Proof-of-Concept Development of a Point-of-Care Direct Quantification of Glycated Albumin Using a Dual Electrochemical Method Approach
Authors: Rescalli Andrea , Hatada Mika , Cellesi Francesco , Cerveri Pietro , Sode Koji 
Published: December 2024

Diabetes mellitus is a prevalent chronic metabolic condition characterized by inadequate insulin production or utilization, leading to elevated blood sugar levels (i.e., hyperglycemia). Effective glycemic control is crucial for preventing long-term complications like cardiovascular disease, kidney disease, neuropathy, and retinopathy. Glycated albumin (GA) is a promising biomarker for glycemic control due to its shorter lifespan and broader reliability compared to glycated hemoglobin (HbA1c), which is the gold standard glycated biomarker widely used in clinical practice. By reflecting the average blood glucose level of the previous 2-3 weeks, as opposed to the 2-3 months of HbA1c, GA allows for better tracking of rapidly evolving conditions. Moreover, it is not affected by hematologic disorders which can bias HbA1c measurements. GA is defined as the fraction of glycated human serum albumin (GHSA) to total human serum albumin (HSA). At present, GHSA is assessed at central laboratories using enzymatic assay kits that rely on spectroscopical measurements and include fructosyl amino-acid oxidase, which requires the proteolytic hydrolysis of GHSA to release fructosyl amino acid. Nevertheless, the need for a pre-treatment of GHSA poses a big challenge in the translation of this technique outside a laboratory setting. Therefore, a point-of-care testing (POCT) platform that minimizes the time gap between measurement and clinical decision-making, and expands the tools available to diabetic subjects for self-monitoring of their blood sugar levels management is needed [1, 2].

Aptamers are a type of single-stranded oligonucleotide that are able to selectively interact with a wide range of target molecules. They are chemically synthesized, possess great thermal stability and exhibit regenerable binding [3, 4]. The aptamer against intact GHSA has been reported [5], thus eliminating the need for a pre-digestion of the GHSA and empowering POCT applications. Aptamer-based detection of HSA has also been successfully achieved [6].

The clinical relevance for GA as a biomarker relies on the percentage ratio quantification of GHSA to the total HSA present in the plasma. Another problem of current spectroscopic techniques is that GHSA and HSA levels are assessed with two distinct assays (i.e., enzymatic for GHSA and bromocresol purple for HSA), which are difficult to combine into a single platform. Hence, for effective implementation of a POCT or personal sensing device it should be able to measure both GHSA and total HSA on the same unit.

In this work we aim to expand on a recent design of our research group [7], in which two aptamer-based extended gate field effect transistor (EGFET) sensors were developed in parallel: one dedicated to GHSA measurement and a second one to quantify total HSA. GHSA was successfully detected in concentrations from 0.1 to 10 μM within 20 minutes, even in presence of bovine serum albumin. Total HSA concentrations ranging from 1 to 17 μM were correctly measured. Furthermore, the linear calibration curves obtained with each sensor allowed to correctly quantify glycation ratios in samples with known and clinically relevant GA levels.

The aim of this work is to develop a novel GA sensing technique exploiting a single sensor with dual electrochemical modalities, which are based on square wave voltammetry (SWV) and EGFET. In this scenario, separating the signal contribution of each aptamer from the total is of critical importance. To achieve this, we propose to interrogate each recognition element with a different electrochemical technique. SWV is used to detect the change in electron transfer kinetics of the redox probe covalently attached on one aptamer. For the second aptamer, the EGFET configuration that we have already successfully reported is implemented to detect the non-faradaic contribution of charge variations at the surface of the extended gate. The combination of these two techniques could lead to the dual measurement of GHSA and HSA at the single sensor, enabling the development of an all-in-one POCT platform for glycemic control in diabetic subjects.

References

[1] Rescalli et al., Biosensors, 2022, 12(9), 687

[2] Hatada et al., Sensors and Actuators, B: Chemical, 2022, 130914

[3] Xiao et al., Angewandte Chemie International Edition, 2005, 44, 5456-5459

[4] Wu et al., Analytical Chemistry, 2022, 94(23), 8335-8345

[5] Apiwat et al., Biosensors and Bioelectronics, 2016, 82, 140-145

[6] Japrung et al., 2017, U.S. Patent No. 9,834,772

[7] Hatada et al., Biosensors and Bioelectronics, 2024, 116118