Sean Brahim

Design of a subcutaneous implantable biochip for monitoring of glucose and lactate

The design, fabrication, and in-vitro evaluation of an amperometric biochip that is designed for the continuous in vivo monitoring of physiological analytes is described. The 2 /spl times/4 /spl times/0.5 mm biochip contains two platinum working enzyme electrodes that adopt the microdisc array design to minimize diffusional limitations associated with enzyme kinetics. This configuration permits either dual analyte sensing or a differential response analytical methodology during amperometric detection of a single analyte. The working enzyme electrodes are complemented by a large area platinized platinum counter electrode and a silver reference electrode. The biorecognition layer of the working electrodes was fabricated from around 1.0-/spl mu/m-thick composite membrane of principally tetraethylene glycol (TEGDA) cross-linked poly(2-hydroxyethyl methacrylate) that also contained a derivatized polypyrrole component and a biomimetic methacrylate component with pendant phosphorylcholine groups. These two additional components were introduced to provide interference screening and in vivo biocompatibility, respectively. This composite membrane was used to immobilize glucose oxidase and lactate oxidase onto both planar and microdisc array electrode designs, which were then used to assay for in vitro glucose and lactate, respectively. The glucose biosensor exhibited a dynamic linear range of 0.10-13.0 mM glucose with a response time (t/sub 95/) of 50 s. The immobilized glucose oxidase within the hydrogel yielded a K/sub m(app)/ of 35 mM, not significantly different from that for the native, solution-borne enzyme (33 mM). The microdisc array biosensor displayed linearity for assayed lactate up to 90 mM, which represented a 30-fold increase in linear dynamic lactate range compared to the biosensor with the planar electrode configuration. Preliminary in vitro operational stability tests performed with the microdisc array lactate biosensor demonstrated retention of 80% initial biosensor response after five days of continuous operation in buffer under physiologic conditions of pH and temperature.

Interferent Suppression Using a Novel Polypyrrole-Containing Hydrogel in Amperometric Enzyme Biosensors

Amperometric biosensors for three clinically important analytes; glucose, cholesterol and galactose were prepared using a novel polymer composite material consisting of a poly(hydroxyethylmethacrylate) [p(HEMA)] hydrogel intimately combined with polypyrrole (PPy), the appropriate enzyme and fabricated on platinum electrodes. These biosensors were evaluated for their sensitivity to two very common electrooxidizable interferents, ascorbic acid and L-cysteine. The composite polymer films showed effective suppression of these interferents in a serum matrix with a deviation of <5% of the biosensor response being observed at ascorbic acid and L-cysteine concentrations that were twice as high as the normal physiological levels found in serum (10 mg L−1 and 4 mg L−1 respectively). In contrast, biosensor films containing an external layer of peroxidase or Nafion were found to be less effective than the p(HEMA)/PPy composite films in screening these two interferents. The sieving properties of the crosslinked hydrogel and the anion exchange properties of the cationic polypyrrole together with its “dopant” anion effectively inhibit transport of these anion interferents to the electrode.

DESIGN AND CHARACTERIZATION OF A GALACTOSE BIOSENSOR USING A NOVEL POLYPYRROLE-HYDROGEL COMPOSITE MEMBRANE

ABSTRACT A rapid, two-step method for constructing galactose biosensors by entrapment of galactose oxidase within a polymeric composite has been developed. The composite material is formed as an interpenetrating network of polypyrrole grown within a UV cross-linked poly(2-hydroxyethyl methacrylate) [p(HEMA)] hydrogel. The optimized galactose biosensor exhibited a linear response range from 5.0 × 10−5 to 1.0 × 10−2 M and detection limit of 25 µM toward galactose. The response time of the biosensor was 70 s. The analytical recovery of galactose in serum samples ranged from 97 to 105% with mean coefficients of variation of 3.8% (within-day analyses) and 4.4% (day-to-day analyses). The biosensor was effective in screening up to twice the physiological levels of ascorbate, urate and acetaminophen interferents and retained 70% of initial enzyme activity after 9 months when stored desiccated in the absence of buffer at 4°C.

COMPOSITE HYDROGELS CONTAINING POLYPYRROLE AS SUPPORT MEMBRANES FOR AMPEROMETRIC ENZYME BIOSENSORS

Conducting polymers and redox hydrogels are shown to be attractive materials for biocompatible electrodes in amperometric biosensors. We have combined electrically conducting polypyrrole (PPy) with crosslinked poly(2-hydroxyethylmethacrylate) (p-HEMA) to produce a novel composite hydrogel membrane. The high water content of these materials provides a biocompatible environment for the long-term immobilization of enzymes and a more favorable medium for the rapid movement of charge neutralizing ions. Electrode-supported composite films were prepared by UV polymerization of the hydrogel component (containing dissolved enzyme) followed immediately by electrochemical polymerization (+0.7V vs. Ag/AgCl) of the pyrrole component within the interstitial spaces of the pre-formed hydrogel network. Typical monomer compositions consisted of HEMA:TEGDA:pyrrole in an 85:10:05 vol%. (TEGDA = tetraethyleneglycol diacrylate). An optimized glucose biosensor displayed a wide linear response range of 5.0 × 10−5 to 2.0 × 10−2 M, a detection limit (3Sy/x/sensitivity) of 25 μM and a response time of 35–40 seconds. The analytical recovery of glucose in serum samples ranged from 98 to 102% with mean coefficients of variation of 4.4% (within-day analyses) and 5.1% (day-to-day analyses). The optimized cholesterol and galactose biosensors also displayed wide linear response ranges (5.0 × 10−4 – 1.5 × 10−2M and 1.0 × 10−4 – 1.0 × 10−2M, respectively) towards their respective substrates. All three biosensors retained > 70% of initial activity after 9 months when stored desiccated in the absence of buffer. An attractive feature with all the biosensors was their ability to effectively screen the endogenous interferents ascorbic acid, uric acid, L-cysteine and acetaminophen. This characteristic, coupled with the high biocompatibility of the polymeric hydrogel composites make these mate rials potential candidates for in-vivo biosensors.

