Christian N. Kotanen

Implantable enzyme amperometric biosensors

The implantable enzyme amperometric biosensor continues as the dominant in vivo format for the detection, monitoring and reporting of biochemical analytes related to a wide range of pathologies. Widely used in animal studies, there is increasing emphasis on their use in diabetes care and management, the management of trauma-associated hemorrhage and in critical care monitoring by intensivists in the ICU. These frontier opportunities demand continuous indwelling performance for up to several years, well in excess of the currently approved seven days. This review outlines the many challenges to successful deployment of chronically implantable amperometric enzyme biosensors and emphasizes the emerging technological approaches in their continued development. The foreign body response plays a prominent role in implantable biotransducer failure. Topics considering the approaches to mitigate the inflammatory response, use of biomimetic chemistries, nanostructured topographies, drug eluting constructs, and tissue-to-device interface modulus matching are reviewed. Similarly, factors that influence biotransducer performance such as enzyme stability, substrate interference, mediator selection and calibration are reviewed. For the biosensor system, the opportunities and challenges of integration, guided by footprint requirements, the limitations of mixed signal electronics, and power requirements, has produced three systems approaches. The potential is great. However, integration along the multiple length scales needed to address fundamental issues and integration across the diverse disciplines needed to achieve success of these highly integrated systems, continues to be a challenge in the development and deployment of implantable amperometric enzyme biosensor systems.

The effect of the physicochemical properties of bioactive electroconductive hydrogels on the growth and proliferation of attachment dependent cells

The physicochemical properties of soft electrode materials for the abio-bio interface of advanced biosensors and next generation bionic devices in the form of electroconductive hydrogels (ECH) of interpenetrating networks of polypyrrole formed within poly(hydroxyethylmethacrylate)-based hydrogels were examined. The 1.5 mol% UV-crosslinked tetraethyleneglycol diacrylate (TEGDA) (step 1) poly(HEMA) and the electropolymerized (step 2) polypyrrole co-networks were covalently joined by the inclusion of a bifunctional monomer (1.5 mol%), 2-methacryloyloxyethyl-4(3-pyrrolyl)butanate (MPB) that served to covalently link the two networks. The optical absorbance, degree of hydration, the frequency dependent electrical impedance and the elastic modulus were examined as a function of electropolymerization charge density (step 2) (1-900 mC/cm(2)) used to prepare the linked, interpenetrating co-networks. The absorption at 430 nm showed a monotonic increase with electropolymerization charge density and correlated with the increase in elastic modulus [56 (± 32)-499 (± 293) kPa], the decrease in % hydration (68-0%) and the decrease in membrane electrical resistance. Polypyrrole (PPy) grows initially from the gel-electrode interface to fill voids within the hydrogel and ultimately onto the surface of the hydrogel. Growth of attachment dependent Rhabdomyosarcoma (RMS13) and pheochromocytoma (PC 12) cells reflects this evolution, showing an increase to a maximal value and then to decrease again at high electropolymerization charge density.

Amperometric glucose biosensor based on electroconductive hydrogels.

Fabrication of an enzyme amperometric biosensor for glucose via electropolymerization of pyrrole in the presence of glucose oxidase onto a hydrogel coated platinum electrode is hereby established as a viable biotransducer fabrication method. Platinum micro- (φ=25 μm) and macro- (φ=100 μm) electrodes were electrochemically activated and chemically modified with 3-aminopropyl-trimethoxysilane (APTMS), functionalized with acryloyl(polyethyleneglycol)-N-hydroxysuccinamide (ACRL-PEG-NHS), dipped into a polyHEMA based hydrogel cocktail and UV cross-linked. Electropolymerization of Py in the presence of GOx produced glucose responsive biotransducers that showed; (i) a 4-fold reduction in sensitivity compared with directly electropolymerized PPy films, (ii) an electropolymerization charge density dependence of biotransducer sensitivity and enzyme activity that was maximal at 1.0 mC/cm(2) with an apparent K(M) of 33 mM, (iii) interference screening of ascorbic acid and (iv) a temporal increase in sensitivity with storage over a 17 days period. This method has the ability to precisely and quantitatively add enzyme catalytic bioactivity to metal or semiconductor biointerfaces for applications in biosensors, bioelectronics and bionics.

Characterization of a Wireless Potentiostat for Integration With a Novel Implantable Biotransducer

Wireless potentiostats are being developed and commercialized for use in the development of implantable electrochemical biosensors for the monitoring of physiological markers in a wide range of pathologies. The merits and drawbacks of the Pinnacle 8151 dual potentiostat were investigated for use with a novel implantable biotransducer, the dual responsive microdisc electrode array (MDEA 5037) of ABTECH Scientific. The potentiostat was evaluated with R and RC dummy cells and with MDEA transducers modified with polypyrrole-poly(2-hydroxyethyl methacrylate) electroconductive hydrogels that were all qualified by calibrated ac impedance spectroscopy. In a laboratory setting, the 8151 potentiostat maintained a steady and unbroken signal as far away as 75 ft (23 m), and when unobstructed, up to 100 ft (30 m). The error between the two channels was determined to be 6.4 (±0.3)% and 0.7 (±0.1)% for resistor-capacitor dummy cells ( RC=nominally 10 MΩ and 1 μF) and resistor dummy cells (R=nominally 10 MΩ), respectively. The intra-channel variability was judged to be too large for exacting analyte determinations.

Monitoring systems and quantitative measurement of biomolecules for the management of trauma.

