Foreign Body Reaction to Implantable Biosensors: Effects of Tissue Trauma and Implant Size.

BACKGROUND: Implantable biosensors for continuous glucose monitoring can greatly improve diabetes management. However, their applications are still associated with some challenges and one of these is the gradual functionality loss postimplantation as a consequence of the foreign body response (FBR). Sensor miniaturization in combination with drug-eluting biocompatible coatings is a promising strategy to enhance in vivo performance. However, limited study has been performed to understand the effect of initial trauma and implant size on foreign body reaction as well as in vivo performance of implantable glucose sensors. METHODS: Different initial trauma was induced by implanting composite coated dummy sensors into rats using various sized needles and 3 different-sized dummy sensors were implanted to examine the size effect. Histological evaluation was performed to relate the inflammatory cell counts and foreign body capsule thickness with the implantation needle size and sensor size respectively. The effect of biocompatible coating on the performance of implantable glucose sensors was determined using both coated amperometric glucose sensors and microdialysis probes. RESULTS: The results revealed that the degree of acute inflammation was mainly controlled by the extent of the initial trauma: the greater the trauma, the greater the acute inflammatory response. Implant size did not affect the acute inflammatory phase. However, the extent of chronic inflammation and fibrous encapsulation were affected by sensor size: the smaller the size the less the extent of chronic inflammation and fibrous encapsulation. Glucose sensors implanted using 14 gauge needles showed significantly lower initial in vivo response compared to those implanted using 16 gauge needles. This was not observed for sensors with dexamethasone-eluting biocompatible coatings since inflammation was suppressed. CONCLUSIONS: The results of the current study indicate that the extent of the inflammatory response post-sensor implantation varies as a function of the initial tissue trauma as well as the sensor size. Accordingly, miniaturization of implantable biosensors together with the utilization of a drug-eluting biocompatible composite coating may be a promising strategy to achieve long-term reliable continuous glucose monitoring.

Low-cost photolithographic fabrication of nanowires and microfilters for advanced bioassay devices.

Integrated microfluidic devices with nanosized array electrodes and microfiltration capabilities can greatly increase sensitivity and enhance automation in immunoassay devices. In this contribution, we utilize the edge-patterning method of thin aluminum (Al) films in order to form nano- to micron-sized gaps. Evaporation of high work-function metals (i.e., Au, Ag, etc.) on these gaps, followed by Al lift-off, enables the formation of electrical uniform nanowires from low-cost, plastic-based, photomasks. By replacing Al with chromium (Cr), the formation of high resolution, custom-made photomasks that are ideal for low-cost fabrication of a plurality of array devices were realized. To demonstrate the feasibility of such Cr photomasks, SU-8 micro-pillar masters were formed and replicated into PDMS to produce micron-sized filters with 3-4 µm gaps and an aspect ratio of 3. These microfilters were capable of retaining 6 µm beads within a localized site, while allowing solvent flow. The combination of nanowire arrays and micro-pillar filtration opens new perspectives for rapid R&D screening of various microfluidic-based immunoassay geometries, where analyte pre-concentration and highly sensitive, electrochemical detection can be readily co-localized.

Enhancing the sensitivity of needle-implantable electrochemical glucose sensors via surface rebuilding.

OBJECTIVE: Needle-implantable sensors have shown to provide reliable continuous glucose monitoring for diabetes management. In order to reduce tissue injury during sensor implantation, there is a constant need for device size reduction, which imposes challenges in terms of sensitivity and reliability, as part of decreasing signal-to-noise and increasing layer complexity. Herein, we report sensitivity enhancement via electrochemical surface rebuilding of the working electrode (WE), which creates a three-dimensional nanoporous configuration with increased surface area. METHODS: The gold WE was electrochemically rebuilt to render its surface nanoporous followed by decoration with platinum nanoparticles. The efficacy of such process was studied using sensor sensitivity against hydrogen peroxide (H2O2). For glucose detection, the WE was further coated with five layers, namely, (1) polyphenol, (2) glucose oxidase, (3) polyurethane, (4) catalase, and (5) dexamethasone-releasing poly(vinyl alcohol)/poly(lactic-co-glycolic acid) composite. The amperometric response of the glucose sensor was noted in vitro and in vivo. RESULTS: Scanning electron microscopy revealed that electrochemical rebuilding of the WE produced a nanoporous morphology that resulted in a 20-fold enhancement in H2O2 sensitivity, while retaining >98% selectivity. This afforded a 4-5-fold increase in overall glucose response of the glucose sensor when compared with a control sensor with no surface rebuilding and fittable only within an 18 G needle. The sensor was able to reproducibly track in vivo glycemic events, despite the large background currents typically encountered during animal testing. CONCLUSION: Enhanced sensor performance in terms of sensitivity and large signal-to-noise ratio has been attained via electrochemical rebuilding of the WE. This approach also bypasses the need for conventional and nanostructured mediators currently employed to enhance sensor performance.

