A two electrode assembly in a polarographic cell includes a sensor electrode and a silver/silver chloride reference electrode in which silver metal is in contact with a substantial reservoir of silver chloride.

BACKGROUND OF THE INVENTION 
This invention relates to polarographic cells including a silver/silver 
chloride reference electrode. 
In polarographic cells having two electrodes, one electrode is polarized in 
the presence of the substance being measured and is known as the sensor 
electrode. The second electrode is depolarized in the presence of the 
substance being measured and is known as the reference electrode. An 
electrolyte generally provides an electrical path between the two 
electrodes, which otherwise are in an electrically insulating relation. In 
the presence of the substance being measured a current passes between the 
electrodes, the current being, at a given applied voltage, proportional to 
the concentration of the substance. The voltage applied to the cell is the 
potential of the sensor relative to that of the reference electrode; the 
potential of the reference electrode should therefore be constant. The 
current passing between the electrodes may result in undersirable 
potential changes at the reference electrode. To counter this effect a 
third, counter, electrode is often used so that the current generated will 
pass between the sensor and counter electrode. 
SUMMARY OF THE INVENTION 
In general, the invention features a two electrode assembly for use in a 
polarographic cell. One electrode in the assembly is a standard sensor 
electrode. The second electrode is a silver/silver chloride reference 
electrode in which silver metal is in contact with a reservoir of silver 
chloride. During the operation of the assembly a current passes between 
the two electrodes and causes silver ion to be reduced to silver metal. 
The silver chloride reservoir supplies sufficient silver ion so that the 
effect of the consumption of the silver ion on the potential of the 
reference electrode is minimal, thereby avoiding the necessity of using a 
counter electrode. 
By reservoir, it is meant that enough silver chloride is present so that 
the assembly can be used for at least 1,000 assays, preferably 2,000 or 
more, without using up the supply; more than just a thin (less than 
10.mu.) layer of silver chloride must be present. 
In a preferred embodiment, the reference electrode is a silver ring 
surrounded (at any position on the ring) by a concentric ring of silver 
chloride that is at least 25.mu. thick, more preferably at least 0.01 cm 
thick; and the sensor electrode (e.g., a standard platinum electrode) is 
disposed inside the concentric rings. 
A preferred use of this assembly is in enzyme electrodes in which a 
laminated membrane covers the solution-contacting face of the sensor and 
reference electrodes. The laminated membrane generally includes an inner 
membrane adjacent the solution-contacting face; an outer 
solution-contacting membrane; and an adhesive enzyme layer between the 
membranes holding them together. The substance being assayed (e.g., 
glucose) is oxidized by the enzyme to generate a second substance, e.g., 
H.sub.2 O.sub.2, to which the sensor electrode is sensitive. A preferred 
construction of the laminated membrane is described in Young et al., U.S. 
Ser. No. 152,836, filed Feb. 5, 1988, of which the present application is 
a continuation in-part and which is hereby incorporated by reference 
herein. 
The electrode assembly is easy to construct, easy to handle, and can be 
used for many assays without needing to replenish the supply of silver 
chloride. Advantageously, only two electrodes need be used in the cell.

Referring to the FIG. 1, a glucose electrode 10 comprises an electrically 
insulating support body 12 which may be of elongated cylindrical shape 
carrying at its end a platinum sensor electrode or anode 14 having an 
active or exposed face 16 and a conductor 18. The lower end of the support 
body 12 also carries a silver/silver chloride reference electrode 20 
having an exposed face 22 and a conductor 24. Conductors 18 and 4 lead to 
an amperometer (not shown). Disposed across the exposed faces of the 
electrodes is a laminated membrane including an outer membrane 26 and an 
inner membrane 28 adhesively secured together by an intermediate layer 30 
comprising the enzyme glucose oxidase, preferably a mixture of the enzyme 
and a cross-linking or binding agent such as glutaraldehyde. The laminated 
membrane is sealed in liquid-tight relation to the lower face of support 
body 12 by O-ring 32 or any other suitable means. 
The outer membrane 26, inner membrane 28, and intermediate layer 30, and a 
procedure for constructing the laminated membrane, are described in depth 
in Young et al., supra. 
