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%0 Thesis
%A Zhou, Lei
%T Development of novel electrochemical aptamer biosensors for point-of-care glycemic control
%I RWTH Aachen University
%V Dissertation
%C Aachen
%M RWTH-2025-03678
%P 1 Online-Ressource : Illustrationen
%D 2024
%Z Veröffentlicht auf dem Publikationsserver der RWTH Aachen University 2025
%Z Dissertation, RWTH Aachen University, 2024
%X Diabetes mellitus is a disease mainly induced by insulin deficiency and/or insulin resistance, which can cause several complications and seriously reduce the life quality of patients. The early-stage diagnosis of this disease is beneficial not only to diabetes monitoring but also to treatment evaluation. The acute and long-term glycemic control for diabetes patients commonly rely on the assessment of the levels of blood glucose and glycated hemoglobin A1C (HbA1C), respectively. Glycated human serum albumin (GHSA) can be considered an alternative to HbA1C, its lifespan is four times shorter and its concentration in blood is independent of hemic diseases. Besides, the insulin level of patients is also an important factor for diabetes monitoring through glucose metabolism control, thus it is crucial to determine the insulin concentration. Although it is important to assess the blood glucose level, there is also a huge necessity for insulin and GHSA determination. In this thesis, we aim to establish aptamer-based electrochemical sensing platforms to detect insulin and GHSA biomarkers in high selectivity and sensitivity. Firstly, a ratiometric electrochemical aptasensor was proposed by using gold rod electrodes for insulin detection, then the gold rod electrodes were used again for the determination of human serum albumin (HSA) and GHSA, respectively. Finally, the HSA and GHSA detection were transferred from the gold rod electrode to a flexible polymer-MEAs chip to facilitate the dual-detection of the two biomarkers. Firstly, a ratiometric electrochemical aptasensor is proposed to recognize the insulin biomarker by square wave voltammetry (SWV) measurement with a gold rod electrode, which was immobilized with insulin-capturing aptamer in hybridization with partially complementary ssDNA. The capturing aptamer and hybridized ssDNA were modified with redox tags of anthraquinone (AQ) and methylene blue (MB) at the side either far or close to the gold surface, respectively. Moreover, the capturing aptamer was self-assembled on the gold surface through functional groups of double dithiol-phosphoramidite ((DTPA)2) thus a considerably superior aptasensor durability can be obtained. Monofunctional methoxy-polyethylene glycol thiol (PEG) was used as a blocking material to passivate the gold surface undecorated by aptamer molecules. The detection of insulin biomarkers can cause the ssDNA dehybridization from the capturing aptamer which leads to the formation of a typical G-quadruplex structure during the specific binding of insulin. Therefore, the target detection results in an increased AQ current and decreased MB current, both of which can be utilized to evaluate the insulin concentration. The fabrication process as well as the target binding of this aptasensor were characterized by different physical or chemical techniques such as quartz crystal microbalance with dissipation (QCM-D), atomic force microscopy (AFM), electrochemical impedance spectroscopy (EIS), and cyclic voltammetry (CV). The optimal aptamer concentration and target incubation time were sequentially obtained by SWV measurements and used in all further tests. Then, the reliability of this aptasensor could be further improved by operating an AND logic gate, in which the obtained current signal only makes sense when both AQ and MB-induced signals are different from zero. Consequently, this insulin dual-signal aptasensor possesses a comprehensive detection limit as low as 0.15 nM and a detection range of 0.5 nM - 2.5 µM, which is promising for the PoC diagnosis.Then, the gold rod electrode was further employed to establish another electrochemical aptasensor for either HSA or GHSA detection. HSA aptamer and GHSA aptamer were accordingly applied. The commonly used blocking material of 6-mercapto-1-hexanol (MCH) was utilized as backfills. The aptasensor production and target binding processes were also characterized with AFM and EIS techniques, followed by the optimization of the experimental conditions including the aptamer concentration as well as the incubation time of targets. Afterwards, the detection of HSA and GHSA were all transferred from gold rod electrodes to the flexible multielectrode arrays (flex⎯MEAs) facilitating the immobilization of respective aptamer receptors. MCH was replaced with PEG backfills which is beneficial to reduce the unspecific adsorption in real blood sample detection. Therefore, the polymer chip is promising for the detection of the two biomarkers only in one mixed sample combined with the cost analysis. In this work, the flex⎯MEAs polymer chip contains two individual sets of gold electrodes, which are independently decorated with respective aptamer receptors. Therefore, this electrochemical aptasensor is capable of simultaneously detecting the HSA and GHSA biomarkers. In this work, the established dual-target aptasensing platform has detection limits of 13 nM and 25 nM for HSA and GHSA, respectively, followed by a comprehensive dynamic detection range of 40 nM - 10 µM, covering the clinically required concentrations.In summary, both the insulin and HSA (GHSA) aptasensors exhibit high selectivity, durability, sensitivity as well as real blood sample detection performance. Benefited from several techniques such as electrochemical impedance spectroscopy, cyclic voltammogram, atomic force microscopy, chronocoulometry, square wave voltammetry, differential pulse voltammetry, and quartz crystal microbalance with dissipation monitoring, the insulin aptasensor for selective quantification of its analyte in diluted real blood samples, in combination with the simultaneous and quantitative detection of HSA and GHSA ratios in concentration, allows a reliable and cheap long-term glycemia control in PoC. Thus, more biomarkers can be included so that a more complete and comprehensive inspection of the glycemic surveillance becomes realistic.
%F PUB:(DE-HGF)11
%9 Dissertation / PhD Thesis
%R 10.18154/RWTH-2025-03678
%U https://publications.rwth-aachen.de/record/1009798