Aptamer-based protein detection using a bioluminescent fusion protein

Area of Science:

• Biochemistry
• Molecular Biology
• Assay Development

Background:

• Thrombin detection is crucial for diagnosing and monitoring various hemostatic and thrombotic disorders.
• Existing detection methods often require labeled analytes or aptamers, increasing complexity and cost.
• Development of simpler, cost-effective, and sensitive detection strategies is needed.

Purpose of the Study:

• To develop a novel aptamer-based bioluminescent immunoassay for human thrombin detection.
• To utilize a fusion protein combining Escherichia coli single-stranded DNA binding protein (SSB) and firefly luciferase (fLuc) as a detection probe.
• To demonstrate the feasibility and effectiveness of this approach for thrombin quantification.

Main Methods:

• Construction and expression of a fusion protein (SSB-fLuc) linking a thrombin aptamer to firefly luciferase via SSB.
• Purification and characterization of the fusion protein, confirming retained biological activities.
• Gel mobility shift assay to verify the binding ability of the SSB fragment with the thrombin aptamer and thrombin-thrombin aptamer complex.
• Development and validation of the bioluminescent immunoassay for thrombin detection.

Main Results:

• The SSB-fLuc fusion protein was successfully expressed and purified, with both SSB and fLuc components retaining their functions.
• The SSB fragment demonstrated specific binding to the thrombin aptamer, forming a triplex in the presence of thrombin.
• The aptamer-based bioluminescent immunoassay detected human thrombin in the nanomolar range.
• The assay proved to be simple, cost-effective, and did not require labeling of analytes or aptamers.

Conclusions:

• A novel and efficient aptamer-based bioluminescent immunoassay for human thrombin detection has been successfully developed.
• The fusion protein strategy simplifies the assay design and eliminates the need for separate labeling steps.
• This versatile detection scheme holds potential for broader applications in detecting various biomolecular targets.