Molecularly "wired" cholesterol oxidase for biosensing

Area of Science:

• Biochemistry
• Enzyme Engineering
• Biosensor Technology

Background:

• Cholesterol oxidase (ChOx) activity is crucial for biosensor applications.
• Modifying enzymes can enhance their stability and performance.
• Ionic liquids offer a greener alternative for chemical modifications.

Purpose of the Study:

• To investigate the influence of room temperature ionic liquids (RTILs) and flavin adenine dinucleotide (FAD) on cholesterol oxidase (ChOx) activity.
• To develop a molecular wiring strategy for ChOx using RTILs and FAD.
• To evaluate the performance of the modified enzyme (ME) in an amperometric cholesterol biosensor.

Main Methods:

• Transient exposure of ChOx to RTILs with varying FAD presence, water content, and FAD:RTIL ratios.
• Spectrophotometric measurement of enzyme activity before and after modification.
• Amperometric detection of cholesterol using the ME in a biosensor setup.

Main Results:

• The RTIL-based modification procedure, incorporating FAD, was enzyme- and environmentally-friendly.
• The modified enzyme (ME) retained a significant portion of FAD and initial activity.
• The ME demonstrated catalytic activity, linearity, and stability in cholesterol detection via amperometric biosensing.

Conclusions:

• FAD incorporation during RTIL exposure acts as a molecular wire, enhancing electron transfer.
• The developed molecular wiring technique offers a stable and efficient method for cholesterol biosensor development.
• This approach provides a greener and potentially more effective way to engineer enzymes for biosensing applications.