Protein-based biosensors for diabetic patients

Area of Science:

• Biochemistry
• Biosensor Technology
• Analytical Chemistry

Background:

• Developing stable and non-consuming biosensors is crucial for accurate glucose monitoring.
• Enzymes and proteins are promising probes for fluorescence biosensing applications.
• Traditional glucose sensors often face limitations such as consumption of the analyte or instability.

Purpose of the Study:

• To develop a new methodology for glucose sensing using inactive enzyme forms.
• To create stable and non-consuming fluorescence biosensors for glucose detection.
• To explore the potential of apo-enzymes as reversible sensors for various analytes.

Main Methods:

• Utilized inactive glucose oxidase (apo-GOx), glucose dehydrogenase (apo-GDH), and glucokinase (apo-GK).
• Employed fluorescence spectroscopy to monitor changes upon glucose binding to apo-enzymes.
• Developed a fluorescence competitive assay using thermostable apo-glucokinase.

Main Results:

• Apo-glucose oxidase showed decreased tryptophan fluorescence upon glucose binding.
• 8-Anilino-1-naphthalene sulfonic acid (ANS) fluorescence intensity decreased by 25% upon glucose binding to apo-GOx.
• ANS-labeled apo-GDH also exhibited a 25% decrease in emission intensity with glucose binding.
• Apo-GDH demonstrated stability in organic solvents, enabling glucose detection in acetone.
• A fluorescence competitive assay for glucose using apo-GK was successfully developed.

Conclusions:

• Inactive apo-enzymes can function as reversible, non-consuming glucose biosensors without co-factors.
• The use of apo-enzymes significantly broadens the scope of proteins applicable for sensing.
• This approach is extendable for the detection of a wide range of biochemically relevant analytes beyond glucose.