Bio-smart materials: Kinetics of immobilized enzymes in p(HEMA)/p(Pyrrole) hydrogels in amperometric biosensors

Various strategies are being pursued to confer the highly specific molecular recognition properties of bioactive molecules to the transducer action of inherently conductive polymers. We have successfully integrated inherently conductive polypyrrole within electrode-supported, UV cross-linked hydroxyethyl methacrylate (HEMA)-based hydrogels. These electroactive composites were used as matrixes for the physical immobilization of several oxidase enzymes to fabricate clinically important biosensors. Measurements were made of the amperometric responses via H 2 O 2 oxidation for each biosensor. Apparent Michaelis constants, K m(app) , for glucose oxidase immobilized in p(HEMA) membranes and in p(HEMA)/p(Pyrrole) composite membranes were 13.8 and 43.7 mM respectively compared to 33 mM in solution. The inclusion of polypyrrole in the hydrogel network increased the thermal stability of the immobilized enzyme at 60°C by 30% and 40% compared to p(HEMA) membranes and solution phase respectively. The composite also yielded larger I max values (19 μA/cm -2 ) for glucose biosensors compared to similar glucose biosensors fabricated without the conducting polymer (15 μA). K m(app) values for cholesterol oxidase immobilized in the same composite films were ca. three orders of magnitude higher than the K m for the soluble enzyme. The polypyrrole component is shown to reduce diffusive transport but to confer thermal stability to these biosensors.

Molecularly engineered hydrogels for implant biocompatibility

The biocompatibility of biosmart polymer membranes synthesized from cross-linkable (2-hydroxyethyl methacrylate) (HEMA) and tetraethylene glycol diacrylate and containing different mole-percent polyethylene glycol methacrylate (PEGMA) and methacryloyloxyethyl phosphorylcholine (MPC), a phosphorylcholine-containing co-monomer, was investigated. The cytotoxicity (cell viability and proliferation) and the adhesion of extra cellular matrix proteins to these hydrogel surfaces were separately tested. Cell proliferation assays were conducted by cultivating human skeletal muscle fibroblasts onto the surfaces of these polymeric membranes prepared by in-situ polymerization in chemically derivatized 8-well cell-culture plates. The compositions containing MPC and PEGMA concentrations greater than 1.0 and 0.05 mole% respectively demonstrated good protein adhesion and cell viability (>90%) of human muscle fibroblast cells. Morphological deviances and partial colonization of the hydrogel surface has been noticed and suggests good compatibility of hydrogels for cellular viability but restricted proliferation. It is well known that the adsorption of proteins onto biomaterial surfaces modulates the cellular interaction with these surfaces. The extent of adsorption of fluorescein labeled proteins (laminin, collagen, and fibronectin) onto these polymer membrane surfaces was evaluated by measuring the resultant fluorescence intensity using a confocal fluorescence scanner.

Carbon-nanotube-modified electrodes for the direct bioelectrochemistry of pseudoazurin

The bioelectrochemistry of the blue copper protein, pseudoazurin, at glassy carbon and platinum electrodes that were modified with single-wall carbon nanotubes (SWNTs) was investigated by multiple scan rate cyclic voltammetry. The protein showed reversible electrochemical behavior at both bare glassy carbon electrodes (GCEs) and SWNT-modified GCEs (SWNT|GCEs); however, direct electrochemistry was not observed at any of the platinum electrodes. The effect of the carbon nanotubes at the GCE was to amplify the current response 1000-fold (nA at bare GCE to µA at SWNT|GCE), increase the apparent diffusion coefficient Dapp of the solution-borne protein by three orders of magnitude, from 1.35 × 10−11 at bare GCE to 7.06 × 10−8 cm2 s-1 at SWNT|GCE, and increase the heterogeneous electron transfer rate constant ks threefold, from 1.7 × 10−2 cm s−1 at bare GCE to 5.3 × 10−2 cm s−1 at SWNT|GCE. Pseudoazurin was also found to spontaneously adsorb onto the nanotube-modified GCE surface. Well-resolved voltammograms indicating quasi-reversible faradaic responses were obtained for the adsorbed protein in phosphate buffer, with Ipc and Ipa values now greater than corresponding values for solution-borne pseudoazurin at SWNT|GCEs and with significantly reduced ΔEp values. The largest electron transfer rate constant of 1.7 × 10−1 cm s−1 was achieved with adsorbed pseudoazurin at the SWNT|GCE surface in deaerated buffer solution consistent with its presumed role in anaerobic respiration of some bacteria.