Note: Descriptions are shown in the official language in which they were submitted.
~20~
TITLE
ION-SELECTIVE LAYERED HALF CELL
FIELD OF THE IN~.ION
The inven~ion is directed to an elec~ro-
chemical sensor and, in particular, to an ion-~elec-
tive layered half cell which i8 u3eful or clinical
applications.
BACKGROUND 0~ THE lNvr;NlION
Electrochemical devices are used extensively
in clinical applications to detect electrical activ-
ity $n living ~ystems as in the ca~e o~ the electro-
c~rdiogram (ECG) and the electroencephalogram (EEG).
They are al~o widely used in the d inical analysis of
biological fluid~.
El~ctrochemical ~ensor~ can be devised to
sense a wide range of ~pecies of in~erest (analy~e)
including electrolytes, ~lood gases, metaboli es,
drugs and hormones. This, together with the relative
ease Q~ their fabrication and use, ~akes them ideally
~uited to he~lth care applications. They are, ~or
~x~ le, sen~itive, selective, compatible with whole
blood samples~ require relatively ~mall sample sizes
and are less prone to interferences, than other com-
monly used method~. ~he small size of such devices,
the fact that ~ew, if any, externaI reagent~ are
re~uired and the relatively simple instrumentation
involved make~ them desirable for use in emergency,
critical c~re and ~urgical settings.
other applications of electroc~e~ical
~ensors include per~onal dosimetry for monitoring
occupational exposure to toxic substances~ chemical
proce~s monitoring and control, microbial proce~
monitoring and the control b pro~thetic devices.
~ here is a need for improved-~lectrochemical
IP-0339 35 sensors which are dependable, versatile, stable on
, .~ .
~2~7027
standing and in storage and inexpensive to manufac-
ture ~o that ~any of the traditional disadvantages of
such sensor~ (i.e., poisoning, dri~ts and offsets)
can be minimized or even eliminated by ~i~posing of
5 the device after a singla use~
BRIEF SUMM~Y OF THE INV~W 1 IO~
~ he invention i8 there~ore directed primar-
ily to a layered hal~ cell compxising:
(a) a dimen~ionally stable chemically inert
10 electrically in~ulating substrate layer having coated
thereon
(b) a terminated layer of electroconductive
~aterial, the conductivity of which is at least 1 x
102 ~ohms-cm) 1 having coa~ed thereon
(c~ a layer of finely divided particle~ of
carbon uniformly dispersed in a ma~rix of organic
polymeric binder having coated thereon
(d) an ion-selective ~embrane layer compris-
ing an ionophoric material uniformly dispersed in a
20 matrix of dielectxic organic polymer, the layer hav
ing an i _ePA~ce at least two order~ of magnitude
greater ~an the i ~e~ce o~ the carbon layer, the
inter~ace between the ion-selective membrane layer
and the carbon dispersion layer compri~lng a zone in
25 w~ich the layers are part~ally interdiffu~ed. In
use, layers (b) and (c~ being shielded ~rom both
che~ical and ~lsctroconductive contact with any
analyte which ma~ be pre~ent.
- As used herein, the term "half cell" re~exs
30 to an assemblage of interfaces at which ioni~ motion
i5 converted to electron motion. A complete ~ensor
i~ compri~ed of two such half ~ells which ~ay also be
referred to a_ ~lectrode~, one of which i~ an indi-
cator and the o her a re~erence hal~ cell.
o
~V~)2~
In other aspects~ the inve~tion is directed
to sensors which employ the layered half cell~ of ~he
invention as sensing and reference electrodes a~d to
the u3e of such ~ensors for mea8uring the relative
5 concentrations of selected ions contained in a liquld
analyte.
BRIEF DESCRIPTION OF T~E DR~WI~G
The drawing, which consists of two sheets~
contain~ three igure~. Figure 1 is a schematic
10 ovexhead view o~ ~ sen~or incorporating two hal
ce~l~ o~ the invention and Figure 2 is a echema~ie
sec~ional view o~ the same sen-~or ~howing the various
layer~ thereo taken along a section defined by ~he
lines A-A. Figure 3 is a response curve for a sensor5 made in accordance with ~he invention.
