Substrate monolayers as electrochemical sensing elements for alpha-chymotrypsin.

Abstract

A disulfide, which carried two L-phenylalanyl p-nitroanilide (Phe-pNA) moieties at both ends, was prepared by the coupling of 11,11'-dithiodiundecanoic acid (DTUA) with Phe-pNA. The compound obtained (DTUA-Phe-pNA) formed a self-assembled monolayer (SAM) on a gold electrode and vacuum-evaporated gold thin film as proven by cyclic voltammetry, and reflection absorption spectroscopy, respectively. Incubation of alpha-chymotrypsin with the SAM-modified electrode induced both a decrease in anodic and cathodic peak currents (-DeltaIa and -DeltaIc) and an increase in potential difference (DeltaEp) in the cyclic voltammogram of potassium ferricyanide as a probe, which suggested the attack of the enzyme at the amide group between Phe and pNA groups of the SAM, resulting in the formation of an intermediate. The linear relationship between the initial rate of increase in the amount of enzyme bound to the SAM and in both DeltaIa (absolute value of the decrease in anodic peak current) and DeltaEp values was confirmed by the quartz crystal microbalance method. The binding rate of the enzyme to the Phe-pNA SAM was dependent on the surface density of the Phe-pNA group in the SAM. The alpha-chymotrypsin-induced increases in the DeltaIa and DeltaEp values were inhibited by the addition of N-acetyl-D-phenylalanine methyl ester (N-Ac-D-Phe-OMe). In contrast with alpha-chymotrypsin, trypsin did not show a significant increase in the DeltaIa and DeltaEp values upon incubation with Phe-pNA-carrying SAM. These results could be attributed to the specific attack of alpha-chymotrypsin to the amide group in the SAM. The inhibition constant for N-Ac-D-Phe-OMe in the SAM system was quite similar to that in the free substrate system, showing that the enzymatic reaction above the SAM proceeds in a similar way to that in the homogeneous solution system.