Phenol red-silk tyrosine cross-linked hydrogels.

Area of Science:

• Biomaterials Science
• Biochemistry
• Cell Biology

Background:

• Phenol red is a pH indicator used in cell culture media but can interfere with estrogen signaling and live cell imaging.
• Existing methods for incorporating phenol red into hydrogels lack covalent binding, leading to leakage and unreliable pH sensing.
• There is a need for cytocompatible, degradable pH sensors for real-time monitoring in cell culture environments.

Purpose of the Study:

• To develop a cytocompatible and degradable hydrogel system for pH sensing using phenol red covalently linked to silk tyrosine.
• To overcome the limitations of phenol red leaching and spectral interference in cell culture applications.
• To create a novel sensor for real-time pH monitoring in cellular microenvironments and bioreactors.

Main Methods:

• Silk tyrosine was cross-linked with phenol red using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2).
• Phenol red entrapment and covalent bonding were confirmed using diffusion studies, UV-Vis spectroscopy, and LC-MS analysis.
• Cytocompatibility was assessed via live-dead staining and alamar blue assays with encapsulated fibroblasts.

Main Results:

• Phenol red was successfully and covalently incorporated into silk-tyrosine hydrogels, forming leak-proof sensors at lower concentrations.
• The developed hydrogels exhibited pH-dependent spectral changes and were cytocompatible with encapsulated cells.
• The sensor demonstrated dual absorbance and fluorescence-based pH sensing capabilities with an average pKa of 6.8.

Conclusions:

• Phenol red-silk tyrosine hydrogels offer a simple, reliable, and cytocompatible solution for pH sensing in phenol red-free cell culture systems.
• These novel hydrogels can be utilized for real-time pH monitoring in cellular microenvironments and bioreactors.
• The covalent linkage strategy overcomes previous limitations, enabling advanced applications in cell biology and tissue engineering.