Electrochemical biosensor modified with dsDNA monolayer for restriction enzyme activity determination

Area of Science:

• Electrochemistry
• Molecular Biology
• Biosensor Technology

Background:

• Restriction endonucleases are crucial enzymes in molecular biology.
• Accurate determination of their activity is essential for various applications.
• Existing methods can be costly and complex.

Purpose of the Study:

• To develop a simple, cost-effective method for determining restriction endonuclease activity.
• To utilize a label-free electrochemical biosensor for enzyme activity assessment.
• To optimize the biosensor for sensitive and reliable measurements.

Main Methods:

• Immobilization of double-stranded DNA (dsDNA) on a gold electrode surface.
• Voltammetric measurements using an external cationic redox probe (methylene blue).
• Electrochemical impedance spectroscopy to assess monolayer stability.

Main Results:

• A decrease in current signal correlates with restriction endonuclease activity.
• Covalent attachment of label molecules is not required, reducing costs.
• Optimal conditions were established, avoiding surfactants and tuning sensitivity via restriction site location.
• Linear response to PvuII activity was observed from 0.25 to 1.50 U/μL.

Conclusions:

• The developed electrochemical biosensor offers a simple and cost-effective approach for restriction endonuclease activity determination.
• The label-free design simplifies the procedure and reduces analysis costs.
• The biosensor demonstrates tunable sensitivity and reliable performance for enzyme activity quantification.