Continued high morbidity and complications due to trauma related hemorrhage underscores the fact that our understanding of the detailed molecular events of trauma are inadequate to bring life-saving changes to practice. The current state of efficacy and advances in biomedical microdevice technology for trauma diagnostics concerning hemorrhage and hemorrhagic shock was considered with respect to vital signs and metabolic biomarkers. Tachycardia and hypotension are markers of hemorrhagic shock in decompensated trauma patients. Base deficit has been predicative of injury severity at hospital admission. Tissue oxygen saturation has been predicative of onset of multiple organ dysfunction syndrome. Blood potassium levels increase with onset of hemorrhagic shock. Lactate is a surrogate for tissue hypoxia and its clearance predicts mortality. Triage glucose measurements have been shown to be specific in predicting major injuries. No vital sign has yet to be proven effective as an independent predictor of trauma severity. Point of care (POC) devices allow for rapid results, easy sample preparation and processing, small sample volumes, small footprint, multifunctional analysis, and low cost. Advances in the field of in-vivo biosensors has provided a much needed platform by which trauma related metabolites can be monitored easily, rapidly and continuously. Multi-analyte monitoring biosensors have the potential to explore areas still undiscovered in the realm of trauma physiology.

Biofabrication Using Pyrrole Electropolymerization for the Immobilization of Glucose Oxidase and Lactate Oxidase on Implanted Microfabricated Biotransducers

The dual responsive Electrochemical Cell-on-a-Chip Microdisc Electrode Array (ECC MDEA 5037) is a recently developed electrochemical transducer for use in a wireless, implantable biosensor system for the continuous measurement of interstitial glucose and lactate. Fabrication of the biorecognition membrane via pyrrole electropolymerization and both in vitro and in vivo characterization of the resulting biotransducer is described. The influence of EDC-NHS covalent conjugation of glucose oxidase with 4-(3-pyrrolyl) butyric acid (monomerization) and with 4-sulfobenzoic acid (sulfonization) on biosensor performance was examined. As the extent of enzyme conjugation was increased sensitivity decreased for monomerized enzymes but increased for sulfonized enzymes. Implanted biotransducers were examined in a Sprague-Dawley rat hemorrhage model. Resection after 4 h and subsequent in vitro re-characterization showed a decreased sensitivity from 0.68 (±0.40) to 0.22 (±0.17) µA·cm-2·mM-1, an increase in the limit of detection from 0.05 (±0.03) to 0.27 (±0.27) mM and a six-fold increase in the response time from 41 (±18) to 244 (±193) s. This evidence reconfirms the importance of biofouling at the bio-abio interface and the need for mitigation strategies to address the foreign body response.

Wireless System with Multianalyte Implantable Biotransducer

Wireless potentiostats are being developed and commercialized for use in the development of implantable electrochemical biosensors for the monitoring of physiological markers in a wide range of pathologies. The merits and drawbacks of the Pinnacle Technology 8151 dual potentiostat are investigated for use with a novel implantable biotransducer, the dual responsive MDEA 5037 of ABTECH Scientific. In a laboratory setting, the 8151 potentiostat maintained a steady and unbroken signal as far away as 75 ft (23 m) and, when unobstructed, up to 100 ft (30 m). The percentage error between the two channels was determined to be 6.4 (±0.3) and 0.7 (±0.1) for resistor-capacitor dummy cells (RC = 10 MΩ and 1 μF) and resistor dummy cells (R = 10 MΩ), respectively. The intrachannel variability may be too large for exacting analyte determinations.

Enzyme biotransducers formed from conductive electroactive polymers

Three types of biotransducer devices were studied: an enzyme Field Effect Transistor (Enz-FET), an interdigitated microsensor electrode (IME) array for impedimetric detection, and a microdisc electrode array for amperometric detection. Each device type was microlithographically fabricated from a pattern of sputter deposited metal (10 nm Ti/W| 100 nm Pt or Au) on an insulating substrate that was either glass or oxidized p-type silicon. Insulating regions were fashioned from Si3N4 and the gate dielectric of the FET was a SiO2 layer of 100 nm thick spin-on glass. For each biotransducer device type, the enzyme glucose oxidase (GOx) was immobilized using a process of electropolymerization of pyrrole (0.8 V vs. Ag/AgCl) in the presences of glucose oxidase (1 mg/ml) in deionized water to achieve a total of 100 mC/cm2. The dose-response characteristics of each of the biotransducers were tested in muta-rotated glucose solutions (PBS 7.2) at RT that ranged from 0.0 mM to 20 mM. The sensitivity (m=slope), linear dynamic range and limits of detection [3(SDblank/m)] were calculated from replicate measures of the dose response curve.

Frontiers of More than Moore in Bioelectronics and the Required Metrology Needs

Silicons intersection with biology is a premise inherent in Moores prediction. Distinct from biologically inspired molecular logic and storage devices (more Moore) are the integration of solid state electronic devices with the soft condensed state of the body (more than Moore). Developments in biomolecular recognition events per sq. cm parallel those of Moores Law. However, challenges continue in the area of “More than Moore”. Two grand challenge problems must be addressed—the biocompatibility of synthetic materials with the myriad of tissue types within the human body and the interfacing of solid state micro‐ and nano‐electronic devices with the electronics of biological systems. Electroconductive hydrogels have been developed as soft, condensed, biomimetic but otherwise inherently electronically conductive materials to address the challenge of interfacing solid state devices with the electronics of the body, which is predominantly ionic. Nano‐templated interfaces via the oriented immobilization of si...