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.
The pharmaceutical industry is comprised of a myriad of active pharmaceutical ingredients, all applied to increasing human health and quality of life. One of the requirements of reaping the health benefits of these drug products is maintaining them in a native or desired form. Such non-native forms can include, aggregated or fragmented structures. Unfortunately, the most widely used methods to detect non-native or denatured proteins require trained technicians, bulky instrumentation and large amounts of reagents. Deviation from the native structures can occur at all stages; from manufacturing and processing to storage. With these limitations in mind, a simplistic and highly sensitive in solution detection method was evaluated to visually detect denatured insulin proteins, utilizing gold nanoparticle aggregation via 3-Aminopropyltreithoxysilane. The insulin in this study was heat stressed using an 80 • C water bath to create an accelerated heat stressed environment. The insulin, gold nanoparticle and aminosilane solution was then characterized utilizing, UV-Vis spectroscopy, dynamic light scattering and scanning electron microscopy. Captured images and resulting absorbance spectra of the trials 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 proteins that have experienced heat stress.
The presence of denatured proteins within a therapeutic drug product can create a series of serious adverse effects, such as mild irritation, immunogenicity, anaphylaxis, or instant death to a patient. The detection of protein degradation is complicated and expensive due to current methods associated with expensive instrumentation, reagents, and processing time. We have demonstrated here a platform for visual biosensing of denatured proteins that is fast, low cost, sensitive, and user friendly by exploiting the plasmonic properties of noble metal nanoparticles. In this study we have exposed artificially heat stressed ferritin and gold nanoparticles to 3-aminopropyl triethoxysilane, which degrades the protein by showing a systematic blue shift in the absorbance spectra of the gold nanoparticle/ferritin and aminosilane solution. This blue shift in absorbance produces a detectable visual color transition from a blue color to a purple hue. By studying the Raman spectroscopy of the gold nanoparticle/ferritin and aminosilane solution, the extent of ferritin degradation was quantified. The degradation of ferritin was again confirmed using dynamic light scattering and was attributed to the aggregation of the ferritin due to accelerated heat stress. We have successfully demonstrated a proof of concept for visually detecting ferritin from horse spleen that has experienced various levels of degradation, including due to heat stress.
When administered to a patient, the presence of denatured proteins within a therapeutic drug product can induce a series of adverse effects ranging from mild irritation, immunogenicity, anaphylaxis, or instant death. Current methods to detect protein degradation are complicated and expensive due to costly instrumentation, reagents and technician processing time. Therefore this paper exploits plasmonic nanoparticles to create a platform for visual biosensing of denatured proteins that is fast, low cost, sensitive and user friendly. In this study artificially heat stressed ferritin and gold nanoparticles are exposed to 3-aminopropyl triethoxysilane. Upon degradation of the protein, systematic blue shifts were observed in the absorbance spectra of the gold nanoparticle/ferritin and aminosilane solution. The blue shifts in absorbance translate visually into a detectable color transition from a blue color to a purple hue. The extent of ferritin degradation was quantified using Raman spectroscopy. The mode of degradation was confirmed using dynamic light scattering and was attributed to aggregation of the ferritin due to accelerated heat stress. We have demonstrated a proof of concept which describes a system to visually detect ferritin from horse spleen that has experienced various levels of degradation. Keywords: Biosensor, Gold nanoparticles, Denatured Protein, Visual Detection, Ferritin, Degraded Protein Figure 1
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