Phosphate binding protein as the biorecognition element in a biosensor for phosphate

Area of Science:

• Biochemistry and Biotechnology
• Biosensor Development
• Analytical Chemistry

Background:

• Inorganic phosphate (Pi) is crucial in biological systems, necessitating accurate detection methods.
• Periplasmic binding proteins (PBPs) offer high specificity for target analyte recognition.
• The phosphate binding protein (PBP) from Escherichia coli naturally binds and translocates Pi.

Purpose of the Study:

• To engineer and develop a sensitive and selective biosensor for inorganic phosphate (Pi) detection.
• To utilize a modified phosphate binding protein (PBP) as the core biorecognition element.
• To demonstrate the sensor's applicability across different analytical platforms.

Main Methods:

• Site-directed mutagenesis was used to create a cysteine-containing mutant PBP (MPBP).
• MPBP was expressed in E. coli, purified, and site-specifically labeled with a fluorophore (MDCC).
• Fluorescence intensity changes of the MPBP-MDCC conjugate upon Pi binding were measured using spectrofluorometry, microtiter plates, and fiber optics.

Main Results:

• The MPBP-MDCC conjugate exhibited a detectable change in fluorescence intensity upon Pi binding.
• Calibration curves demonstrated a linear relationship between fluorescence intensity and Pi concentration.
• The developed sensing system achieved sub-micromolar detection limits for Pi across all tested platforms and showed selectivity over similar anions.

Conclusions:

• A novel fluorescence-based biosensor for inorganic phosphate was successfully developed using a modified periplasmic binding protein.
• The PBP-based sensor offers high sensitivity, selectivity, and versatility, adaptable to microtiter plate and fiber optic formats.
• This work paves the way for a new generation of biosensors leveraging the specific binding properties of periplasmic proteins for various analytes.