Hydrogen Peroxide → N-(tert-Buloxycarbonyl)-3,4-dihydroxy-L-phenylalanine

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Before the surface modification of silicon nitride (Si3N4) tips, cleaning procedures were performed using O2 plasma (name of a machine) for 3 min and subsequently they were transferred to a piranha solution (sulfuric acid:H2O2=8:2) for 30 min. They moved to 20% (v/v) 3-aminopropyltrimethoxysilane in toluene to be functionalized with amines for 30 to 60 min after rinsing with H2O. Two polyethylene glycol (PEG) derivatives were chosen for PEGylation on the AFM tips: mPEG-N-hydroxy succinimide (NHS) (Mw 2000) and Fmoc-PEG-NHS (Mw 3400) (Nektar Inc.). Mixture (Fmoc-PEG-NHS:mPEGNHS=1:5-10, 5 mM) of PEGs were prepared in 50 mM sodium phosphate buffer, 0.6 M K2SO4, pH 7.8 and chloroform. PEGylation reactions were conducted sequentially first in a sodium phosphate buffer at 40° C. and subsequently in chloroform for 3 hrs in each step. The reason to use PEG mixture is to prevent multiple DOPA binding on TiO2. Fmoc-PEG-NHS provides an amine for Boc-DOPA conjugation after Fmoc cleavage.Piperidine (20% v/v in NMP) was used to deprotect Fmoc for 5 min and subsequently cantilevers were transferred to BOP/HOBt/DOPA (a molar ratio of 1:1:1, final 8 mM in NMP) solution with 10 μL DIPEA. The same procedure was used for Tyrosine modification.

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