An enzyme substrate binding aptamer complex based time-resolved fluorescence sensor for the adenosine deaminase...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Adenosine deaminase (ADA) is a crucial enzyme in purine metabolism.
• Accurate detection of ADA activity is vital for diagnosing various diseases.
• Existing methods for ADA detection may lack sensitivity or require complex procedures.

Purpose of the Study:

• To develop a sensitive and reliable fluorescence sensor for adenosine deaminase (ADA) activity detection.
• To utilize an enzyme substrate binding aptamer complex for enhanced sensor performance.
• To validate the sensor's efficacy in both buffer solutions and biological samples like serum.

Main Methods:

• A DNA probe with an adenosine aptamer, dual-labeled with biotin and digoxigenin (DIG), was designed.
• The probe was immobilized on a streptavidin-coated microplate.
• Enzyme activity was detected by monitoring the accessibility of the DIG tag to an anti-DIG-HRP conjugate, leading to a fluorescence signal via a Eu³⁺-labeled antibody.

Main Results:

• The developed sensor demonstrated accurate reflection of ADA enzyme activity.
• A low detection limit of 0.5 U L⁻¹ for ADA was achieved.
• The sensor performed effectively in both buffer solutions and human serum samples.
• The study successfully investigated the effect of an ADA inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride, at concentrations as low as 0.01 nM.

Conclusions:

• The aptamer-based fluorescence sensor provides a sensitive and accurate method for ADA activity detection.
• The sensor's ability to function in serum expands its potential diagnostic applications.
• This approach offers a promising tool for biochemical analysis and enzyme activity monitoring.