Visual Detection of Denatured Glutathione Peptides: A Facile Method to Visibly Detect Heat Stressed Biomolecules

Abstract

Every year pharmaceutical companies use significant resources to mitigate aggregation of pharmaceutical drug products. Specifically, peptides and proteins that have been denatured or degraded can lead to adverse patient reactions such as undesired immune responses. Current methods to detect aggregation of biological molecules are limited to costly and time consuming processes such as high pressure liquid chromatography, ultrahigh pressure liquid chromatography and SDS-PAGE gels. Aggregation of pharmaceutical drug products can occur during manufacturing, processing, packaging, shipment and storage. Therefore, a facile in solution detection method was evaluated to visually detect denatured glutathione peptides, utilizing gold nanoparticle aggregation via 3-Aminopropyltreithoxysilane. Glutathione was denatured using a 70 °C water bath to create an accelerated heat stressed environment. The peptide, gold nanoparticle and aminosilane solution was then characterized via, UV-Vis spectroscopy, FTIR spectroscopy, dynamic light scattering and scanning electron microscopy. Captured images and resulting absorbance spectra of the gold nanoparticle, glutathione, and aminosilane complex demonstrated visual color changes detectable with the human eye as a function of the denaturation time. This work serves as an extended proof of concept for fast in solution detection methods for glutathione peptides that have experienced heat stress.

Figure 1

Displays the UV-vis absorbance spectra for the gold nanoparticle and glutathione solutions in the presence of 0.1–0.2% APTES. (A) 3.07 mg/mL GSH. (B) 30.7 pg/mL GSH. Resulting trials hues from left to right: AuNps, AuNps/GSH, 0.1%, 0.12%, 0.14%, 0.16%, 0.18%, 0.2% (APTES). (C) 3.07 mg/mL GSH trial and (D) 30.7 pg/mL GSH trial.

Figure 2

Absorbance spectra of the AuNps, GSH, and APTES solutions over a 3.5 hour GSH peptide denaturation, via a 70 °C accelerated heat stress study. (A) 0.1% APTES. (B) 0.12% APTES. (C) 0.14% APTES. (D) 0.16% APTES. (E) 0.18% APTES. (F) Shows the change in the max peak wavelength as a function of denaturation time for each concentration of APTES.

Figure 3

Captured images of the AuNps/GSH/APTES solutions over a 3.5 hour GSH peptide denaturaation via 70 °C accelerated heat stress. From left to right: AuNps, 0.1% APTES, 0.12% APTES, 0.14% APTES, 0.16% APTES, 0.18% APTES. (A) 0 minute, (B) 1 hour, (C) 2.5 hours, (D) 3 hours, (E) 3.5 hours, and (F) 4 hours no APTES.

Figure 4

Size and size distribution plots of the AuNps/GSH/APTES solutions as determined by dynamic light scattering measurements. (A) Control consisting of just gold nanoparticles. (B) Gold nanoparticles and non-denatured glutathione complex. (C) Gold nanoparticles and non-denatured glutathione complex in the presence of 0.16% APTES. (D) Gold nanoparticles and denatured glutathione complex in the presence of 0.16% APTES.

Figure 5

SEM images of the gold nanoparticles, glutathione and 0.16% APTES trials on a p-type silicon. The solutions were mixed, then deposited using a drop cast method and allowed to dry overnight. GSH 2.5 hour heat stress trial (A) 20.0 um, (C), 3.0 um, (E) 400 nm, (F) 200 nm. GSH 0 hour heat stress trial (B) 20.0 um and (D) 3.0 um. The samples were imaged using the HITACHI FE-SEM SU8010 instrument at 10.0 kV accelerating voltage and 5 milliamps.

Figure 6

FTIR absorbance spectra of the glutathione peptide as a function of the accelerated heat stress exposure time.

Figure 7

Size and size distribution plots of the glutathione trial samples over a 3 hour accelerated heat stress study at 70 °C, as measured by dynamic light scattering measurements.

Figure 8

Proposed interactions of the citrate capped gold nanoparticles with denatured and non-denatured GSH in the presence of 0.1% APTES. The change in hue from the original red solution is modeled via gold nanoparticle clustering and aggregation as a function of the GSH accelerated heat stress.