Improvement of a glucose sensor electrode by coating of composite membranes

Ramon Garca, Carlos Rubio, Marc Seco. Universitat autnoma de Barcelona. Introduction The body has several ways to increase the glucose concentration in blood, but there is only one protein that commands the concentration decrease, insulin. Diabetic patients cannot express insulin. However, this deficit can be corrected injecting an appropriate dose of synthetic insulin. The necessary dose of this hormone depends on the glucose concentration in blood. Nowadays, patients must poke themselves to extract a blood sample several times a day. It is awkward for most patients. In addition these results can bring to a mistaken interpretation of the data. It is because glucose concentration is a fast changing value and the data of a punctual moment may be insufficient for a correct determination. Because of that, the development of glucose sensors for monitoring the glucose concentration has been an important research field. Time depending data can be obtained by using implantable sensors. The implantation of sensors for monitoring still finds several limitations although there are already three commercial devices [1]. The lack of biocompatibility due to the toxicity of the sensors, their components and leachables, the potential infection of the tissue and its regeneration are the major problems [2]. Another limitation is the stability of the sensor, which depends on the response of the body against it. In addition, precision of the sensor and the removal of interfering signals from molecules of the body have to be enhanced. Nowadays, the enzymatic electrochemical sensors are focusing most of the attention. The work electrode is covered by a membrane functionalized by glucose oxidase enzyme. In that membrane the following reaction is produced [1]: glucose + oxigen gluconic cid +H2O2 Thus, the electrochemical signal detected is given by the oxidation of the hydrogen peroxide: H2O2 2H++O2 + 2eOne of the most common strategies adopted in order to improve the performance of the sensors is to recover the electrodes with a membrane which can give a good biocompatibility, enzyme stability or structural stability. Some combinations of materials can be implemented for a better working even a synergic effect. In this project we want to determine the properties of a two-membrane coated electrode. The first layer is of PPD (poly (ophenylenediamine)). This material is a good filter for the usual interfering molecules in the subcutaneous matrix, as ascorbic acid, uric acid, L-cysteine, urea or fructose acetaminophen [4]. The second layer is based on polypyrrole grown within UV cross-linked hydroxyethyl methacrylate (HEMA)-based hydrogels. The GOx (glucose oxidase) is bonded to that membrane [3]. The hydrogel is a biocompatible material besides it is functionalized by bonding to it surface a cellular membrane lipid (2methacrylooyloxyethyl phosphorylcholine (MPC). It improve the correct cicatritzation of the wound. If the body doesnt recognize the implant as a tissue it will produce clots around the sensor and it will decrease the sensitivity of the device. In addition the membrane is also modified changing the pyrrol component of the monomer formulation for a unique bifunctional pyrrol monomer, 2methacrylooyloxyethyl pyrrolyl bytyrate.

Polymerization of this derivative will covalently secure the electroactive polypyrrole component to the hydrogel network, thus preventing any possible in vivo leeching of the conductive polymer component from the composite membrane. Furthermore, the membrane is functionalized with polyethylene glycol methacrylate (PEGMA) in order to provide long-term stabilization of membraneimmobilized oxidoreductases. The composite material obtained is expected to provide the biocompatibility and stability of the trifuntionalized membrane at the same time as the interfering signals filtration. Moreover we find a synergic effect between both membranes. The PPD is partially impermeable against H2O2 so the sensibility of the signal can be drastically reduced as the PPD layer thickness increases. Despite of that, if the extern membrane is also permeable to H2O2 the concentration between both membranes will increase and the sensitivity will be better. We experimentally compare the permeability of the hydrogel membrane with a Nafion membrane which had been previously tested as a biocompatible coating for a PPD coated electrode giving promising results.

Solutions of interfering species in pH 7.4 buffer were prepared just before use, as was 5 mM o-phenylenediamine (o- PD) (Aldrich) in an acetate buffer (pH 5.5). Equipment. Amperometry was performed using a Pine RDE-4potentiostat (Pine Instrument Company, Grove City, PA). A Pine MSR rotator and Pt rotating disk electrode (0.5-cm diameter), were also used. A three-neck, round-bottom flask served as the electrochemical cell. 2.2 Permeability measures We wanted to experimentally compare the permeability of the different external membranes to H2O2 (Nafion and functionalized hydrogel). The H2O2 oxidation occurs in a microelectrode coated with a Nafion membrane and also in a microelectrode coated with the functionalized hydrogel. Both electrodes are immersed in a solution of H2O2 increasing concentration. Comparing both signals given by the peroxid oxidation we have been able to know the relative permeability of the two materials.

2. Experimental section The materials and procedure followed for the microfabrication of the electrodes is descrived in previous manuscript [4]. The synthesis of the hydrogel membrane is descrived in the articles [6], [7].
Material. A pH 7.4 phosphate buffer solution (PBS) was prepared from phosphate salts (p = 0.05 M) with sodium benzoate (5 mM) and ethylene diaminetetraacetic acid (1 mM) as preservatives and NaCl(O.1 M) as electrolyte. Glucose (0.1 M) was added, allowed to mutarotate overnight at room temperature, and then stored at 4 C.

Fig 1. Current vs Hydrogen Peroxid concentration for Nafion coated sensor and Hydrogel coated sensor.

The hydrogel membrane gave lower values than the Nafion one. That is because the Nafion membrane is more permeable to H2O2 than the Hydrogel one. Because of that, the H2O2 can pass through the membrane and give a more intense signal. Higher permeability means greater sensibility since the concentration of H2O2 between the outer membrane and the PPD membrane increases.

2.3 Sensibility testing

We proceeded to test the sensor sensibility by an amperometry in vitro. We analyzed the response of the sensor for several PPD thicknesses. The thickness of the PPD layer was determined by the electrodeposition time of the polymer (15 min, 5 min, 2 min and 0 min deposition time membranes were tested). These four microsensors were tested in growing concentration of glucose and PBS buffer, Ph=7,4.

