Molecular wire injection sensors

Details Molecular wire injection sensors Molecular wire injection sensors

2001-09-20

2004-03-02

Chin, Christopher L. (Department: 1641)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C204S400000, C204S403060, C422S082010, C422S082020, C435S004000, C435S287100, C435S287200, C436S149000, C436S150000, C436S151000, C436S518000, C436S524000, C436S525000, C436S531000, C436S806000

Reexamination Certificate

active

06699667

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to biosensors and chemical sensors. More particularly, it relates to sensors having a chemical or biochemical species detection group connected to an electronic circuit by electrically conducting polymer strands.
Biosensors employing enzymes have been applied to the detection of numerous analyte species concentrations including glucose, cholesterol, or both glucose and cholesterol concentrations in whole blood samples. Such sensors and associated instruments employ an enzyme capable of catalyzing a reaction at a rate representative of the selected compound concentration in an assay mixture.
There are three general detection approaches employing a glucose enzyme electrode. The first and earliest measures oxygen consumption. The oxygen-sensing probe is an electrolytic cell with a gold (or platinum) cathode separated from a tubular silver anode by an epoxy casting. The anode is electrically connected to the cathode by electrolytic gel, and the entire chemical system is isolated from the environment by a thin gas-permeable membrane (often Teflon). A potential of approximately 0.8V (from solid-state power supply) is applied between the electrodes. The oxygen in the sample diffuses through the membrane and is reduced at the cathode with the formation of the oxidation product, silver oxide, at the silver anode. The resultant current is proportional to the amount of oxygen reduced. The analyzer unit operates over the range from 0.2 to 50 ppm of dissolved oxygen. Gases that reduce at 0.8V will interfere; these include the halogens and SO
2
. H
2
S contaminates the electrodes.
A second approach detects H
2
O
2
production but requires an applied potential of approximately 0.65V (from solid-state power supply) applied between the electrodes, one of which is inside a permselective membrane. The H
2
O
2
in the sample diffuses through the permselective membrane (if one is present) and is oxidized at the anode. Many metal, metal complexes, nonmetal, organic and biochemical species that oxidize at approximately 0.65V will interfere; such as ascorbic acid, amines, hydrazines, thiol compounds, catechols, hydroquinones, ferrocenes, and metalloporphyrins. The inside permselective membrane is not always capable of removing the complicated mix of possible interferences from the analyte matrix.
A third approach takes advantage of the fact that the enzymatic reaction requires two steps. First, the enzyme glucose oxidase (GOD) (EC 1.1.3.4) is reduced by glucose, then the reduced enzyme is oxidized to its initial form by an electron acceptor, i.e., a mediator. In natural systems, the mediator is oxygen. In biosensors, another mediator compound may be employed to transfer electrons between the enzyme and a conductive surface of an electrode at a rate representative of the enzyme catalyzed reaction rate when an appropriate potential is applied to the particular redox mediator in use. Such biosensors may employ amperometric measurements to determine glucose concentration in a whole blood sample. This involves an integrated sample measurement of the area under the ampere versus time curve, corresponding to the amount of glucose in the sample.
The mechanism by which a common amperometric sensor works is depicted in
FIG. 1. A
sensor
2
employs glucose oxidase (GOD), for example, as a molecular recognition group. Glucose oxidase catalyzes the oxidation of glucose to gluconolactone in analyte
4
. This reaction involves the FAD/FADH
2
redox center of the enzyme. Sensor
2
includes a molecular recognition group, region
6
, attached to an electrode
8
. When glucose in analyte
4
contacts GOD-FAD (glucose oxidase including the FAD redox center) in region
6
, it is oxidized to gluconolactone. At the same time, the GOD-FAD is reduced to GOD-FADH
2
. This involves two electrons and two hydrogen ions being transferred to the FAD. Normally, in the absence of a sensor mediator, the GOD-FADH
2
is reoxidized by atmospheric oxygen to GOD-FAD to complete the catalytic reaction. In the presence of a mediator, however, the GOD-FADH
2
is sometimes reoxidized by a mediator (M
OX
). In this case, the GOD-FADH
2
releases two hydrogen ions to analyte
4
and two electrons to the mediator. The resulting reduced mediator (M
red
) may then be reoxidized by electrode
8
at an appropriate potential. The reoxidation of the mediator is accompanied by the transfer of an electron or electrons to electrode
8
. This is the current that is monitored to provide a concentration of glucose.
In theory, a mediator may be any small molecule inorganic, organometallic or organic compounds, which are reduced by the enzyme, and oxidized by an appropriate applied potential at the electrode surface. The mediator should be designed to rapidly and efficiently transfer electrons between the enzyme and the electrode. Otherwise, ambient oxygen would oxidize nearly all of the reduced GOD and the desired signal would be very weak. The mediator should also transfer a total charge proportional to the glucose or cholesterol concentration in the sample. The current which results from the mediator oxidation is known as the Cottrell current which, when integrated with respect to time, gives the number of coulombs associated with the sensor reaction. The total coulombs passed is proportional to the amount of analyte.
Unfortunately, mediators are commonly provided as mobile “reagents” which diffuse to the enzyme where they are oxidized or reduced (depending upon the reaction catalyzed by the enzyme). The oxidized or reduced mediator then diffuses to the electrode surface where it gains or loses an electron. Unfortunately, such mechanism is dependent upon the continuing presence of recycled mobile mediators. As such compounds can leak from the electrode surfaces, there may be a gradual depletion in available mediator and a consequent reduction in sensor sensitivity. Examples of diffusing redox mediators include dyes (e.g., methylene blue), ferrocene derivatives (Cass, A E G; Davis, G; Francis, G D; Hill, H A O; Aston, W J; Higgins, I J; Plotkin, E V; Scott, L D L; Turner, A P F: Ferrocene-Mediated Enzyme Electrode for Amperometric Determination of Glucose. Anal. Chem. 56:667-671, 1984), components of conducting organic metals and quinones.
Also, available sensors applying the above amperometric approach to the detection of glucose, cholesterol, lactate, H
2
O
2
, NAD(P)H, alcohol, and a variety of other compounds in whole blood samples, can have other serious complicating problems. For example, the percentage of sensor surface area covered by blood can vary; sometimes the blood sample does not cover the entire electrode. This may be caused by a poorly adherent enzyme (often applied by spraying) thus allowing leakage of blood or other analytes along the edges of the electrode. A related problem results from hydration of the reaction area prior to test. This dilutes the ligand (e.g., glucose) concentration and therefore gives a lower reading than would be accurately given by an unhydrated surface.
Further, the partial pressure of molecular oxygen (O
2
) may complicate the interpretation of sensor data. Molecular oxygen is the natural electron acceptor mediator of the enzyme glucose oxidase (GOD). Following oxidation of D-(+)-glucose by GOD, reduced glucose oxidase (GOD
red
) will transfer electrons to O
2
forming H
2
O
2
in the absence of other mediators. In amperometric glucose biosensors described above, the unwanted O
2
side reaction competes with synthetic chemical mediators for electrons supplied by the GOD
red
enzyme. Calibration of GOD-based biosensors at different altitudes (i.e., different partial pressures of O
2
) may be a problem if electron transfer rates of selected synthetic chemical mediators are not orders of magnitude faster than the O
2
side reaction.
Humidity (i.e., H
2
O) may be another potential problem if mass action of H
2
O and O
2
present drives the enzyme catalyzed oxidation product of D-gluconolactone in reverse back to the reduced starting material, D-(+

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