PRIOR ART
Ion 3~1ective electrode hal~ cells are, o~
course, well known in the prior art, as is illuc-
trat~d by the pate~ts which are summarized below:
U.S. 4,020,830 ~ohnson et al.
Thi8 patent is direc~ed to a field effect
transistor (FET~ tra~sducer comprising a~ ion selec-
tive membrane deposited over th~ gate regio~ of ~he
FE~. ~en the "gate" i8 exposed to a test solution~
the electrochemical yotential modulates the gate
voltage and thus the source-to-drain current.
U.S. 4,053,381, Hamblen et al.
This patent i~ directed to a layered ele~-
trode comprising an ion-selective membrane layer
coated over an internal re~erence element supported
on a substra~e. ~he reference element can itsel~
con~i~t of several layers such a~ a metal layer in
contact with a soluble salt layer which, in turn, is
in contact with a layer o~ electrolyte dissolved in a
~olid hydrophilic bind~r such as polyvinyl alcohol,
agarose or deionized gelati~.
~2~02~
U.SO 4,133,735, A~ramowitz
The Af~amowitz patent is directed to a
layered ion-sensitive electrode co~pri~in~ a p~anar
sub~trate to which are bound separate first and
5 secona regions o~ conductive material. An ion-sen-
~itive membrane i3 bonded to a substrate wafer and
the first conductor region and the electrical output
~eans are connecte~ to the second conductor re~ion~
The wafer, the ~econd conductor region and the ou~put
10 ~eans are ~n~Apsul~ted to ~aXe the~ fluid-tight.
U.S. 4,225,410 Pace
The Pace pate~t iB directed primarily to an
array o electrodes ~or analyzi~g di~erent analytes
supported on a c: .o~ sub~trate. At least one of the
layered electrodes i8 for the purpose of calibration
and con ists o~ a ~ir~t electrode layer containing a
given co~e~tration of analyte and a ~econd electrode
layer ~ontaining a ~iven but different concen~ration
of analyte.
DETAILED DESCRIPTIO~ OF TH~ lhv~l~TO~
A. Ion-Selective ~embrane
Ion selective msmbranes have ~een u~ed
extensively in liquid membrane electrQdes, especially
in recent year~. The~e membranes basically are com-
2S prised o~ either an electrically charged or neutral
ligan~ ~ionophore) dispersed in an inert matrix.
A wide variety of io~horic materi~ls is
available for di~erent ion selectivity such as those
listed in Table.l which ~llow~
J
~Z~7~)2'7
s
TABLE 1
IO~OPHORIC MAT~RT~T..~ POR
MEMBRANE ELECTRODES
Analyte Ion Ionophore
Valinomycin
Crown ethers, e4g.,
dimethyldibenzo 30-
crown-10, dicyclo-
hexyl-18-crown,
dimethyldicy~lo~
hexyl-18-crown-6.
~a~ Methyl ~onensin,
Ca2l ~idecyl phosphoric acid
- dioctyl phenyl phosphate
Thenoyltri~luoroacetone +
tributylphosphate
Mono- and di-esters o~
phosphori~ acid ~ di~2-
ethylhexyl~2-ethylhexyl-
pho~phonate
Calcium dide~ylphosphate
di-(octylphenyl)
pho.phonate
Cal~ium di~octylphenyl)
phvsphate ~ dioctylphenyl-
phosphonate
B~2+ Calcium di-(2 ethylhexyl)
phosphate ~ decan-l-ol
Barium complex of nonyl-
p~enoYypoly ~ethylene
- - - oxy) ethanol in o-nitro-
. diphenyl ethar
Cl- Ag/AsCl with halide
scavenger Aliquat 33~S
in decan-l-ol shaken with
j aqueous solu~io~ of
appropriate sodium salt
1~C)702~7
TABL~ 1
HCO or total~(CO2~ Quaternary ammonium ion
3 exchanger p-octodecyloxy
m-chlorophenyl-hydrazone-
mesoxalonitrile (OCPH)
N~4~ Nonactin
Monactin
, NO3- Tridodecylhexadecylammonium
: nitrate I n-octyl-o-
nitrophenyl
1:10 phenanthxoline nickel
~II) nitrate ~ p-nitxo-
cymene
H~ Trioctylami~e
~5 As is shown in Examples 26-32, the concen-
tration of ionophore in the ion selective lay~r is
not highly critical. Typically, from about 0.5% by
weight of the layer can be used, with about 3.0% by
j weight ionophore being preferred to obtai~ maximum
sensitivity for a~y givsn system. ~till hi~her con-
centrations of ionophore can be used without any par-
ticular disadvantage, but no further benefit appears
to be obtained. However, it is im~ortant ~hat the
ion carrying capacity of She ionophoric layer not be
aturated. Therefore, it may be nece~sAry to in-
crease the ionophore level for analytes which havevery high co~centrations of the selec~ed ions.
