An aptamer assay using rolling circle amplification coupled with thrombin catalysis for protein detection

Area of Science:

• Biochemistry
• Molecular Biology
• Analytical Chemistry

Background:

• Protein detection assays are crucial in diagnostics and research.
• Existing methods may lack sensitivity or require complex procedures.
• A need exists for novel, highly sensitive protein detection strategies.

Purpose of the Study:

• To develop a sensitive aptamer-based sandwich assay for protein detection.
• To utilize rolling circle amplification (RCA) coupled with thrombin catalysis for signal amplification.
• To demonstrate the assay's efficacy using platelet-derived growth factor-BB (PDGF-BB) as a model target.

Main Methods:

• A microplate-based sandwich assay format was employed.
• Rolling circle amplification (RCA) generated DNA oligonucleotides with repeat thrombin-binding aptamer sequences.
• Thrombin molecules were labeled onto the RCA product, and its enzymatic activity was used for signal generation via chromogenic or fluorogenic peptide substrates.

Main Results:

• The assay achieved sensitive detection of PDGF-BB down to 3.1 pM using a fluorogenic peptide substrate.
• The combination of RCA and thrombin catalysis provided significant signal amplification.
• The method successfully detected the model protein target, PDGF-BB.

Conclusions:

• This aptamer-based assay offers a novel approach for signal generation in RCA-involved assays through direct thrombin labeling.
• The method circumvents the need for time-consuming preparation of enzyme-conjugates and affinity probes.
• The developed assay shows promise for various analytical applications requiring sensitive protein detection.