Aptamer-modified monolithic capillary chromatography for protein separation and detection

Area of Science:

• Analytical Chemistry
• Biochemistry
• Materials Science

Background:

• Protein separation and detection are crucial in various scientific fields.
• Existing methods may face challenges with specificity and complex matrices.
• Aptamers offer specific molecular recognition capabilities.

Purpose of the Study:

• To develop a novel capillary chromatography technique for protein separation and detection.
• To utilize aptamer affinity and monolithic column properties for enhanced separation.
• To validate the method for specific protein targets like cytochrome c and thrombin.

Main Methods:

• Immobilization of a biotinylated DNA aptamer targeting cytochrome c onto a streptavidin-modified polymer monolithic capillary column.
• Utilizing the G-quartet structure of the aptamer for binding both cytochrome c and thrombin.
• Employing changes in ionic strength of the mobile phase for protein elution.
• Testing the column with various proteins including human immunoglobulin G (IgG), hemoglobin, transferrin, and human serum albumin.

Main Results:

• Successful separation of cytochrome c and thrombin from each other and other proteins.
• Demonstrated selective capture and preconcentration of thrombin using the porous monolithic column.
• Achieved rapid elution of bound proteins by increasing ionic strength.
• Showed no interference from a dilute serum matrix when determining spiked cytochrome c and thrombin.

Conclusions:

• The developed aptamer-modified monolithic columns are effective for protein detection and separation.
• The combination of aptamer affinity and porous monolithic properties offers a powerful tool for biochemical analysis.
• This technique shows promise for analyzing proteins in complex biological samples.