Active Analyte Import Improves the Dynamic Range and Sensitivity of a Vitamin B₁₂ Biosensor

Area of Science:

• Biotechnology
• Biosensor Development
• Molecular Diagnostics

Background:

• Cell-free systems offer a versatile platform for low-cost biosensor development.
• Improving sensor affinity for enhanced sensitivity in biosensing remains a challenge.
• Vitamin B12 is a critical micronutrient, necessitating sensitive detection methods.

Purpose of the Study:

• To develop a sensitive biosensor for vitamin B12 detection.
• To investigate the sensitivity differences between cell-free and whole-cell biosensor systems.
• To establish a field-deployable diagnostic tool for vitamin B12.

Main Methods:

• Utilized a vitamin B12-responsive transcription factor for B12-dependent output.
• Compared sensor performance in cell-free reactions versus whole-cell systems.
• Investigated the role of regulated import in enhancing cytoplasmic B12 accumulation.
• Overexpressed importers to further improve sensor sensitivity.

Main Results:

• Cell-free biosensor showed limited sensitivity, responding only to high vitamin B12 concentrations.
• Whole-cell biosensors exhibited significantly higher sensitivity to vitamin B12.
• Regulated cellular import was identified as a key factor in enhanced sensitivity.
• Overexpression of importers further boosted sensitivity in whole-cell systems.

Conclusions:

• Whole-cell biosensors possess inherent advantages over cell-free systems for sensitive analyte detection.
• The developed whole-cell biosensor is responsive to vitamin B12 in serum and can be lyophilized.
• This technology holds potential for sensitive, field-deployable diagnostics in resource-limited settings.