A miniaturized transcutaneous system for continuous glucose monitoring.

Implantable sensors for continuous glucose monitoring hold great potential for optimal diabetes management. This is often undermined by a variety of issues associated with: (1) negative tissue response; (2) poor sensor performance; and (3) lack of device miniaturization needed to reduce implantation trauma. Herein, we report our initial results towards constructing an implantable device that simultaneously address all three aforementioned issues. In terms of device miniaturization, a highly miniaturized CMOS (complementary metal-oxide-semiconductor) potentiostat and signal processing unit was employed (with a combined area of 0.665 mm(2)). The signal processing unit converts the current generated by a transcutaneous, Clark-type amperometric sensor to output frequency in a linear fashion. The Clark-type amperometric sensor employs stratification of five functional layers to attain a well-balanced mass transfer which in turn yields a linear sensor response from 0 to 25 mM of glucose concentration, well beyond the physiologically observed (2 to 22 mM) range. In addition, it is coated with a thick polyvinyl alcohol (PVA) hydrogel with embedded poly(lactic-co-glycolic acid) (PLGA) microspheres intended to provide continuous, localized delivery of dexamethasone to suppress inflammation and fibrosis. In vivo evaluation in rat model has shown that the transcutaneous sensor system reproducibly tracks repeated glycemic events. Clarke's error grid analysis on the as-obtained glycemic data has indicated that all of the measured glucose readings fell in the desired Zones A & B and none fell in the erroneous Zones C, D and E. Such reproducible operation of the transcutaneous sensor system, together with low power (140 µW) consumption and capability for current-to-frequency conversion renders this a versatile platform for continuous glucose monitoring and other biomedical sensing devices.

Theoretical analysis of the performance of glucose sensors with layer-by-layer assembled outer membranes.

The performance of implantable electrochemical glucose sensors is highly dependent on the flux-limiting (glucose, H(2)O(2), O(2)) properties of their outer membranes. A careful understanding of the diffusion profiles of the participating species throughout the sensor architecture (enzyme and membrane layer) plays a crucial role in designing a robust sensor for both in vitro and in vivo operation. This paper reports the results from the mathematical modeling of Clark's first generation amperometric glucose sensor coated with layer-by-layer assembled outer membranes in order to obtain and compare the diffusion profiles of various participating species and their effect on sensor performance. Devices coated with highly glucose permeable (HAs/Fe(3+)) membranes were compared with devices coated with PSS/PDDA membranes, which have an order of magnitude lower permeability. The simulation showed that the low glucose permeable membrane (PSS/PDDA) sensors exhibited a 27% higher amperometric response than the high glucose permeable (HAs/Fe(3+)) sensors. Upon closer inspection of H(2)O(2)diffusion profiles, this non-typical higher response from PSS/PDDA is not due to either a larger glucose flux or comparatively larger O(2) concentrations within the sensor geometry, but rather is attributed to a 48% higher H(2)O(2) concentration in the glucose oxidase enzyme layer of PSS/PDDA coated sensors as compared to HAs/Fe(3+) coated ones. These simulated results corroborate our experimental findings reported previously. The high concentration of H(2)O(2) in the PSS/PDDA coated sensors is due to the low permeability of H(2)O(2) through the PSS/PDDA membrane, which also led to an undesired increase in sensor response time. Additionally, it was found that this phenomenon occurs for all enzyme thicknesses investigated (15, 20 and 25 nm), signifying the need for a holistic approach in designing outer membranes for amperometric biosensors.

