Phosphate sensing by fluorescent reporter proteins embedded in polyacrylamide nanoparticles

Area of Science:

• Biotechnology
• Nanotechnology
• Biochemistry

Background:

• Fluorescent reporter proteins (FLIPPi) are valuable tools for biological sensing.
• Developing robust methods for protein stabilization and activity preservation is crucial for in vivo applications.
• Polyacrylamide nanoparticles offer a potential matrix for encapsulating sensitive biomolecules.

Purpose of the Study:

• To develop and optimize polyacrylamide nanoparticles for embedding fluorescent reporter proteins (FLIPPi) as phosphate sensors.
• To investigate the impact of nanoparticle composition on sensor activity and protein loading efficiency.
• To assess the protective effect of the nanoparticle matrix against protease degradation.

Main Methods:

• Synthesis of polyacrylamide nanoparticles with varying total monomer content (% T) and cross-linker content (% C).
• Embedding of fluorescent reporter proteins (FLIPPi) within the nanoparticle matrix.
• Characterization of nanoparticle size (40-120 nm), protein loading efficiency, and sensor activity.
• Evaluation of FLIPPi stability against soluble proteases within the nanoparticle matrix.

Main Results:

• Optimal nanoparticle composition identified as 28% T and 20% C, yielding high loading efficiency (50.6%) and protein activity (50%).
• The polyacrylamide matrix provided a degree of protection to FLIPPi against degradation by soluble proteases.
• Nanoparticle size ranged from 40 to 120 nm, influenced by composition.

Conclusions:

• Polyacrylamide nanoparticles are a promising platform for developing functional fluorescent biosensors.
• The developed embedding method offers a versatile approach for protecting and utilizing fragile bioactive elements in nanostructures.
• This technology holds potential for in vivo metabolite studies, biosensing, biological catalysis, and gene delivery applications.