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 method of sensing glucose in a solution comprising:
(a) contacting the solution with 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; and
an agent that enhances the hydrophilicity of the dextran; and
a quenching agent, wherein the assay fluorophore and quenching agent form a Förster Resonance Energy Transfer (FRET) pair;
(b) observing the glucose sensing complex for signals indicative of the presence of glucose; and
(c) correlating observed signals with the concentration of glucose; wherein
the glucose sensing complex exhibits a sensor dose response (DR) loss of less than 2.5% per day.
2. The method of claim 1 , wherein the solution comprises interstitial fluid or blood.
3. The method of claim 1 , wherein the glucose sensing complex is disposed in vivo.
4. The method of claim 1 , wherein the agent that enhances the hydrophilicity of the dextran is the quenching agent.
5. The method of claim 1 , wherein the quenching agent is hexamethoxy crystal violet-1 (HMCV1).
6. The method of claim 1 , wherein the dextran is modified by an agent selected to enhance its hydrophilicity.
7. A method of sensing glucose in a solution comprising:
(a) contacting the solution with 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
(b) observing the glucose sensing complex for signals indicative of the presence of glucose; and
(c) correlating observed signals with the concentration of glucose.
8. The method of claim 7 , wherein the agent that enhances the hydrophilicity of the dextran is the quenching agent.
9. The method of claim 8 , wherein the quenching agent is hexamethoxy crystal violet-1 (HMCV1).
10. The method of claim 7 , wherein the reference fluorophore is Alexa Fluor 647 (AF647) or Alexa Fluor 700 (AF700).
11. The method of claim 7 , wherein the fluorophores and/or quenching agent are water soluble.
12. The method of claim 7 , wherein the dextran is modified by an anhydride compound.
13. The method of claim 7 , wherein the dextran comprises less than 1500 glucose units.
14. The method of claim 7 , wherein the Degree of Labeling (DOL) of the fluorophore is 4.1 and the DOL of the quencher dye is 5.7.
15. The method of claim 7 , wherein the agent that enhances the hydrophilicity of the dextran comprises a cyclic anhydride or a tartaric anhydride derivate.
16. The method of claim 7 , 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.
17. A method of sensing glucose in a solution comprising:
(a) contacting the solution with 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 Førster Resonance Energy Transfer (FRET) pair;
(b) observing the glucose sensing complex for signals indicative of the presence of glucose; and
(c) correlating observed signals with the concentration of glucose.
18. The method of claim 17 , wherein the Degree of Labeling (DOL) of the fluorophore is 4.1 and the DOL of the quencher dye is 5.7.
19. The method of claim 17 , wherein the dextran comprises about 600 glucose units.
20. The method of claim 17 , wherein the reference fluorophore is Alexa Fluor 647 (AF647) or Alexa Fluor 700 (AF700).