On-line green fluorescent protein sensor with LED excitation

Area of Science:

• Biotechnology
• Biophysics
• Analytical Chemistry

Background:

• Green fluorescent protein (GFP) is a vital reporter for monitoring gene expression.
• Accurate, real-time quantification of protein expression is crucial for bioprocess optimization.
• Existing methods for monitoring protein expression can be time-consuming and labor-intensive.

Purpose of the Study:

• To develop and validate an intensity-based sensor for on-line monitoring of green fluorescent protein (GFP) expression.
• To assess the sensor's performance in a real-time fermentation process.
• To compare the sensor's on-line data with traditional off-line measurement techniques.

Main Methods:

• An intensity-based sensor utilizing a blue light emitting diode and optical fiber bundle was designed.
• Steam-sterilizable components (fiber bundle, stainless steel housing with quartz window) were incorporated.
• Long-pass and band-pass filters were used to isolate excitation and emission wavelengths.
• A photomultiplier tube detected fluorescence, with data recorded on a computer.
• The sensor was tested during an Escherichia coli fermentation expressing GFP.

Main Results:

• The on-line sensor signal closely paralleled off-line fluorescence spectrophotometer measurements.
• Western blot analysis confirmed the sensor's ability to quantitatively monitor GFP expression.
• The sensor provided continuous, real-time data throughout the fermentation process.

Conclusions:

• The developed sensor enables continuous and quantitative on-line monitoring of green fluorescent protein expression.
• This technology offers a significant advancement for real-time bioprocess control and optimization.
• The sensor's robust design and accuracy make it suitable for industrial fermentation applications.