Table1. Sensor response to Various Interferences

Fig 2. Relation I vs [Glucose] obtained from the devices prepared with different deposition times. The sensibility value is the slope of the linear signal.

We could observe a sensibility decrease as we increased the membrane thickness due to the difficulty of H2O2 to diffuse across the membrane. 2.4 PPD effect in interferent molecules filtration We evaluated the PPD effect in the interferent molecules filtration for the range of membrane thickness. The results were obtained from the measurements of different membranes in a 5.6mM constant glucose concentration media. We performed the analysis for different interfering molecules per separate. The interfering molecules tested were ascorbic acid, uric acid, L-cysteina, urea, fructose acetaminophen in their maximum physiological concentration in blood. The experimental part was done with two electrodes for each PPD membrane thickness. The effect of the interferents is expressed as the percenctual variation of the signal relative to the 5.6mM glucose signal

The main interferences have been produced by uric acid, L-cysteine and acetaminophen. We observed the five-minute PPD membrane exhibits good filtering properties in comparison with the two-minute PPD membrane. Differences between the filtering of the fifteen-minute PPD membrane and the five-minute PPD membrane are less meaningful. Taking into account the previous results about the relation between sensitivity and membrane thickness we concluded that the five-minute PPD membrane is the optimal option in order to get the best compromise between sensitivity and filtering properties. 2.5 Enzymatic stability in hydrogel To test the enzymatic stability in the hydrogel membrane we studied the amperometric signal given at an in vitro media to a constant glucose concentration at 37C and pH=7,4. The analysis is realized for the four thicknesses of the PPD membranes as in the previous tests. We obtained data about the enzymatic stability in our hydrogel membrane and also the effect of peroxide concentration between the membranes that depends on PPD thickness [4]. As it is shown in the figure 3 the intensity decreases in time more sharply as we increase the PPD membrane thickness. It may be due to the increase of H2O2 concentration between the layers

since the peroxide improve the enzyme degradation.

Fig 3. Stability of enzyme is shown by the graph Intensity vs time.

Fig 4. Chronic in vivo evaluation. Sensor output vs glycemia measured in blood samples and the glycemia measured in an active, healthy dog after a rapid glucose injection (a) immediately after sensor implantation and (b) after 7 days. Surgery during implantation led to some bleeding at the sensor location.

2.6 In vivo testing The experiments in vivo have been carried out with the five-minute PPD deposition sensor because it showed the best results in relation to sensibility, filtration of interfering molecules and enzyme stability. To conduct the experiment in vivo we chose the most suitable animal model in terms of similarity with human tissues biology and size. The dog is a good model used in previous studies to test implantable electrodes. The study consisted in the implantation of the subcutaneous sensor and the collection of glucose concentration data. Microsensors signal needs a stabilization time of 30-40 minutes due to the variation of microvascular perfusion after causing the injury [5].

We took measurements after injecting a glucose dose to the animal. We performed the monitoring the first day after implantation and seven days later. The comparison of the data gives us information about the degradation of the glucose oxidase during time. The signal proportional to the glucose oxidation rate decreased in time in both graphs due to the animal consume of glucose. We could also observe that the curves obtained the first day and the seventh day were similar. We could conclude that the degradation of the enzyme is not significantly improved in the in vivo media. Conclusions:

The improvement of implantable glucose microsensors characteristics depends on the development of more complex composites. This would allow increasing biocompatibility, sensibility, interfering molecules filtering as well as long term stability. In this study we designed and characterized a new functional electrode based on the mixing of a trifunctionalized hydrogel membrane and a PPD layer,

which had been previously tested with a biocompatible Nafion coating [4]. The results showed a synergic effect due to the fact that the hydrogel membrane exhibits a lower permeability to H2O2 than the Nafion membrane. Besides we

obtained the best compromise between the filtering properties of the PPD membrane and the sensitivity of the sensor varying the PPD coating thickness. We also proved that the new sensor exhibits a good in vivo response and stability.

[1] Implantable glucose sensors for diabetes monitoring [2] Biocompatibility of an Enzyme-Based, Electrochemical Glucose Sensor for Short-Term Implantation in the Subcutis. [3] Design of a Subcutaneous Implantable Biochip for Monitoring of Glucose and Lactate [4] Performance of Subcutaneously Implanted Needle-Type Glucose Sensors Employing a Novel Trilayer Coating [5]Advances and prospects in glucose assay technology [6] S. Brahim, D. Narinesingh, and A. Guiseppi-Elie, Polypyrrole-hydrogel composites for the construction of clinically important biosensors, Biosens. Bioelectron., vol. 17, pp. 5359, 2002. [7] A. Guiseppi-Elie, S. Brahim, and D. Narinesingh, Composite hydrogels containing polypyrrole as support membranes for amperometric enzyme biosensors, J. Macromol. Sci., Pure Appl. Chem, vol. A38, no. 12, pp. 15751591, 2001. E. Renard et al. Implantable closed-loop glucose-sensing and insulin delivery: the future for insulin pump therapy. Gough, David A, and Jon C Armour et al . "Development of the implantable glucose sensor: What are the prospects and why is it taking so long?." Diabetes 44.9 (1995): 1005-1009. Ahyeon Koh, Scott P. Nichols, B.S, Mark H. Schonfisch Ph.D. et al. Gl ucose Sensor Membranes for Mitigating the Foreign Body Response. Journal of Diabetes Science and Technology. Volume 5, Issue 5, September 2011. Gough, DA, JC Armour, and DA Baker. "Advances and prospects in glucose assay technology." Diabetologia 40.2 (1997): S102-S107. .