By far th~ mo t widely used me~brane matri~
materials are noncrys~alline, hydrophobic polymers
3uch as those listed in Table 2 below:
lZ070~f
TABLE 2
HYDROPHOBIC POLYM~RS FOR
MEMB~ANE ELECTRODE MATRICE~
Poly(vinylchloride) ~PVC]
.' 5
~ Cellulose Acetate
Poly~bi3phenol-A carbonate)
Poly~iloxane/Poly(bisphenol-A carbonate3 block
copolymer
10 Poly ( methylmethacrylate ~
Poly(vinylidene chloride)
Polystyrene
LGwer alkyl acrylate and ~ethacrylate polymers a~d
15 copolymers
- Polyurethane
~ilicona rubber
The matri~ polymer must be a chemically
L 20 re~istant, high dielectric material and preferably
somewhat ~ore conductive.than an electrical insulator~
Of the foregoing li~ted polymer~ which are
~uitable for use a~ a membrane ele~rode ma.rix, PVC
i~ by ~ar the most widely used.
Generally, the ~atrix polymer i5 combined
with a p~a~ticizer to effect a certain amount of
swelling of the polymer. ~hi~ is in many in~tances
neces3ary to allow for sufficient ~obility o~ ion
carriers through the membrane.
Among the plasticizers which have been used
in this application are dioctyl adipate, tris(2-ethyl-
hexyl) pho3phate, dibutyl sebacate, o-nitrophenyl
o~tyl ether, diphenyl ether t dinonyl phthalate~ di-
pentyl phthalate, di-2-nitrophenyl ether, glycerol
triacetate9 tributyl pho~phate, and dioc~yl p~enyl
phosphate.
~7(~
Ion-selective membrane-~ of this type are
usually made by ~orming a solution of the pol~mer and
plasticizer (if ~ne i~ u~ea) in a volatile organic
zolvent, casting the ~olution onto ~he de~ired sur-
S face or into the desired shape and then removing thesolvent by evaporation.
ln addition to the above-m~ntioned criteria,
the materials which constitute the ionophoric layer
m~st be chosen o that the electrical impedance of
that layer is at least two orders of magnitude and
preferably at least five orders of magnitude higher
than the electrical 1 _e~A~ce of the underlying dis-
persea carbon layer. I~ the impedance is substan-
tially le~ than this value, ion and~or ionophore
~obility i excessiv~ for normal electrcde opera-
tiOh On the other hand, the i _)e~nce of the
; ~embrane layer mus not be too high lest it approach
the impedance o~ the electrometer an~ incur an impe-
dance loading e~ect which will result in ~ubstantial
error~ of ~easurement. For thi3 reason, it i~ pre-
ferred that ~he impedance o the ionophoric layer be
at least about two orders of magnitude le~ than the
ce of the electrometer uRed to ~ea~ure the
potential ~etween the conductive layer o~ the half
2~ cell~ of he invention.
