Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
2001-11-21
2004-11-09
King, Roy (Department: 1742)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S489000, C204S507000, C205S106000, C205S149000, C205S198000, C205S317000, C435S176000, C435S817000
Reexamination Certificate
active
06814845
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is broadly concerned with improved implantable biosensors and methods of production thereof. More particularly, the invention pertains to biosensors having enhanced sensitivity and stability characteristics coupled with relative ease of manufacture. The biosensors are preferably fabricated using enzymes, and especially oxidase enzymes such as glucose oxidase.
2. Description of the Prior Art
The enzyme electrode, and especially the glucose electroenzymatic biosensor, has served for more than 25 years as a valuable clinical tool for detecting and monitoring diabetes. A majority of glucose sensors, especially those used in in-vivo applications, are based on the rate of glucose oxidase-catalyzed oxidation of glucose by dioxygen, where the rate of the reaction is measured by monitoring the formation of hydrogen peroxide or the consumption of oxygen. The fabrication of such a sensor involves the controlled deposition of a permselective polymer layer used to eliminate interferences such as ascorbate, urate and acetaminophen, an enzyme layer, and an outer layer that renders the sensor response mass transfer rather than kinetically controlled and which also provides a biocompatible interface with the surrounding environment. The use of thick film techniques, including screen printing, has been demonstrated to be successful in the preparation of sensors with reasonably reproducible characteristics, and this approach has been applied, for example, in the electrochemically-based sensors used for self-monitoring of blood glucose as marketed by Abbott Laboratories (Medisense) and others. If, however, it is desired to employ a cylindrical geometry or to prepare a sensor array, then the reproducible deposition of the various functional layers becomes significantly more complicated. Thus it would be of considerable advantage to control the preparation of the sensor electrochemically, especially when the sensing elements in an array are themselves electrochemically addressable. This would also allow for the deposition of different enzymes in various parts of the array.
Electropolymerization makes it possible to generate a coating on small electrodes of complex geometry and to do so precisely in one or two rapid and simple steps. In general, electrochemically-mediated fabrication of biosensors has been accomplished in two ways. First, a polymer layer is formed directly on the electrode, and polymers formed from such monomers as pyrrole, aniline, tyramine, o-aminophenol and o-phenylenediamine have been used to create a permselective layer before or after the application of enzyme solution and cross-linking with glutaraldehyde. A second approach involves the entrapment of enzyme in a growing polymer network by co-polymerization of enzyme and monomer. In some cases a monomer unit is attached to the enzyme to facilitate this process. Yacynych employed a copolymer of 1,3-diaminobenzene and resorcinol as the preferred film for blocking interferences from the surface of carbon or partially platinized carbon electrodes (Geise, R. J.; et al.,
Biosens. Bioelectron
., 1991, 6, 151-160). Vadgama and coworkers found that electropolymerized 4-aminophenol and then phenol constituted an exceptionally selective film against acetaminophen and ascorbate in glucose biosensors (Eddy, S.; et al.,
Biosens. Bioelectron
., 1995, 10, 831-839). Curulli et al (Carelli, I.; et al.,
Electrochim. Acta
, 1996, 41, 1793-1800 reported the results that poly(1,3-diaminobenzene/catechol) was the most efficient polymer to prevent the interference of acetaminophen. It has also been pointed out that electropolymerized films have significantly different characteristics when formed on different electrode materials and under different electropolymerization conditions. Experience has shown that these approaches typically give sensors of moderate activity but often high selectivity, but that both of these essential characteristics deteriorate quickly over a period of several days. The fact that the diffusion of enzyme and monomer cannot proceed at the same rate makes it difficult to enrich the composite layer with enzyme without at the same time degrading the permselective properties of the polymer.
U.S. Pat. Nos. 5,540,828, 5,286,364, 5,165,407, 5,310,469, 5,411,647, 5,166,063 and 4,721,677 describe various types of electrochemical biosensors.
SUMMARY OF THE INVENTION
The present invention overcomes many of the problems outlined above and provides biosensors such as implantable glucose sensors which can be economically prepared and which have excellent selectivity and stability characteristics far in excess of typical prior art sensors. Selectivity, or the ability to exclude electroactive interferants, is defined as, in the case of a glucose sensor, the percent change in the electrical signal at 5 mM glucose according to the relationship % Interf=((I
Tot
−I
Glu
)/I
Glu
)×100, where I
Glu
is the current response for glucose and I
Tot
is the current response of the glucose and interferant. Current response is defined as the current produced by the sensor in response to the analyte. The current response produced by the sensor in response to the analyte divided by the analyte concentration is herein referred to as the sensor sensitivity. For a biosensor prepared in accordance with this invention, the ratio of the current response at 5 mM glucose to that for 0.1 mM acetaminophen should be no less than about 100 in vitro.
Broadly speaking, a method of preparing a biosensor in accordance with the invention includes providing an electrically conductive biosensor electrode, which is immersed with a reference electrode in an aqueous conductive dispersion containing an enzyme and a non-ionic surfactant, the latter being present in an amount at least equal to the critical micelle concentration for the surfactant in the dispersion. A potential is then applied across the electrodes, causing the enzyme to deposit on the biosensor electrode. Next, the enzyme-deposited biosensor electrode is immersed in synthetic monomer and an electropolymerization procedure is carried out to create a polymer layer on the electrode which is intermingled with the initially deposited enzyme. In preferred forms, the electrode is next coated with a silane and then a polyurethane to complete the biosensor.
In more detail, the biosensor electrode (or more broadly an electrically conductive substrate) is typically formed of a metal alloy such as Pt—Ir or other noble metal; however, other conductive electrodes such as graphite electrodes can also be used. Normally, the electrode is in the form of a thin wire (having a diameter of from about 0.01 to 0.3 millimeters), and may have insulation over the majority of the length of the wire, leaving only a small stripped section to serve as an enzyme-receiving zone.
Virtually any enzyme can be deposited on the biosensor electrode, depending upon the nature of the analyte to be detected. The most useful enzymes are the oxidase enzymes, such as those selected from the group consisting of glucose, lactate, oxalate, D-aspartate, L-amino acid, D-amino acid, galactose, sarcosine, urate, ethanol, lysine, glutamate, cholesterol, glycerol, pyruvate, choline, ascorbate, and monoamine oxidases. Of these, the glucose, lactate, pyruvate, glutamate, cholesterol and choline oxidase enzymes are the most important.
During the initial deposition of the enzyme, it is preferred to employ an aqueous dispersion (normally a solution) which is buffered to a pH of about 7-8, more preferably about 7, containing the enzyme and a compatible surfactant (i.e., a surfactant which will facilitate enzyme deposition and not otherwise interfere with the desired electrodeposition). A variety of surfactants can be used in this context, although the nonionic surfactants are most preferred. One class of surfactants has been found to be particularly useful, namely the Triton surfactants, which are octylphenol ethoxylates, produced by the polymerization of octylphenol with ethy
Chen Xiaohong
Hu Yibai
Matsumoto Norio
Wilson George S.
King Roy
Leader William T.
Stinson Morrison & Hecker LLP
University of Kansas
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