochemistry
• Cancer Research

Background:

• Tyrosine phosphorylation (pTyr) is a critical regulator of cellular signaling, governing cell functions, and its dysregulation is linked to diseases, particularly cancers.
• Accurate sensing of pTyr is vital for understanding disease mechanisms and developing targeted anticancer therapies, but it is challenging due to its similarity to serine (pSer) and threonine phosphorylation (pThr).
• Existing methods face difficulties in distinguishing pTyr from other phosphorylation types, hindering precise molecular diagnostics and therapeutic development.

Purpose of the Study:

• To develop a novel sensing platform capable of specifically detecting tyrosine phosphorylation (pTyr) in complex biological environments.
• To overcome the challenge of differentiating pTyr from pSer and pThr using a nanotechnology-based approach.
• To demonstrate the utility of the developed sensor for monitoring kinase activity and screening kinase inhibitors.

Main Methods:

• Modification of nanochannels with polyethylenimine-g-phenylguanidine (PEI-PG) to enhance interactions with phosphorylated residues.
• Utilizing the conformational change of PEI-PG upon binding to phosphorylated peptides, leading to an 'OFF-ON' ion flux signal.
• Implementing a logic operation with Ca2+ as an interferent to achieve specific pTyr sensing.

Main Results:

• The PEI-PG-modified nanochannels successfully distinguished phosphorylated peptides from non-modified peptides.
• The nanochannels demonstrated the ability to recognize peptides with pSer, pThr, or pTyr residues, and peptides with varying numbers of identical residues.
• Specific sensing of pTyr peptides was achieved with high accuracy, even in the presence of Ca2+ as an interferent, and real-time monitoring of c-Abl kinase activity on a peptide substrate was demonstrated.

Conclusions:

• Polyethylenimine-g-phenylguanidine (PEI-PG)-modified nanochannels offer a robust and specific platform for sensing tyrosine phosphorylation (pTyr).
• The developed sensing technology enables precise detection of pTyr peptides in biosamples and real-time monitoring of kinase activity.
• This approach holds significant potential for advancing cancer diagnostics, understanding disease states, and facilitating the screening of kinase inhibitors.