Enzymatic cleavage and mass amplification strategy for small molecule detection using aptamer-based fluorescence...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Fluorescence polarization (FP) assays with labeled aptamers are valuable but struggle with small molecule detection due to minimal molecular weight changes.
• Existing FP aptamer assays face challenges in sensitivity and applicability to complex biological samples without extensive pretreatment.

Purpose of the Study:

• To develop a highly sensitive fluorescence polarization aptamer assay for small molecule detection.
• To introduce a novel signal amplification strategy to overcome the limitations of traditional FP aptamer assays for small molecules.

Main Methods:

• A dual-amplified, aptamer-based strategy was developed using a FAM-labeled aptamer and a streptavidin complex.
• Nuclease digestion was employed: in the absence of the target, the aptamer is cleaved; in the presence of the target, the aptamer-target complex is protected.
• Streptavidin acted as a molar mass amplifier, increasing the signal upon target binding.

Main Results:

• The assay successfully detected adenosine within a concentration range of 0.5 μM to 1000 μM.
• A low detection limit of 500 nM for adenosine was achieved, surpassing previously reported aptamer-based FP methods.
• The developed FP assay demonstrated robustness against environmental interferences, enabling analysis in complex biological samples.

Conclusions:

• The novel dual-amplified aptamer-based strategy offers a sensitive and selective method for small molecule detection.
• This approach significantly enhances the capabilities of fluorescence polarization assays for analyzing small molecules in intricate biological environments.
• The method provides a promising platform for developing rapid and sensitive diagnostic tools for various applications.