Rational design of direct electron transfer type l-lactate dehydrogenase for the development of multiplexed biosensor

Area of Science:

• Biomedical Engineering
• Electrochemistry
• Enzyme Engineering

Background:

• Wearable biosensors are crucial for real-time metabolite monitoring, particularly in sweat.
• Direct electron transfer (DET) principles are key for efficient biosensing.
• Developing multiplexed sensors for simultaneous detection of multiple analytes remains a challenge.

Purpose of the Study:

• To construct a fusion enzyme for multiplexed direct electron transfer (DET)-type lactate and glucose sensors.
• To enhance the operational range and performance of the lactate sensor.
• To develop and evaluate a simultaneous lactate and glucose monitoring system for wearable applications.

Main Methods:

• Engineered a fusion enzyme (AvLOx A96L/N212K mutant and b-type cytochrome) for lactate sensing.
• Introduced mutations to create "b2LOxS" for improved lactate detection range and substrate inhibition elimination.
• Developed a multiplexed sensor system using flexible thin-film electrodes with b2LOxS and glucose dehydrogenase.

Main Results:

• The fusion enzyme demonstrated DET to a gold electrode but had a limited operational range (<0.5 mM).
• The b2LOxS enzyme, with an outer membrane, expanded the lactate detection range up to 10 mM.
• The dual sensor system achieved simultaneous lactate (0.5-20 mM) and glucose (0.1-5 mM) detection without cross-talk, with high sensitivity and low limits of detection.
• Electrochemical interferants showed negligible impact on sensor performance.

Conclusions:

• A novel DET-type enzyme-based dual sensing system for simultaneous lactate and glucose detection was successfully developed.
• The engineered fusion enzyme and multiplexed electrode system offer a promising platform for wearable sweat analysis.
• This work represents the first report of a DET-type enzyme-based dual sensing system for lactate and glucose.