B. Carbon Layer
~ he dispersed carbon layer con~i~ts e~en-
tially of a aisper-qion o~ ~inely dlvided particles of
carbon in a matrix of dielectric organic hydrophobic
polymer. The precise con~iguration of the carbon
part$cles is not critical. For example, carbon black
a~ well a~ finely divided pellet~ and charcoal par-
ticles can be u~ed. ~owevert it i~ essential that
the amount of carbon be sufficient that the carbon
polymer dispersion layer has an electrical impedance
~L2~7027
o~ 1~8~ than about 1% of the i~re~nce of the ion-
~elective membxane.
However, in view of the fa~t that the
di3persed carbon layer mu~t be partially diffu~ed
into the ion-~elective membrane, it will be apparent
that the caxbon particles must be ~inely divided;
that i8, they must b~ ~ub~tantially smaller than the
thickness of either the membrane layer or the carbon
layer. Dependin~ on the thickae~s o~ the layer,
which iB not ~y it~elf critical, a particle ~ize of
from 1 to 20 ~m i8 preferred, ~rom 1 to 10 ~m bein~
particularly preferred.
The ~articular organic polymer used as
matri~ for ~he carbon particles i8, likewise, not
particularly critical 80 long as the poly~er is a
di21ectric and forms a matrix which i~ nonreactive with
bo~h thç carbon and the ion-selective membrane
layer. For example, all of the polymers whic~ are
suitable for the matri~ of the membraae layer are
also suitable as the matrix for ~he ai~persed carbon
layer. In addition, ~ore highly crystalline polymers
~uch aæ polyethylene and polypropylene can be u~ed.
Suitable matrix polymQrs ~or the disper~ed
carbon layer also include fil~-~or~ing oleophilic
polymer~ ~uch a~ poly(caprolactone); polyacrylate and
alpha-alkyl polyacrylate ester3, e.g., polymethacryl-
ate, polymethyl ~etha~rylate and polyethyl methacryl-
ate7 v ~y1idene ~hloride copolymers, e.g., vinylidene
chloride/acrylonitrile, vinylidene chloride/metha-
cryla~e and vinyliaene chloride/vinyl~cetate copoly-
mer~, ethyleae/vinyl acetate copolymers; polyethyl-
ene; polyvinyl esters, e.g., polyvinyl acetate/acryl-
ate, polyvinyl acetate/methacrylate and polyvinyl
acetate: copolye~texs, e.y~, those prepared ~rom the
r~a~tion product o~ a polym~thylene glycol of the
)7~7
formula HO(C~) 0~, wherain n is a whole number 2
to 10 incluaive, and (1) hexahydroterephthalic,
s~bacic and terephthalic acids~ (2) terephthalic,
isophthalic and ebacic acids, (3~ terephthalic and
5 sebacic acids~ terephthalic and isophthalic
acids, and ( 5 ) mixtures of copoly2~ters prepared from
~aid glycols and (i) terephthalic, isophthalic,
sebacic and adipic acids~ nylons or polyamid~s, e.g.,
N~methoxymethyl polyhexamethylene adipamide; syn-
thetic rubbers, e.g., butadiene/acrylonitrile co-
poly~ers, and chloro-2-butadiene-1,3-polymers; block
copolymers, e.g., poly~tyrene-polybutadiene-polysty~
rene and polystyrene polyi-Qopxene; poly (vinyl chlor-
ide) and copoly~ers, e.g., poly(vinyl chlorideJace-
tate); polyvinyl acetal, e.g., poly(vinyl butyral),poly(vinyl formal) polyure~nes; polycarbonates;
polystyrene; phenolic re~ins; and melamine-formalae-
hyde resins.
¦ In some ins~ances, plasticization of the
matri~ polymer for the di~persed carbon layer may not
be need~d. However, to minimi~e the ~ormation o~
unwanted concentration gradients between the ion-
s~lective ~ dne and the dispersed carbon layer,
use o~ the sam~, or at least similar, polymers in the
~5 two layers is prererred. Thus, when PVC plasti¢ized
with dioctyl phthalate is used a~ the ~atrix for the
membrane layer, a most convenient and economical way
of minimizing migration of speeies between the layers
is to use the ~ame plasticized PVC composition as the
matxix ~or the dispersed carbon layer. Furthermore,
interdi~fusion o the layers is facilitated by the
mutual solubility of the matrices.
