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A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay
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Tunable stringency aptamer selection and gold nanoparticle assay for detection of cortisol
Jennifer A Martin1, Jorge L Chávez, Yaroslav Chushak
1Air Force Research Laboratory, Human Effectiveness Directorate, 711th Human Performance Wing, Wright-Patterson Air Force Base, Dayton, OH, 45433, USA.
Analytical and Bioanalytical Chemistry
|June 2, 2014
Summary
Researchers developed a new method to select aptamers, which are DNA molecules that bind to specific targets. This new strategy successfully identified an aptamer for the stress biomarker cortisol, showing improved binding and potential for diagnostic applications.
Area of Science:
- Biochemistry
- Molecular Biology
- Analytical Chemistry
Background:
- Aptamer selection is crucial for developing molecular diagnostic tools.
- Cortisol is a key stress biomarker with diagnostic relevance.
- Optimizing aptamer selection stringency is essential for high-affinity binders.
Purpose of the Study:
- To select the first aptamer for cortisol using a tunable stringency magnetic bead strategy.
- To investigate the correlation between sequence copy number and binding affinity during aptamer selection.
- To develop a cortisol-targeting aptamer for potential point-of-care diagnostic applications.
Main Methods:
- Utilized a tunable stringency magnetic bead selection strategy with systematically lengthened DNA capture probes.
- Employed next-generation sequencing to analyze aptamer pools across selection rounds.
- Quantified aptamer-target binding affinity using equilibrium dialysis and microscale thermophoresis.
- Applied the selected aptamer in a gold nanoparticle assay for biomarker discrimination.
Main Results:
- Identified a dominant aptamer sequence (15-1) with enhanced binding affinity for cortisol (K(d) ≈ 6.9–16.1 μM).
- Demonstrated that extended selection under higher stringency yields aptamers with enhanced binding, contrary to previous assumptions.
- The aptamer successfully discriminated cortisol from norepinephrine, epinephrine, and cholic acid in a gold nanoparticle assay.
- High copy number in sequencing data does not always correlate with enhanced affinity.
Conclusions:
- The tunable stringency magnetic bead selection strategy is effective for identifying high-affinity aptamers.
- Extended selection rounds with increased stringency can improve aptamer performance.
- The selected cortisol aptamer shows promise for developing rapid, point-of-care diagnostic tests.
- This approach advances aptamer selection methodologies for biomarker detection.
