Abbreviations: (AP) acetaminophen, (BSA) bovine serum albumin, (FAD) flavin adenine dinucleotide, (GO x ) glucose oxidase, (PBS) phosphatebuffered saline, (PPh) polyphenol, (PU) polyurethane, (PVA) polyvinyl alcohol Keywords: bienzymatic, biosensor lag time, drug delivery coatings, foreign body response, implantable glucose sensors, outer membranes, selectivity Development of electrochemical sensors for continuous glucose monitoring is currently hindered by a variety of problems associated with low selectivity, low sensitivity, narrow linearities, delayed response times, hysteresis, biofouling, and tissue inflammation. We present an optimized sensor architecture based on layer stratification, which provides solutions that help address the aforementioned issues. Method:The working electrode of the electrochemical glucose sensors is sequentially coated with five layers containing: (1) electropolymerized polyphenol (PPh), (2) glutaraldehyde-immobilized glucose oxidase (GO x ) enzyme, (3) dip-coated polyurethane (PU), (4) glutaraldehyde-immobilized catalase enzyme, and (5) a physically cross linked polyvinyl alcohol (PVA) hydrogel membrane. The response of these sensors to glucose and electroactive interference agents (i.e., acetaminophen) was investigated following application of the various layers. Sensor hysteresis (i.e., the difference in current for a particular glucose concentration during ascending and descending cycles after 200 s) was also investigated. Results:The inner PPh membrane improved sensor selectivity via elimination of electrochemical interferences, while the third PU layer afforded high linearity by decreasing the glucose-to-O 2 ratio. The fourth catalase layer improved sensor response time and eliminated hysteresis through active withdrawal of GO x -generated H 2 O 2 from the inner sensory compartments. The outer PVA hydrogel provided mechanical support and a continuous pathway for diffusion of various participating species while acting as a host matrix for drug-eluting microspheres. Conclusions:Optimal sensor performance has been achieved through a five-layer stratification, where each coating layer works complementarily with the others. The versatility of the sensor design together with the ease of fabrication renders it a powerful tool for continuous glucose monitoring.

Highly sensitive, long-term stable and reusable microfluidics electrodes have been fabricated and evaluated using H 2 O 2 and hydroquinone as model analytes. These electrodes composed of a 300 nm Pt-black layer situated on a 5 μm thick electrodeposited Au layer, provide effective protection against electrooxidation of an underlying chromium adhesion layer. Using repeated cyclic voltammetric (CV) sweeps in flowing buffer solution, highly sensitive Pt-black working electrodes were realized with five-(four-) decade linear dynamic range for H 2 O 2 (hydroquinone) and low detection limit of 10 nM for H 2 O 2 and 100 nM for hydroquinone. Moreover, high sensitivity for H 2 O 2 was demonstrated at low (0.3 V vs. Ag/AgCl) oxidation potentials, together with long-term stability and reusability for at least 30 days. Microfluidic flow was employed for desorption and reactivation of the nominally planar Pt-black electrodes. Such electrocatalytic surface architecture should be appropriate for long-term electrochemical detection of various molecules and biomolecules as well as in reusable immunoassay configurations.

The promise of implantable electrochemical sensors is often undermined by the critical requirement of device miniaturization that inadvertently degrades sensor performance in terms of sensitivity and selectivity. Herein, we report a novel miniaturized and flexible amperometric sensor grown at the ‘edge plane’ of a 25-µm gold wire. Such geometry affords extreme miniaturization along with ease of fabrication, minimal iR drop and 3-D diffusion for effective mass transfer. This together with electrochemical rebuilding of the Au working electrode and subsequent Pt nanoparticles deposition resulted in the highest H2O2 sensitivity (33 mA·mM−1·cm−2), reported thus far. Concurrent electrodeposition of o-phenylenediamine with glucose oxidase afforded glucose detection at these edge-plane microsensors with a six fold improvement in sensitivity (1.2 mA·mM−1·cm−2) over previous reports. In addition, these sensors exhibit low operation potential (0.3 V), high selectivity (more than 95%) against in vivo interferences, and an apparent Michealis-Menten constant (Kmitalicapp) of 17 and 75 mM of glucose in the absence and presence of an outer polyurethane coating, respectively. These features render the edge-plane sensor architecture as a powerful platform for next-generation implantable biosensors.

Biofouling and tissue inflammation present major challenges toward the realization of long-term implantable glucose sensors. Following sensor implantation, proteins and cells adsorb on sensor surfaces to not only inhibit glucose flux but also signal a cascade of inflammatory events that eventually lead to permeability-reducing fibrotic encapsulation. The use of drug-eluting hydrogels as outer sensor coatings has shown considerable promise to mitigate these problems via the localized delivery of tissue response modifiers to suppress inflammation and fibrosis, along with reducing protein and cell absorption. Biodegradable poly (lactic-co-glycolic) acid (PLGA) microspheres encapsulated within a poly (vinyl alcohol) (PVA) hydrogel matrix, presents a model coating where the localized delivery of the potent anti-inflammatory drug dexamethasone has been shown to suppress inflammation over a period of 1-3 months. Here it is shown that the degradation of the PLGA microspheres provides an auxiliary venue to offset the negative effects of protein adsorption. This was realized by: 1) the creation of fresh porosity within the PVA hydrogel following microsphere degradation (which is sustained until the complete microsphere degradation); and 2) rigidification of the PVA hydrogel to prevent its complete collapse onto the newly created void space. Incubation of the coated sensors in PBS buffer led to a monotonic increase in glucose permeability (50%), with a corresponding enhancement in sensor sensitivity over a one-month period. Incubation in serum resulted in biofouling and consequent clogging of the hydrogel microporosity. This however, was partially offset by the generated macroscopic porosity following microsphere degradation. As a result of this, a two-fold recovery in sensor sensitivity for devices with microsphere/hydrogel composite coatings was observed as opposed to similar devices with blank hydrogel coatings. These findings suggest that the use of macroscopic porosity can reduce sensitivity drifts resulting from biofouling and this can be achieved synergistically with current efforts to mitigate negative tissue responses through localized and sustained drug delivery.

This study demonstrates that the PLGA microsphere/PVA hydrogel composite coatings allow sufficient glucose diffusion and sensor functionality and therefore may be utilized as a smart coating for implantable glucose biosensors to enhance their in vivo functionality.