Effect of dexamethasone-loaded poly(lactic-co-glycolic acid) microsphere/poly(vinyl alcohol) hydrogel composite coatings on the basic characteristics of implantable glucose sensors.

BACKGROUND: Hydrogels alone and in combination with microsphere drug delivery systems are being considered as biocompatible coatings for implantable glucose biosensors to prevent/minimize the foreign body response. Previously, our group has demonstrated that continuous release of dexamethasone from poly(lactic-co-glycolic acid) (PLGA) microsphere/poly(vinyl alcohol) (PVA) hydrogel composites can successfully prevent foreign body response at the implantation site. The objective of this study was to investigate the effect of this composite coating on sensor functionality. METHODS: The PLGA microsphere/PVA hydrogel coatings were prepared and applied to glucose biosensors. The swelling properties of the composite coatings and their diffusivity to glucose were evaluated as a function of microsphere loading. Sensor linearity, response time, and sensitivity were also evaluated as a function of coating composition. RESULTS: The PLGA microsphere/PVA hydrogel composite coating did not compromise sensor linearity (sensors were linear up to 30 mM), which is well beyond the physiological glucose range (2 to 22 mM). The sensor response time did increase in the presence of the coating (from 10 to 19 s); however, this response time was still less than the average reported values. Although the sensitivity of the sensors decreased from 73 to 62 nA/mM glucose when the PLGA microsphere loading in the PVA hydrogel changed from 0 to 100 mg/ml, this reduced sensitivity is acceptable for sensor functionality. The changes in sensor response time and sensitivity were due to changes in glucose permeability as a result of the coatings. The embedded PLGA microspheres reduced the fraction of bulk water present in the hydrogel matrix and consequently reduced glucose diffusion. CONCLUSIONS: 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.

Design and fabrication of a high-performance electrochemical glucose sensor.

OBJECTIVE: 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 (GOx) 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-O2 ratio. The fourth catalase layer improved sensor response time and eliminated hysteresis through active withdrawal of GOx-generated H2O2 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.

Edge-plane microwire electrodes for highly sensitive H2O2 and glucose detection.

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 (K(m)(app)) 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.

Technologies for continuous glucose monitoring: current problems and future promises.

Devices for continuous glucose monitoring (CGM) are currently a major focus of research in the area of diabetes management. It is envisioned that such devices will have the ability to alert a diabetes patient (or the parent or medical care giver of a diabetes patient) of impending hypoglycemic/hyperglycemic events and thereby enable the patient to avoid extreme hypoglycemic/hyperglycemic excursions as well as minimize deviations outside the normal glucose range, thus preventing both life-threatening events and the debilitating complications associated with diabetes. It is anticipated that CGM devices will utilize constant feedback of analytical information from a glucose sensor to activate an insulin delivery pump, thereby ultimately realizing the concept of an artificial pancreas. Depending on whether the CGM device penetrates/breaks the skin and/or the sample is measured extracorporeally, these devices can be categorized as totally invasive, minimally invasive, and noninvasive. In addition, CGM devices are further classified according to the transduction mechanisms used for glucose sensing (i.e., electrochemical, optical, and piezoelectric). However, at present, most of these technologies are plagued by a variety of issues that affect their accuracy and long-term performance. This article presents a critical comparison of existing CGM technologies, highlighting critical issues of device accuracy, foreign body response, calibration, and miniaturization. An outlook on future developments with an emphasis on long-term reliability and performance is also presented.

Highly sensitive and reusable Pt-black microfluidic electrodes for long-term electrochemical sensing.

Highly sensitive, long-term stable and reusable microfluidics electrodes have been fabricated and evaluated using H2O2 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 H2O2 (hydroquinone) and low detection limit of 10 nM for H2O2 and 100 nM for hydroquinone. Moreover, high sensitivity for H2O2 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.

Emerging synergy between nanotechnology and implantable biosensors: a review.

