Self-Assembled Monolayer of a Pepstatin Fragment as a Sensing Element for Aspartyl Proteases

Abstract

A novel disulfide, which carried two pepstatin fragments at both ends, was prepared by the coupling of 11,11'-dithiobisundecanoic acid (DTUA) with a fragment (Val-Val-Sta) carrying a n-hexyl end (Pepsta(h)). The compound obtained (DTUA-Pepsta(h)) formed a self-assembled monolayer (SAM) on a gold electrode and vacuum-evaporated gold thin film as proven by cyclic voltammetry and reflection absorption infrared spectroscopy, respectively. When the SAM-modified gold electrode was incubated with a solution of aspartyl protease, pepsin, a decrease in both anodic and cathodic peak currents and an increase in potential difference were observed in the cyclic voltamogram of hydroquinone as a probe, whereas a coexistence of free pepstatin fragment inhibited these phenomena, indicating the specific binding of pepsin to the fragment at the exterior of the SAM. The binding rate of the enzyme to the SAM was largely dependent on the surface density of the fragment moiety in the SAM. Furthermore, when the SAM of DTUA-Pepsta(h) on a gold colloid array deposited on an amino group-modified glass plate was immersed in a pepsin solution, absorption of the glass plate at 550 nm corresponding to a localized surface plasmon resonance of the gold colloid abruptly increased and slightly red-shifted, and a further addition of pepstatin A gradually decreased the absorbance. From the increasing and decreasing profiles of absorbance, the association constant (K(assoc)) for pepsin with the fragment on the SAM was determined. Similar phenomena were observed upon immersion of the fragment-modified SAM in a solution of HIV-1 protease, suggesting a usability of the pepstatin fragment SAM for the detection and removal of the enzyme from biological fluids.

Carboxybetaine Polymer‐Protected Gold Nanoparticles: High Dispersion Stability and Resistance against Non‐Specific Adsorption of Proteins

“…[72] The detection of protease, lectin, glycoprotein, etc., was also examined with the help of LSPR. [73][74][75][76][77] In this section, the LSPR technique was adopted to follow the adsorption of proteins to the surface of polymer brushes (PCMB and PMMA) on the gold colloid. Figure 3 clearly indicates that BSA, HSA, lysozyme, and cytochrome c did not adsorb to the surface of the PCMB brush significantly, whereas substantially adsorbed to the surface of both bare gold colloid and PMMA brush.…”

Cleavage-Sensing Redox Peptide Monolayers for the Rapid Measurement of the Proteolytic Activity of Trypsin and α-Thrombin Enzymes

“…To date, electrochemical detection methods are one of the rare methodological approaches able to meet the ongoing health-care market requirements for molecular diagnostics testing in blood in a sensitive, rapid, cost-effective, compact, easy-to-use format. Applications are exemplified in the established field of blood glucose monitoring and in the emerging area of electrochemical peptide-based sensors for protease activity monitoring. − …”

Kinetic study on the binding of lectin to mannose residues in a polymer brush

“…[20][21][22][23][24][25]. For the detection of enzymes, a SAM of a specific substrate or inhibitor of the enzymes was also constructed on a gold electrode [26,27]. * Moreover, -mercapto-polymer chain with many pendent glucose residues were accumulated on silver colloids as a "polymer brush" [28].…”