One-step homogeneous protein detection based on aptamer probe and fluorescence cross-correlation spectroscopy

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biophysics

Background:

• Protein detection assays are crucial for diagnostics and research.
• Existing methods may lack sensitivity, specificity, or speed.
• Fluorescence cross-correlation spectroscopy (FCCS) offers potential for sensitive molecular detection.

Purpose of the Study:

• To develop a novel protein assay utilizing aptamer probes and FCCS.
• To demonstrate the assay's capability for detecting specific proteins, using thrombin as a model.
• To achieve sensitive, specific, and rapid protein quantification in a homogeneous format.

Main Methods:

• Development of a protein assay based on fluorescence cross-correlation spectroscopy (FCCS).
• Utilized two spectrally distinct fluorophore-labeled aptamer probes for thrombin recognition via a sandwich reaction.
• Analyzed sandwich complexes diffusing through a confocal detection volume to generate cross-correlation signals.

Main Results:

• Cross-correlation signals were observed specifically in the presence of aptamer probe-thrombin sandwich complexes.
• The assay successfully detected thrombin with low-nanomolar sensitivity.
• High specificity was achieved, with the detection process completed within one hour.

Conclusions:

• A novel aptamer-based FCCS assay for protein detection has been successfully developed.
• The assay provides a simple, rapid, and sensitive method for protein analysis.
• This homogeneous assay format shows significant promise for various protein quantification applications.