Enhanced protein loading on a planar Si(111)–H surface with second generation NTA

A Si(111)–H surface was modified via a direct reaction between Si–H and 1-undecylenic acid (UA) under microwave irradiation to form molecular monolayers with terminal carboxyl groups. After esterifying carboxylic acid being esterified with N-hydroxysuccinimide (NHS), aminobutyl nitrilotriacetic acid (ANTA) was bound to the silicon surface through amidation (pH = 8.0) between its primary amino group and NHS-ester, producing nitrilotriacetic acid (NTA) anions. Then hexa-histidine tagged thioredoxin–urodilatin (his-tagged protein) and FITC-labeled hexa-histidine tagged thioredoxin–urodilatin (FITC-his-tagged protein) can be anchored after NTA was coordinated with Ni2+. Furthermore, the NTA-terminated chip was acidified with 0.1 M HCl and subsequently esterified with NHS and then amidated with ANTA again to produce a second generation NTA. Thus the surface density of nitrilotriacetic acid anions was improved and resultantly that of anchored proteins was also enhanced through the iterative reactions. Both multiple transmission-reflection infrared spectroscopy (MTR-IR) and fluorescence scanning measurements demonstrated a proximate 1.63 times of anchored proteins on the second generation NTA/Ni2+ as that on the first generation NTA/Ni2+ monolayer.