A~ will be made clear in the ~ollowing
discu~sion of the fabrication of the electrode half
cells of the invention, it is preferred ~hat the
~,
~2C)7~
11
disper~ed carbon lay~r matrix polymer be plasticized
to ~ome extent to acilitate interdiffusion of ~he
membrane and carbon layers.
C. Salt Bridge Layer
When the layered half ~ell o~ the invention
- i~ used as a referenca electrode in~tead o~ the pri-
mary sensing or indicating electrode, ~he ionophoric
l~yer will normally be covered with a salt bridge
layer wh~ch i8 a source for a constant concentration
of the measured ion specie~. ~hi~ salt bridge serves
as an ion bridge batween an analyte-containing solu- .
~ion and an ionophoric layer and eonsists of a small
amount of appropriate electrolyte dis~olved in a
water-per~eable hydrophilic polymer.
Suitable hydrophilic pol~ers include poly-
vinyl alcohol ~PVA~, polyethyle~e oxides, polyethyl-
ane oxide ethers, and various polysaccharides. Among
the many polysaccharides suitable for thi~ purpose
are natural gums ~uch as agar and agarose, cellulose
ana cellulose derivatives such as cellulose e~ters,
'` cellulose ethers and ~enzyl cellulose. The hydro-
philic polymer must be ~hosen ~o have at least ~oder-
ate ~i -ncional stability in the pre6ence of, and be
substantially insoluble in, liquid analyte at ambient
temperatures.
To protect the salt bridge layer, it is
covered with an inert substantially water-impe~ --hl e
layer such as Silastic3 or PVC. By and large, the
covering for the 3alt bridge can be ~ade from any
30 imperviou8 hydrophobic polymer, such as the oleo
philic polymers mentioned hereinabove for use as
matrix in forming the dispersed carbon layer.
Only a small edge s~rface of the ~alt bridge
layer i8 exposed to provide a restrict~d area in
contact with an analyte by which ~igration of salt
11
~7~7
between the salt bridge layer and the analyte is
minimized, while at the ~ame time providing an ionic
bridge between the ~en60r and referenc2 cells when
analyte i3 placed ~etween them. When the salt bridge
layer iB prope~ly ~ormulated ~o that the elactrolyte
concentration therein remains substantially constant,
~he reference signal therefrom al~o re~ains constant.
~ he salt bridge material i8 re~dily formu-
lated by di8801ving an electrolyte in an hydrophilic
10 polymer. This ormulation can easil~ be screen-
printed onto an appropriate 6ubstrate and solidified
by heating and/or drying, depending upon ~he parti-
cular type o~ hydrophilic polymer wh;ch is used. In
the case of PVA, drying alone may be ~ufficient.
15 However, in other i~st~nces, it may be desirable to
include in the ~ormulation a small amo~nt o~ a cro~s-
linking agent and an appropriate initiation ~tem
whi~h can be aGtivated by heating the pranted layer
to a ~oderate temperature, e.g., 50C.
20 D~ Conductive Material
~ enerally, a wide variety of conductive
materials can be u. ed i~ the inve~tion half cell~ so
long a~ the inter~ace with the di~per~ed carbon layer
has a stable conductox contact potential dif~rence
25 and i8 free from o~idation. ~hough noble metals such
as silver and gold are often preferred as the condue-
tive ~sterial, beoau~e o~ their ~hemical i~ertne~,
ba~e metals 6~ch as copper, nickel, iron, tin, cad~
~ium and aluminum can al80 be u~ed, because they are
30 not subject to stringent o~idative ~ondition~ in the
devi e of the invention~
In all ca~es, the conductive layer~) of the
hal~ cell o~ the invDntion must be provided with
ter~inations by which the electxode can be connected
to an electrometer-amplifiex to ~easure any potential
~;Z0702'~
13
difference between the hal~ cell~. The~e termina-
tions may merely ba an e~tension of the conductive
layer or they may be made by specially printing a
contiguous con~uctive layer of either the same or a
S different conductive metal. The precise method of
termination i8 not critical and will largely be
determined by fabrication econnm~c~ ~or a.ny given
performance level.