The development of implantable biosensors for continuous monitoring of metabolites is an area of sustained scientific and technological interests. On the other hand, nanotechnology, a discipline which deals with the properties of materials at the nanoscale, is developing as a potent tool to enhance the performance of these biosensors. This article reviews the current state of implantable biosensors, highlighting the synergy between nanotechnology and sensor performance. Emphasis is placed on the electrochemical method of detection in light of its widespread usage and substantial nanotechnology based improvements in various aspects of electrochemical biosensor performance. Finally, issues regarding toxicity and biocompatibility of nanomaterials, along with future prospects for the application of nanotechnology in implantable biosensors, are discussed.

Enhanced glucose sensor linearity using poly(vinyl alcohol) hydrogels.

BACKGROUND: High linearities, sensitivities, and low oxygen dependence constitute prime requisites for electrochemical glucose sensors. However, for implantable sensors the need to control tissue inflammation requires the use outer membranes that permit inward analyte diffusion while continuously releasing anti-inflammatory drugs and other tissue response-modifying (TRM) agents. We have shown previously that while outer membranes based on layer-by-layer (LBL) assembly enhance linearity, poly(vinyl alcohol)(PVA) hydrogels loaded with TRM-containing microspheres enable a significant reduction in tissue inflammation. This article discusses amperometric performance of glucose sensors coated with stacked LBL/PVA hydrogel outer membranes. METHODS: Sensors were fabricated by immobilizing glucose oxidase enzyme on a 50-microm platinum wire followed by deposition of stacked LBL/PVA hydrogel outer membranes. The sensor response to various glucose concentrations was determined by applying 0.7 V vs an Ag/AgCl reference electrode in phosphate-buffered saline (37 degrees C). Michaelis-Menten analysis was performed to quantify sensor performance in terms of linearity (K(m,glu)(app)) and oxygen dependence (K(m,O(2))(app)/[Glucose]). RESULTS: When overlaid onto LBL-assembled outer membranes, PVA hydrogels improved sensor linearity by 60% from 10 to 16 mM of glucose and resulted in a twofold decrease in oxygen dependence. CONCLUSIONS: Enhancement in the performance of a PVA-coated sensor is attributed to the oxygen-storing capability of PVA hydrogel due to the formation of hydrophobic domains during its freezing and thawing employed to physical cross-link the PVA. Such membranes with the capability to release TRMs continuously while storing oxygen constitute a major improvement over current outer membrane technologies.

Layer-by-layer assembled semipermeable membrane for amperometric glucose sensors.

BACKGROUND: The performance of implantable glucose sensors is closely related to the behavior of the outer membrane. Such membranes govern the diffusion characteristics of glucose and, correspondingly, the sensitivity of the sensors. This manuscript discusses the selection of various membrane materials and their effect on the device response. METHODS: Sensors were fabricated utilizing a 50-microm platinum wire followed by immobilization of the glucose oxidase (GO(X)) enzyme. Sequential adsorption of various ionic species via a layer-by-layer process created devices coated with bilayers of humic acids/ferric cations (HAs/Fe(3+)), humic acids/poly(diallyldimethylammonium chloride) (HAs/PDDA), and poly(styrene sulfonate)/poly(diallyldimethylammonium chloride) (PSS/PDDA). The in vitro amperometric response of the sensors was determined at 0.7 V vs an Ag/AgCl reference electrode in phosphate-buffered saline (37 degrees C) for various glucose concentrations. The diffusion coefficients of glucose and hydrogen peroxide (H(2)O(2)) through these membranes were calculated and analyzed. RESULTS: Outer membranes based on the sequential deposition of bilayers of HAs/Fe(3+), HAs/PDDA, and PSS/PDDA were grown successfully on immobilized layers of GO(X). The amperometric response and reversibility upon changing the in vitro concentration of glucose were investigated. CONCLUSIONS: Through alteration of the number of bilayers of the outer membrane, it was possible to modulate the diffusion of glucose toward the sensor as a result of its flux-limiting characteristics. Semipermeable membranes based on five HAs/Fe(3+) bilayers exhibited a superior behavior with a minimum hysterisis response to glucose cycling and a lesser current saturation at hyperglycemic glucose concentrations because of a more balanced inward diffusion of glucose and outward diffusion of H(2)O(2).