A Novel Sensing Technique to Detect Thrombin

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Abstract:

We have developed an optical technique to detect thrombin, an important pathophysiological marker in many cardiovascular diseases. The technique is based on the chemical transduction method, Fluo- rescence Resonance Energy Transfer, FRET, which requires the utilization of special molecular groups (donor and acceptor fluorophores), whose combined purpose is to fluoresce in the presence of a given analyte when subjected to an external photon source. The donor and acceptor fluorophores were covalently attached at either ends of peptide constructs that were synthesized using Fmoc (9-fluorenylmethoxycarbonyl) strategy, a method for solid-phase synthesis of peptides. Three different peptide substrates were synthesized, two positive constructs which were cleaved by the enzyme, throm- bin, and one negative construct. The constructs were exposed to thrombin and the response times were determined as well as the effects of the enzyme concentration and pH. The results showed that when the peptide constructs were exposed to thrombin at a pH of 7.4 and 8.3, the positive constructs were cleaved, separating the distance between the two fluorophores, resulting in detectable changes in fluo- rescence. The higher pH value resulted in faster reaction rates. The peptide constructs demonstrated a limit of detection of 0.02 Units/ml with a 90% steady state value (90) response time within 2 minutes. Keywords:

Keywords: BIOSENSING TECHNIQUE; FRET; PEPTIDE SUBSTRATES; THROMBIN

Document Type: Research Article

DOI: http://dx.doi.org/10.1166/sl.2004.048

Publication date: September 1, 2004

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