It i8, of course, nece~sary that ~he con-
10 ductor pattern which is not in contact with the dis-
persed carbon layer be ~hielded rom analyte liquid
by a layer which i~ both electrically insulating and
chemically inert with respect to the analyte liquid.
~his i~ normally done by covering the nonchemically
functional portion of the conductor pattern with a
layer of chemically inert insulating material such as
a polymer or glass. The polymers which are suitable
for *he matrix of the me~bran~ and carbon layers are
~180 in gen~ral ~uitable as an insulator for those
portions of the conductive layer which ne~d to be
shislded.
E. Fabrication
The electrode half cell of the invention and
the ~ensor~ ~ade therefrom have a further advantage
in that they ca~ be fabricated in very ~mall sizes
and as very thin layers. As is apparent from the
drawingJ the half cell of the invention is essen-
tially a ~imple multilayer laminate, iOe., it i8 a
stack of thin layer~ or films.- A particularly con-
venient method of forming the layers i8 to ~reenprin~ them on whatever substrate i8 used. For exam-
ple, in the case of the conductive material, a dis-
per~ion of fi~ely davided particles o~ ~he conductive
material in an inert organic medium is formed, screen-
printed in the desired pattern onto ~he insulatins
13
~Z/~70;~7
14substrate and ~ired to sinter the conducti~e material
and ~orm a "continuou3" conductive layer.
On the other hand, finely divided particles
of the conductive material can be dispersed in a
photo~en3itive medium, coated on the in~ulating ~ub-
strate, exposed through an appropriate phototool to
harden the di~persion in the de~ired pattern,.and
develvped to remove the uneYpo~ed dispexsion.
Likawise, the ~uccessive carbon and iono-
10 phore layers can be applied lby $he same techlliques orothers whi~h will be well ~own by tho~e ~killed in
~he art.
0~ primary importance, however, i9 the
formation of the interdifu3ion ~one between the
carbon and ionophoric layers. The interdi~usion
zone must not extend completely through either the
ionophoric ~embrane or the carbon layer. However,
,, the zone mu~t be e~te~sive enou~ to difuse any
charge gradient developPd by the passage of current
20 a~ requirea ~y the electrometer-amplifier used or
the measuremen~. The reason for this i~ that there
~ust e~ist zo~es of substantially pure io~ophoric
layer and substantially pure conductive layer to
effect the transitio~ from electron flow to ion flow
25 in support o~ the electrical currentO ~he concen-
tratio~ gradient is d8v~ ope~ at the sample solu-
tion/ionor~oric layer interface and extend~ inwardly
toward the interdiffusion zone and a subætan~ially
.. . . . . . . . . . . . . ................. .. . . . .
zero concentration gradient ~hould re~ide at the car-
30 bon layer side of the zone. On the other hand, ~rom
the carbon layer side o the interdiffusion zone, the
carbon layer provides a conductive path ~or electrons
and also allows the tran~port o~ ions at the inter-
face to support current flow by the electrometer-
35 ampliier.
14
170~15
~ hough ir. theory these criteria might be met
by a large nu~er of separate stacked layer~, each
having appropriate changes in concentration o~
ionophore and carbon particles~ this would be very
S onerous and very e~pensive. A much easiex way to
acco~pli~h this is to plasti~ize both o~ the matrix
polymers whereupon the laminated layer~ will auto
genically i~terdi~use due to mutual ~olubilization.
In addition, interdiffusion o~ the layers, either
with or without plasticization, can be effec~ed by
the application o~ pressure and/or by the application
o~ heat fox a time sufficient to effect the proper
degree of interdi~fusion.
However, by whatever method the interdi~fu-
15 sion zone i~ formed, it is essential that the inter-
diffu~ion zone have a high-to-low dielectric gradient
from the membrane to the carbon layer. In addition,
r it is essential that the interdifused zone be ~ta-
ble. For this last reaso~, it i8 very desirable to
lncorporate a cxoss linking ag~t in the binder com
ponent of either or both of the polymer matrices to
effect both physical and chemical stabilization of
both the ~ ane and carbon lay~rs a~ w~ll as the
interposed interdif~usion layer.
