A 3rd generation Biosensor Towards the Continuous Long-Term Glucose Monitoring without Ag/AgCl Reference Electrode
Authors: Ramašauskas Lukas , Dagys Marius , Ratautas Dalius 
Published: February 2020

Continuous glucose monitoring (CGM) is highly important, especially, for the diagnostics and control of Type I diabetes. [1] In recent years, automatic insulin pumps (artificial pancreas) are being applied and CGM is necessary for their operation. The insulin pump should deliver a correct dose of insulin based on real-time data about glucose levels in blood. Therefore, CGM sensors should be very selective and accurate, as well as reliable and stable. For this reason, CGM sensors are developed using selective enzymes – glucose oxidase or and FAD-dependent glucose dehydrogenase. [2] They usually operate by measuring either glucose oxidation current (amperometric biosensors) or potential (potentiometric biosensors). Historically, those biosensors are divided into three generations according to the electron acceptor: O2 dependent (1st), mediator depended (2nd) and direct electron transfer (3rd). The 3rd generation biosensors are the most attractive since they are most accurate, sensitive, operate at low potential and their response does not depend on the concentration of external mediator (either O2 or synthetic). However, to this date there are multiple unresolved problems which still hold the development of 3rd generation glucose biosensors for the CGM. Some of those problems include low stability (typically from a few hours to a few days) and miniaturization difficulties due to the usage of bulk reference electrodes for the control of the potential (Ag/AgCl, Hg/Hg2Cl2, etc.). Moreover, typical reference electrodes are poorly suited for long-time operation because they leak electrolyte and ions to the media. [3] A prolonged leakage of electrolyte solution changes the potential of reference electrode and may cause health risk if CGM biosensor in directly implanted in the patient.
In this work, we present an open circuit potential (OCP) type 3rd generation glucose biosensor, which addresses the mentioned issues and operates in an electrolyte solution (pH 7.4) which mimics the composition of human serum. Our biosensor is composed of two electrodes: gold nanoparticle-glucose dehydrogenase (AuNPs-GDH) based bioanode and gold nanoparticles-laccase (AuNPs-LAC) based biocathode (Figure A). We have used
AuNPs-LAC biocathode, which we have reported earlier [4], as a counter/reference electrode since its potential depends on oxygen reduction reaction. In the majority of solutions, O2 concentration is constant, thus it has a very stable OCP which is independent on glucose concentration and holds a constant value of 0.48 V vs.Ag/AgCl (Sat.) in an electrolyte solution. We have used AuNPs-GDH electrode as a working electrode since its OCP is dependent on glucose concentration raging from 10-6 M to 0.05 M. The OCP difference between two electrodes was -0.374 V and decreased depending on the concentration of glucose (10-6 M – 0.05 M) according to Nernst model (Figure B and C). The biosensor was calibrated in glucose range of 0.001 M to 0.01 M (typical glucose concentrations present in human blood) and a high correlation calibration curve was obtained (Figure D). Moreover, biosensor had showed an excellent stability and was able to operate at least for two weeks without a significant change in OCP.
In conclusion, we have developed a promising biosensor for a constant long-term glucose monitoring which operates in an electrolyte solution, mimicking human serum conditions. A future challenge is to apply and optimize our biosensor to operate in serum and blood.

References

[1] N. Engl. J. Med. 2008, 359, 1464-1476.
[2] Biosens. Bioelectron. 2019, 124­–125, 216–223.
[3] Anal. Chem. 1994, 66, 674–679.
[4] Energy Environ. Sci., 2017, 10, 498–502.

Figure 1