Electrolysis: processes – compositions used therein – and methods – Electrolytic analysis or testing – Involving enzyme or micro-organism
Reexamination Certificate
2001-03-06
2003-04-22
Warden, Jill (Department: 1743)
Electrolysis: processes, compositions used therein, and methods
Electrolytic analysis or testing
Involving enzyme or micro-organism
C204S403100, C204S403110
Reexamination Certificate
active
06551496
ABSTRACT:
BACKGROUND
The invention relates to sensors and more particularly biosensors based upon a microporous architecture.
One of the earliest patents describing biosensors is U.S. Pat. No. 3,539,455 to Clark, in which a membrane-covered polarographic probe transduces hydrogen peroxide created by entrapped glucose oxidase enzyme. In the thirty years since that patent issued, many other patents have focused upon the layering of materials and reagents to yield better performing sensors. Many of these patents have in common a method of biosensor construction which generally consists of a membrane composed of various functional layers, including an immobilized enzyme layer, all superimposed upon an electrode, or set of electrodes. In this configuration the analytes, chemical co-factors or other influences all approach the electrochemical transducer from one side, the face, or front of the sensor. In its most common manifestation, the face of the electrode probe, covered by the membrane, is situated in a buffer-filled sample chamber into which a sample is injected. Some of the substrate diffuses through the membrane. Using a glucose sensor as an example, when the glucose contacts the immobilized oxidase enzyme, it is rapidly oxidized, producing hydrogen peroxide. Oxygen is necessary for the reaction to proceed; this substrate also must enter the sensor from the front. The oxidase enzyme catalyzes the reaction of glucose and oxygen to produce Glucono-&dgr;-lactone and hydrogen peroxide. The peroxide is, in turn, oxidized at the platinum anode, producing electrons. A dynamic equilibrium is achieved when the rate of H
2
O
2
production and the rate at which H
2
O
2
leaves the immobilized enzyme layer are constant and is indicated by a steady state response. The electron flow is linearly proportional to the steady state H
2
O
2
concentration and, therefore, to the concentration of the analyte, glucose.
In the sensor described above, when the sensor is exposed to high glucose concentrations it is very possible that insufficient oxygen will be present to support the conversion of all of the available glucose in the enzyme layer to the reaction products. For example, if the sensor is implanted, the oxygen levels in the blood are not sufficient to support the glucose reaction. In this case the reaction is considered to be “oxygen limited” and the sensor will not give a linear response to further increases in glucose concentration. Oxygen limitation may be caused by low oxygen concentration in the analyte matrix, e.g., blood, a relatively low diffusivity of oxygen through the materials used in the construction of the sensor and, lastly, relatively long distances (on a molecular scale) which separate the oxygen source and the reaction site. Efforts to circumvent these limitations have focused upon the materials, as well as, the basic sensor chemistry itself. Some of the contemporary development of this type of biosensor has focused upon the selection, or formulation, of materials which limit the diffusion of the analytes (e.g. glucose) more than the secondary substrate, in this case oxygen, so as to reduce non-linearity due to oxygen limitation. This approach is used in U.S. Pat. No. 5,882,494. While extending the typical linear range of the device, this approach also reduces the sensitivity of the device due to the increased diffusional resistance of the outer membrane to the analyte. This tradeoff between sensitivity and linearity has been a major obstacle to the use of these sensors. Sensors with good sensitivity usually exhibit poor linearity and vice-versa. In applications where the sensor is to be employed in-vivo the developer seeks to keep the device as small as possible, so as to minimize it's invasiveness. Smaller dimensions generally tend to restrict electrode area and consequently reduce the available signal. In applications such as these, it is desirable to preserve as much signal as possible, despite the reduced size.
SUMMARY OF INVENTION
One object of the invention seeks to reduce the effect of co-reactant concentration limitations in sensor design by proposing a new sensor microgeometry which, if implemented on the appropriate scale, augments substantially the concentration of oxygen, or other co-reactants or reagents, in the reaction zone of the sensor. This is accomplished in one embodiment by allowing analyte to enter the sensor from one side of the sensor, while allowing a co-substrate to enter from both sides of the sensor. The extreme thinness of the sensor that is feasible can serve to increase the angle of acceptance of the oxygen molecules, but more importantly, it can greatly increase the steepness of the oxygen concentration gradient, which in turn directly affects the rate of oxygen diffusion. The result of this design approach is sensor performance well beyond that obtained from traditional microscale devices employing unilateral orientation. Performance enhancements are especially apparent in the areas of sensitivity, linear range and low-oxygen tolerance.
A microporous biosensor is provided which comprises a substrate having at least one pore therein extending from a front surface of the substrate to the back surface, the pore having an enzyme-containing membrane near the front face of the pore on one surface of the substrate and an electrode in electrochemical conductive contact with the enzyme-containing membrane. The electrode can be deposited on the wall of the pore or adjacent to the perimeter of the pore on the back surface of the substrate. In one embodiment of the invention, a back membrane is provided across the back face of the pore. The back membrane can function as a hydrodynamic stabilizer to prevent fluids from flowing freely through the pore. The back membrane is permeable to a co-substrate, co-reactant or other reagent for the analytical reaction, which occurs as the analyte contacts the enzyme at or near the front face of the pore. In the most typical embodiments of the invention, the biosensor includes a plurality of pores which may be arranged in a random or ordered pattern within the substrate.
The invention is the result of studies directed to improving sensor performance. In one embodiment sensor performance is improved by preferentially supplying a co-substrate for the analytical reaction from the back side of the sensor through the pore. For example, one embodiment of the invention is a glucose sensor in which oxygen is preferentially supplied from the backside of the sensor. The sensor can be implemented in one mode in which the front and back faces of the sensor contact the same medium, e.g., blood, as in the case of an in vivo implanted sensor. The membrane at the front side of sensor is designed to allow an analyte such as glucose to diffuse into the membrane and react with a co-substrate such as oxygen. The back side membrane, however, is different than the front side and is designed to preferentially allow the co-substrate, often in the absence of analyte, to diffuse through the pore. In this way co-substrate supplied through the pore augments co-substrate supplied in the analyte-containing medium at the front face of the pore and overcomes co-substrate concentration limitations on the analysis.
In the second mode in which the sensor can be used, the medium to which the back of the sensor is exposed is different than the medium containing the analyte. The back side medium can be one which is augmented with the co-substrate. Using a microfluidic glucose analyzer as an example, the backside of the sensor can be open to the air, an oxygen gas reservoir or an oxygen-containing liquid so that oxygen preferentially diffuses through the back membrane and through the pore where it augments the oxygen in the blood which contacts the front face of the sensor.
In another manifestation of the invention, instead of preferentially supplying a co-substrate from the back side of the sensor, other reagents can be supplied. For example, in many cases it may be desirable to maintain a predetermined pH at the enzyme membrane. By supplying an acidic,
Leland Quinn
Madaras Marcel
Moles Donald R.
Noguerola Alex
Thompson Hine LLP
YSI Incorporated
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