Abstract
Measuring combination of blood sugar and glycated hemoglobin (HbA1c) is a way to monitor the diabetes progression. However, HbA1c level in the red blood cell keeps constant during its life cycle (120 days), which is somehow too long to be monitored in severe cases. In addition, in condition effecting red blood cell structure or hemoglobin production (hemolytic anemia, thalassemia, and etc.), HbA1c level can be unreliable. Therefore, monitoring the intermediate indicator outside the red blood cell could improve the way to control diabetes progression and treatment.
The percentage of GHSA in normal serum is 0.6-3.0% whereas in diabetic patient serum has been found to be 2-5 folds higher than normal level. It has become clear that GHSA can be a marker of intermediate glycation and also a causative agent of the damage of diabetes complications, such as atherosclerosis, coronary artery disease and kidney failure. GHSA testing has been strongly recommended for monitor glycemic control for diabetes mellitus in hemodialysis patient and a test for complications associated with diabetes, which can also serve as a monthly report card for type 1 diabetes in the USA. Several GHSA detection platforms have been developed, which include enzymatic assay, colorimetric assay, high performance liquid chromatography, affinity chromatography and immunoassay. Enzymatic and chromatography based assay have demonstrated higher %GHSA due to nonspecific enzymes and imprecise of the binding site on the resin bead. The lower %GHSA has been observed in the immunoassay using antibody that specifically bind GHSA however using antibody are high cost and easily to be denatured
This research program are focusing on development of aptasensor platform for detection of glycated albumin for diabetes mellitus detection and monitoring. Three platforms, which are graphene oxide-aptamer, electrochemical and nanopore sensors, have been developed.
Considering the graphene oxide-aptamer approach, the fluorescence aptamer was fluorescence labeled and incubated with the graphene oxide (GO) and fluorescence signal was quenched. The fluorescence signal was recovered in the presence of GHSA protein. We tested the approach with three known proteins, which were GHSA, HSA, and streptavidin. HSA structure is similar as GHSA structure but streptavidin structure is totally different from the GHSA structure. These results indicated that GO-aptamer approach is specific method for in vitro GHSA detection. After optimization of the serum dilution, we investigated LOD of the GO-aptamer approach at serum dilution 1:5000. We found the LOD of 50 µg/ml at serum dilution 1:5000 with the linear correlation between 0.05 µg/ml and 3 µg/ml
For the electrochemical sensor development, the aptamer was coated on carbon electrodes and electrochemical signal was detected when the aptamer bind glycated albumin. By using this approach, we could detect glycated albumin in PBS buffer and also in serum with the LOD of 30 µg/ml
For the nanopore analysis, we have set nanopore measurement and prepared all components for the analysis. Next study, all components will be assembled and the sensitivity and specificity of the system will be tested.
These results demonstrated that our GO-aptamer and electrochemical approach could potentially be used for simple and sensitive detection and monitoring of GHSA in human, which is an intermediate protein marker for diabetes mellitus. Next study, we will do field test for both GO-aptamer and electrochemical approach.
