In vitro selection of human cerebrospinal fluid-specific aptamers using clinical samples.

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Biomarker Discovery

Background:

• Cerebrospinal fluid (CSF) leaks are serious conditions with significant morbidity.
• Current diagnostic methods in the U.S. involve protein electrophoresis, lacking point-of-care (POC) solutions.
• There is a need for rapid, accessible CSF leak detection.

Purpose of the Study:

• To discover single-stranded deoxyribonucleic acid (ssDNA) aptamers that selectively bind to CSF biomarkers.
• To lay the groundwork for an aptamer-based POC CSF detection device.

Main Methods:

• Systematic Evolution of Ligands by EXponential enrichment (SELEX) was employed using a randomized DNA library.
• Quantitative polymerase chain reaction (qPCR) and fluorescence anisotropy (FA) were used to assess aptamer binding.
• Binding affinity and kinetics were evaluated for selected aptamer candidates.

Main Results:

• Two dominant ssDNA aptamers (C2 and C3) were identified after 14 SELEX cycles.
• Aptamers C2 and C3 exhibited significantly higher affinity for CSF compared to serum (~586x and ~82x, respectively).
• The dissociation constants (K1/2) for C2 and C3 were determined to be 5.0% and 14.1% respectively, indicating strong binding.

Conclusions:

• In vitro selection successfully identified two ssDNA aptamers with high selectivity for CSF biomarkers.
• These aptamers show potential for integration into a rapid, point-of-care electrochemical diagnostic system.
• The developed aptamers represent a promising advancement for CSF leak diagnostics.