Modified-dextrans for use in optical glucose assays
The invention is directed to a competitive glucose binding affinity assay comprising a glucose receptor (typically mannan binding lectin) labeled with an assay fluorophore and a modified glucose analog (typically dextran) labeled with a reference fluorophore. In certain embodiments, the glucose analog is dextran and is coupled to both a reference fluorophore and a quencher dye (e.g. hexamethoxy crystalviolet-1). Optionally the reference fluorophore is blue shifted relative to the assay fluorophore.
1. A glucose sensing complex comprising:
mannan binding ligand, wherein the mannan binding ligand is coupled to an assay fluorophore;
a reference fluorophore;
dextran, wherein the dextran functions in the assay as a glucose analog and is coupled to:
the reference fluorophore;
an agent that enhances the hydrophilicity of the dextran;
a quenching agent, wherein the assay fluorophore and quenching agent form a Förster Resonance Energy Transfer (FRET) pair; and
the glucose sensing assay complex exhibits a sensor dose response (DR) loss of less than 2.5% per day.
2. The glucose sensing complex of claim 1 wherein the agent that enhances the hydrophilicity of the dextran is the quenching agent.
3. The glucose sensing complex of claim 2 wherein the quenching agent is hexamethoxy crystal violet-1 (HMCV1).
4. The glucose sensing complex of claim 1 wherein the reference fluorophore is Alexa Fluor 647 (AF647) or Alexa Fluor 700 (AF700).
5. The glucose sensing complex of claim 1 wherein the fluorophores and/or quenching agent are water soluble.
6. The glucose sensing complex of claim 1 wherein the dextran is modified by an anhydride compound.
7. The glucose sensing complex of claim 1 wherein the dextran comprises less than 1500 glucose units.
8. The glucose sensing complex of claim 1 wherein the Degree of Labeling (DOL) of the fluorophore is 4.1 and the DOL of the quencher dye is 5.7.
9. The glucose sensing complex of claim 1 wherein the agent that enhances the hydrophilicity of the dextran comprises a cyclic anhydride or a tartaric anhydride derivate.
10. The glucose sensing complex of claim 1 wherein the agent that enhances the hydrophilicity of the dextran comprises a succinic acid, a glutaric acid, an adipic acid, a pimelic acid, a suberic acid, an azelaic acid or a sebacic acid.
11. A glucose sensing complex comprising:
mannan binding ligand, wherein the mannan binding ligand is coupled to an assay fluorophore;
a reference fluorophore;
dextran, wherein the dextran functions in the assay as a glucose analog and is coupled to:
the reference fluorophore;
an agent that enhances the hydrophilicity of the dextran, wherein the agent that enhances the hydrophilicity of the dextran comprises a cyclic anhydride, a tartaric anhydride derivate, a succinic acid, a glutaric acid, an adipic acid, a pimelic acid, a suberic acid, an azelaic acid or a sebacic acid; and
a quenching agent, wherein the assay fluorophore and quenching agent form a Förster Resonance Energy Transfer (FRET) pair.
12. The glucose sensing complex of claim 11 , wherein the Degree of Labeling (DOL) of the fluorophore is 4.1 and the DOL of the quencher dye is 5.7.
13. The glucose sensing complex of claim 11 , wherein the dextran comprises about 600 glucose units.