In the embodiment shown in FIG. 1, a flow cell 34 is mounted in 
liquid-tight relation against the lower face of outer membrane 26, being 
sealed thereto by a silicone washer or by O-ring 32. Cell 34 may be 
constructed of polystyrene, polymethacrylate, or any other suitable rigid 
liquid impervious material and includes a chamber 36 exposed to the face 
of outer membrane 26 as well as inlet 38 and outlet 40. In a preferred 
embodiment, the volume of chamber 36 together with inlet 38 and outlet 40 
is approximately 5 to 10 microliters. 
Referring to FIG. 2, the support body 12 has 0.016 inch diameter central 
platinum sensor electrode 14 surrounded by concentric rings including one 
of lead glass (42) (Corning 0120 type; 0.095 inch O.D.); versilok 
structural adhesive (44) (0.005 inch thick); silver (46) (0.105 inch I.D.; 
0.125 inch O.D.); a 60-40 mixture of silver sulfide (Ag.sub.2 S)-silver 
chloride (AgCl) (48) (0.01 inch thick); potting material epoxy (50) (0.02 
inch thick); and Noryl (52) (0.337 inch O.D.). Rings 46 and 48 are the 
silver/silver chloride reference electrode 20, and are about 0.75 cm in 
height. The AgCl ring 48 is the same height as the silver metal ring and 
provides an adequate supply of silver ion so that the changes in potential 
at the reference electrode caused by the current is minimal. A reference 
electrode having an 0.01 inch thick ring can be used for thousands of 
measurements; a counter electrode is not needed with the assembly. In 
general, the AgCl ring should be at least 25.mu., more preferably at least 
0.01 cm thick, to provide the adequate supply of silver ion; there is no 
real upper limit on thickness, although as a practical matter the ring 
probably should not be thicker than about 0.5 cm. Ag.sub.2 S is mixed with 
the AgCl to make the ring easier to work with. 
The amount of silver chloride needed in the reservoir (in any assembly) to 
provide sufficient Ag.sup.+ for 2,000 assays depends on the amount of 
current in a typical assay, and the length of the typical assay. One 
skilled in the art will know how to readily obtain these values. For the 
preferred assembly, the typical current is about 5 nanoamps, and the 
length of a typical assay is 60 sec. This converts to 3.multidot.10.sup.-7 
coulombs per assay. The minimum amount of AgCl that should be in the 
reservoir is readily obtained by dividing the number of coulombs for 2,000 
assays by 96,400 coulombs/mole, and then multiplying the value obtained by 
the molecular weight of AgCl. 
In a typical assay, a body fluid, e.g., whole blood, is flowed through the 
inlet 38 and fills the sample chamber 36. When the outer membrane 26 
contacts the whole blood, glucose molecules and oxygen molecules present 
in the sample pas through it and contact the enzyme in layer 30; the 
enzyme catalyzes the oxidation of glucose to gluconic acid. The hydrogen 
peroxide produced during the oxidation passes through membrane 28 and 
contacts surface 16 of sensor electrode 14, which is poised at +700 mV in 
relation to reference electrode 20, and also contacts the face 22 of 
reference electrode 20, forming an electroconductive path between the two 
electrodes. A current is generated, the magnitude rising to a constant 
(steady state) value (response) related to the equilibrium concentration 
of the hydrogen peroxide. The current causes the consumption of Ag.sup.+ 
(from AgCl) in the reference electrode; because of the reservoir of AgCl, 
however, the effect of this consumption on the potential of the reference 
electrode is minimized for thousands of assays. 
OTHER EMBODIMENTS 
Other embodiments are within the following claims. For example, the 
preferred electrode can be designed to assay other substances besides 
glucose, provided the enzyme in the layer 30 oxidizes the substance to 
generate hydrogen peroxide, as desired in Young et al., supra. 
One skilled in the art will recognize that the AgCl reservoir in contact 
with the silver metal can be one-half or one-quarter the height of the 
silver ring, or can be a half-ring, quarter-ring, etc., so long as 
sufficient AgCl is present to avoid changes in potential. For example, a 
half-ring of AgCl (one-half of a concentric ring) of the same thickness as 
ring 48 can be used. Moreover, the silver metal can be non-ring shapes, 
provided that in whatever configuration is selected the metal is in 
contact with a AgCl reservoir. Furthermore, two electrode assemblies in 
accordance with the invention can, of course, be used in other types of 
polarographic cells, aside from enzyme electrodes having a laminated 
membrane.