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Displacement enzyme linked aptamer assay.

Eva Baldrich1, Josep Lluis Acero, Gunter Reekmans

  • 1Department of Chemical Engineering, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007 Tarragona, Spain. eva.baldrich@urv.net

Analytical Chemistry
|August 2, 2005
PubMed
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This study introduces a novel aptamer-based displacement assay for rapid and sensitive detection. The assay utilizes enzyme-labeled targets, offering improved performance over traditional immunoassays and enabling long-term storage for commercial viability.

Area of Science:

  • Biotechnology
  • Analytical Chemistry
  • Biochemistry

Background:

  • Immunoassays often use displacement strategies, but achieving optimal suboptimal targets for antibodies is challenging.
  • Aptamers, with their high affinity and specificity, present a promising alternative to antibodies in biosensor development.
  • Enzyme-labeled targets can serve as suboptimal molecules in aptamer-based displacement assays.

Purpose of the Study:

  • To demonstrate the first exploitation of aptamers in a rapid and highly sensitive displacement assay.
  • To investigate the binding affinities of a thrombin-binding aptamer to both unmodified and enzyme-labeled thrombin.
  • To establish optimal storage conditions for aptamer-coated microtiter plates for commercial applications.

Main Methods:

  • Surface plasmon resonance (SPR) was used to determine the dissociation constants (K(D)) of the aptamer for thrombin and enzyme-labeled thrombin.

Related Experiment Videos

  • Development and optimization of a displacement assay format utilizing aptamers and enzyme-labeled targets.
  • Evaluation of assay speed, sensitivity (detection limit), and storage stability of precoated assay plates.

    • Main Results:

    • The thrombin-binding aptamer exhibited lower affinity for enzyme-labeled thrombin (K(D) = 1.1 x 10(-8) M) compared to unmodified thrombin (K(D) = 2.9 x 10(-9) M).
    • The developed aptamer displacement assay is extremely rapid, completing in 10 minutes.
    • The assay demonstrated a lower detection limit than competitive enzyme-linked aptamer assays and comparable sensitivity to hybrid aptamer-antibody assays.
    • Precoated microtiter plates showed amenability to long-term storage under optimal conditions.

    Conclusions:

    • Aptamers can be effectively utilized in displacement assays, leveraging their differential binding affinities for modified and unmodified targets.
    • This aptamer-based displacement assay offers significant advantages in speed and sensitivity for biomolecular detection.
    • The assay's stability and ease of use, with a total time of 30 minutes including color development, facilitate its commercial viability for displacement enzyme-linked aptamer assays.