The Development of an Aptamer-based Surface Plasmon Resonance (SPR) Sensor for the Real-time Detection of Glycated Protein

The direct detection of blood proteins can benefit a number of scientific and clinical applications, one of which is glycated protein monitoring for diabetes diagnosis and treatment. The formation of glycated proteins is proportional to the average glucose concentration over a certain past time period. Thus, the protein glycation level can reflect the long-term glycemic compliance according to a specific protein's half-life. Albumin has a short half-life of 2-3 weeks and the glycated albumin (GA) level could indicate the average plasma glucose concentration on a monthly basis, which provides valuable complementary information for more complete assessment of glycemic control in comparison to conventional glucose and HbA1c testing. (HbA1c is a common long-term glucose indicator with a half-life of 7-8 weeks).;According to a literature review, no glycated protein monitor, other than HbA1c, is currently on the commercial market for diabetes guidance. A cost-effective, real-time monitoring approach to assess the HbA1c and other proteins (such as GA) could bring significant advantages for diabetes diagnosis and treatment, whereas the existing monitoring technology does not make this economical.;In this research project, the feasibility of using an aptamer-based surface plasmon resonance (SPR) sensors for the real-time detection of glycated blood proteins (i.e., GA) has been studied. For the purpose of establishment, the well documented thrombin binding aptamers were immobilized onto a gold sensing surface using a two-step amine coupling method. The experimental results show that the aptamer functionalized SPR is a well suited sensing platform for selective blood protein detection applications.;To obtain the specific receptors that enable selective detection, the human serum albumin (HSA) binding aptamer has then been developed using the magnetic beads-based Systematic Evolution of Ligands by EXponential amplification process (MB-SELEX). The experimental protocol of the in vitro selection process have been standardized. The developed aptamers have shown a common pattern in sequence and a similarity in secondary structures.;Furthermore, the glycated albumin (GA) binding aptamers have been identified and the sensor performance has been characterized under different binding conditions. The aptamer functionalized sensor has shown a better performance in both sensitivity and selectivity for the detection of GA compared to the well documented phenylboronate monolayer-based sensor, indicating that aptamers are a better receptor choice for GA detection applications.;Finally, a novel multi-channel aptamer functionalization sensor and a feedback data processing model have been developed based on the glycated and non-glycated albumin binding aptamers. The experimental results demonstrate that the aptamer-based SPR sensor has the ability to detect GA levels within a 25 % error range.;This dissertation provides the essential foundation experiments to demonstrate the feasibility of using aptamer-based SPR sensor for the detection of glycated protein. It is expected that the further engineering and improvement of the sensor will eventually lead to a fast, reliable, label-free, and cost-effective glycated protein detection sensor, which may prove useful in the diagnosis and treatment of diabetes.