In situ fabrication of cleavable peptide arrays on polydimethylsiloxane and applications for kinase activity assays

Analytica Chimica Acta
Huang-Han ChenShu-Hui Chen

Abstract

Polydimethylsiloxane (PDMS) is widely used for microfabrication and bioanalysis; however, its surface functionalization is limited due to the lack of active functional groups and incompatibility with many solvents. We presented a novel approach for in situ fabrication of cleavable peptide arrays on polydimethylsiloxane (PDMS) viatert-butyloxycarbonyl (t-Boc)/trifluoroacetic acid (TFA) chemistry using gold nanoparticles (AuNPs) as the anchor and a disulfide/amine terminated hetero-polyethylene glycol as the cleavable linker. The method was fine tuned to use reagents compatible with the PDMS. Using 5-mer pentapeptide, Trp5, as a model, step-by-step covalent coupling during the reaction cycles was monitored by Attenuated total reflectance-Fourier transform infrared spectrometer (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), or atomic force microscopy (AFM), and further confirmed by mass spectrometry (MS) detection of the cleaved peptides. Using such a method, heptapeptides of the PKA substrate, LRRASLG (Kemptide), and its point mutated analogs were fabricated in an array format for comparative studies of cAMP-dependent protein kinase (PKA) activity. Based on on-chip detection, Kemptide sequence exhibited the highest phosphory...Continue Reading

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Citations

Jan 5, 2019·ACS Applied Materials & Interfaces·Tzu-Yi HungJie-Ren Li
Dec 12, 2020·Analytical Chemistry·Yuri M ShlyapnikovElena A Shlyapnikova

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