Sensitive and selective detection of thrombin by using a cyclic peptide as affinity ligand

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biotechnology

Background:

• Thrombin is a critical enzyme in blood coagulation.
• Sensitive and selective detection of thrombin is essential for diagnosing and monitoring coagulation disorders.
• Existing assays may lack the required sensitivity or specificity.

Purpose of the Study:

• To develop a highly sensitive assay for thrombin detection.
• To utilize a high binding-affinity cyclic peptide as an affinity ligand for thrombin capture.
• To demonstrate the applicability of the assay in biological samples like plasma.

Main Methods:

• Immobilization of a cyclic peptide with high affinity for thrombin onto magnetic beads or microplates.
• Selective capture and enrichment of thrombin from sample solutions.
• Enzymatic cleavage of a specific substrate by captured thrombin to generate a detectable product.
• Detection of the product using fluorogenic or chromogenic substrates.

Main Results:

• The assay achieved femtomolar (fM) detection limits for thrombin using a fluorogenic substrate.
• Picomolar (pM) detection limits were obtained with a chromogenic substrate.
• Successful detection of thrombin in plasma samples was demonstrated.
• The cyclic peptide affinity ligand proved effective for thrombin capture and enrichment.

Conclusions:

• A sensitive and selective assay for thrombin detection has been successfully developed.
• The high-affinity cyclic peptide serves as an effective affinity ligand for thrombin capture.
• The assay demonstrates potential for diverse thrombin analysis applications, including in clinical settings.