NOVEL HEPARIN ENTITIES AND METHODS OF USE
The present invention relates to immobilized biologically active entities that retain a significant biological activity following manipulation. The invention also comprises a medical substrate comprising a heparin entity bound onto a substrate via at least one heparin molecule, wherein said bound heparin entity is heparinase-1 sensitive.
1 . A method of determining the structure of a heparin entity bonded to a substrate, comprising the steps of:
a) providing a substrate comprising a heparin entity,
b) depolymerizing the heparin entity to generate a mixture of soluble heparin fragments,
c) detecting each heparin fragment in said mixture using column chromatography,
d) determining the identity of each detected heparin fragment from step (c), and
e) deriving the structure of the heparin entity from the identities of the detected heparin fragments.
2 . The method of claim 1 , wherein said depolymerizing is by heparinase-1 depolymerization.
3 . The method of claim 1 , wherein said column chromatography is SAX-HPLC.
4 . A method of determining the structure of a heparin entity bonded to a substrate, comprising the steps of:
a) providing a substrate comprising a heparin entity,
b) depolymerizing the heparin entity to generate a surface comprising surface-bonded heparin fragments,
c) reacting the surface with a labeling reagent which introduces a detectable component to said surface-bonded heparin fragments,
d) detecting said surface-bonded heparin fragments via said detectable component,
and
e) deriving the structure of the heparin entity from the presence of the surface-bonded heparin fragments.
5 . The method of claim 4 , wherein said depolymerizing is by heparinase-1 depolymerization.
6 . The method of claim 4 , wherein said labeling reagent is a lanthanoid Michaels-like addition organo-complex.
7 . The method of claim 4 , wherein said organo-complex is terbium tris(4-methylthio)benzoate.
8 . The method of claim 4 , wherein said labeling reagent is a metachromatic dye.
9 . The method of claim 4 , wherein said metachromatic dye is toluidine blue.
10 . The method of claim 4 , wherein said detecting is by epifluoroscence, absorption spectroscopy and visually.
11 . A method of determining the spatial distribution of a heparin entity bonded to a substrate, comprising the steps of:
a) providing a substrate comprising a heparin entity,
b) depolymerizing the heparin entity to generate a surface comprising surface-bonded heparin fragments,
c) reacting the surface with a labeling reagent which introduces a detectable component to said surface-bonded heparin fragments,
d) detecting said surface-bonded heparin fragment via said detectable component,
and
e) deriving the spatial distribution of the heparin entity from the presence of the surface-bonded heparin fragments.
12 . The method of claim 11 , wherein said depolymerizing is by heparinase-1 depolymerization.
13 . The method of claim 11 , wherein said labeling reagent is a lanthanoid Michaels-like addition organo-complex.
14 . The method of claim 11 , wherein said labeling reagent is terbium tris(4-methylthio)benzoate.
15 . The method of claim 11 , wherein said organo-complex comprises chemisorbed gold nanoparticles.
16 . The method of claim 11 , wherein said detecting is by epifluoroscent microscopy or transmission electron microscopy.
17 . A system for determining the structure of a heparin entity bonded to a substrate, comprising:
a) a depolymerization solution,
b) a labeling reagent solution, and
c) a detector.
18 . The system of claim 17 , wherein said depolymerization solution comprises heparinase-1.
19 . The system of claim 17 , wherein said labeling reagent solution comprises toluidine blue, and terbium tris(4-methylthio)benzoate.
20 . The system of claim 17 , wherein said detector comprises SAX-HPLC, an epifluoroscent microscope, and an absorption spectroscope.