Electrochemical and Optical Evaluation of the Stability of Enzymatic Hydrogels (BPEI-GOx) Used in Glucose Biosensors
Authors: Díaz Jan-carlo , Casanova-Moreno Jannu 
Published: July 2022

At present, research and development of a large number of new devices for the detection and quantification of biomedical analytes incorporated into miniaturized platforms of high interest such as organ-on-a-chip (OCC), lab-on-a-chip (LOC), and implantable devices. Together, these systems are characterized by taking continuous measurements for long periods (hours, days, weeks) of the products and/or by-products generated by these platforms[1, 2]. Based on the above, it is essential to detect the failure mechanisms of the systems developed to make improvements in the operational stability of these devices, which could generate biosensing instruments with a longer life span.
In this work, the stability of hydrogels was evaluated for the detection of glucose, made up of the branched polyethyleneimine/glucose oxidase (BPEI/GOx) system using ethylene glycol diglycidyl ether (EGDGE) and glutaraldehyde (GA) as crosslinking agents. The stability of these systems was evaluated by light, fluorescence, and electrochemical microscopy (SECM).
First, the hydrogels formed were deposited by drop-casting on glass surfaces, gold, and a combination of both (polycrystalline gold chips deposited on glass substrates). The surfaces were treated with different cleaning methodologies and the impact they had on the adhesion of the hydrogels was subsequently evaluated. For their part, the hydrogels were subjected to different conditions such as exposure to aqueous media and potential disturbances to measure their adhesion to the surfaces described. The results showed that, when cleaning the surfaces with the RCA process for 15 minutes, both the hydrogels with EGDGE and GA show better adhesion. Likewise, both hydrogels showed better adherence on gold surfaces compared to glass ones. This can be attributed to electrostatic interactions between the surface and the amino groups present in BPEI and GOx.
Subsequently, the spatial arrangement of the BPEI and GOx of both systems was evaluated by fluorescence microscopy and SECM. For this, the GOx was labeled with the fluorophore fluorescein-5-isothiocyanate (FITC) and the BPEI with tetramethylrhodamine-5-Isothiocyanate (5-TRITC). The hydrogels were irradiated with blue, green, and UV light. In the fluorescence images of the controls, unlabeled hydrogels, it was observed that when cross-linked with GA, there was an intense emission of fluorescence at the three wavelengths evaluated. According to the literature, this fluorescence can be attributed to the mechanism proposed by Liu et al.[3] which is based on the formation of Schiff bases from the reaction between aldehydes and amino acid residues. The above-described mechanism was used to confirm the crosslinking reaction between BPEI and enzyme with the crosslinking agent. On the other hand, in hydrogels with EGDGE, heterogeneous structures are seen, in which the distribution of each of the labeled components can be observed. Which facilitated the measurement of stability to time. The spatial arrangement of the enzyme and its stability to time was also evaluated by SECM using the generation of the substrate/collection at the tip (SG/TC) method. A 15 µm Pt UME was used, applying a potential of 0.45 V vs Ag│AgCl to follow the oxidation of H2O2 generated by the enzymatic reaction.
As a final step, the concentration of EGDGE and GA was varied to observe the behavior of the hydrogels for the electrochemical detection of different concentrations of glucose in 0.1 M phosphate buffer pH 7.4, following the oxidation of H2O2. Calibration curves were made by chronoamperometry of solutions with concentrations of 0-50 mM of glucose. For EGDGE, the concentration of the solutions ranged from 7.5 to 20%. The results obtained showed that the variation in the concentration of the crosslinker does not affect the diffusion of glucose or H2O2 through the hydrogel and therefore the currents obtained are very similar. In the case of GA, the concentration of the solutions ranged from 4.5 to 0.45%. In this system, it was observed that the concentration of crosslinker significantly affects the diffusion of glucose and/or H2O2 through the hydrogel, reflecting on the currents obtained. In this way, it was determined that the GA concentration of 0.45% allows efficient diffusion through the hydrogel, allowing an increase in the current as of the glucose concentration increases.
References

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