Santhisagar Vaddiraju

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.

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.

The role of H2O2 outer diffusion on the performance of implantable glucose sensors

The performance of an implantable glucose sensor is strongly dependent on the ability of their outer membrane to govern the diffusion of the various participating species. In this contribution, using a series of layer-by-layer (LBL) assembled outer membranes, the role of outwards of H(2)O(2) diffusion through the outer membrane of glucose sensors has been correlated to sensor sensitivity. Glucose sensors with highly permeable humic acids/ferric cations (HAs/Fe(3+)) outer membranes displayed a combination of lower sensitivities and better linearities when compared with sensors coated with lesser permeable outer membranes (namely HAs/poly(diallyldimethylammonium chloride) (PDDA) and poly(styrene sulfonate) (PSS)/PDDA). On the basis of a comprehensive evaluation of the oxygen dependence of these sensors in conjunction with the permeability of H(2)O(2) through these membranes, it was concluded that the outer diffusion of H(2)O(2) is crucial to attain optimized sensor performance. This finding has important implications to the design of various bio-sensing elements employing perm-selective membranes.

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 of 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 the 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-μm 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°C). Michaelis—Menten analysis was performed to quantify sensor performance in terms of linearity (K app m,glu) and oxygen dependence (K app m,O2 /[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.

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.

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.

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-μm platinum wire followed by immobilization of the glucose oxidase (GOx) enzyme. Sequential adsorption of various ionic species via a layer-by-layer process created devices coated with bilayers of humic acids/ferric cations (HAs/Fe3+), 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°C) for various glucose concentrations. The diffusion coefficients of glucose and hydrogen peroxide (H2O2) through these membranes were calculated and analyzed. Results: Outer membranes based on the sequential deposition of bilayers of HAs/Fe3+, HAs/PDDA, and PSS/PDDA were grown successfully on immobilized layers of GOx. 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/Fe3+ 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 H2O2.

Microsphere erosion in outer hydrogel membranes creating macroscopic porosity to counter biofouling-induced sensor degradation.

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, present 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 phosphate buffered saline (PBS) led to a monotonic increase in glucose permeability (50%), with a corresponding enhancement in sensor sensitivity over a 1 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 2-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.

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, H2O2, O2) 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 Clarks 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/Fe3+) 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/Fe3+) sensors. Upon closer inspection of H2O2 diffusion profiles, this non-typical higher response from PSS/PDDA is not due to either a larger glucose flux or comparatively larger O2 concentrations within the sensor geometry, but rather is attributed to a 48% higher H2O2 concentration in the glucose oxidase enzyme layer of PSS/PDDA coated sensors as compared to HAs/Fe3+ coated ones. These simulated results corroborate our experimental findings reported previously. The high concentration of H2O2 in the PSS/PDDA coated sensors is due to the low permeability of H2O2 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.

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.