The following procedure i~ typical of the
way in which the electrode half cells of the inven-
tion and 3ensor~ made ~herefrom are fabricated in
layers using qcreen-printing techniques~
- -- The various layers are-built up on a pla~ar --
6ub3trate which typically ha3 ~ ion~ 1" x 2" x
0.025" (2.5 x S.0 x 0.006 cm)~ The re~ultant struc-
ture is common~y re~exred to as a chip. The sub-,
~trate must be c~emically inert with re~pect to
analyte and the layers placed th~reon. I~ must also
be dimensionally stable and immiscible with respect
:~20~02~7
16
to the analyt~ and/or overlying layer~. The dimen
sions, however, are not critical and may be variedO
The composition of the sub~trate i8 chosen on the
basis o~ cost, ea~e of manufacture, and durahili~y.
Althou~h ~or the purposes of ~his discussion ceramic
chosen as the sub6trate, other ~ubstratas, for
e~ample, Mylar~ polyester ~il~, can be employed.
The first or bottommo5~ functio~al layer of
the device i8 the conductor, usually silver. A 8il-
ver pasta is 3creened over a ceramic substrate usinga 250 me~h screen and allowed to dry for 5 to 10 min-
utes at rcom temperature. It i~ then dried ~or an
additional 10 minute~ at 150C and finally fired for
30 minutes at 850~C.
The ~econd functional layer of the device is
the insulator, usually glass, whi~h i8 printed over
the conductor pattern using a 165 mesh screen in such
~anne~ that the conductor paths are protected~ bu~
the conta~t area ~f the electrode is exposedO It is
dried for appro~imately 5 minu~es at room temperature
and for an additional 10 minutes at 150C4 Finally,
it i8 fired for 30 minut88 at 850C. I~ a poly~eric
substrate is used then the sa~e function ~an be
servea by uQe of . dielectric photosensitive polymer
~ystem (e.g., a dry ilm photore~i3t) to form the
appropriate prote~tive patter~0
The third functional layer of tha device,
the carbon layer, i8 printed above the conductor
- -pattern. A-1~5 mesh screen is employedO The carbon
layer is dried for appro~imately 5 minute~ at roo~
temperature and cured for another 20 minute~ at 160C.
The fourth layer o~ the device, the poly-
vinylchloriae (PVC)/iono~hore layer, i8 printed above
the car~on pattern using a 165 mesh screen. The com
35 po3ition o this layer i~ variable, depending on ~he
spacies to be detected.
16
!
~;~C)7V;~
~ he PVC typically has an inherent ViSC06ity
o~ about 0.65 and compriseR about ll. 3 weight percent
of the ormulation. The ionop~ore comprises about
1.1 weight percent of the formulation and imparts
S ion-selectivity to the device. Various plasticizers
can al~o be pre~ent. The PVC/ionop~ore is dried for
abs~ut 10 minutes at room temperature and then cured
or 30 mim~tes at 35~C.
The choice of ionophore varies depending on
the electrolyte to be detectedO For example, valino-
~ycin i~ selective for potassium, methylmonensin ~or
sodium: and nonactin for ~ ~niumO
U3ing the matsrials de~cri~ed above, it was
not nece~&ary to ca~ry out any special procedures to
~orm the interdiffusion zone at the interface of the
aonophoric and carbon layers. The reason ~or this
was that in both layers the ~ame plasticizer a~d 6a~e
'' polymer were used, whi~h gives to the layers a sub-
~tantial degree of mutual solubility which, without
further manipulation, result3 in formation and
stabiliza~ion of the zone within a very ~hor~ time
after fabric~tion, BecauQe the interdif~usion zone
can be formed 80 easily by this ~athod, it iY a pre-
ferred w~y of ~abricating this ~egment o the elec-
trode half cells of the invention. ~he ex~ent and
rate o~ interdi~usion can be adjusted by increasing
or decrea~ing the mutual 301ubility of t~ layers,
e.g~, by changing the c~ ition and/or concentra-
tion of the plasticizer in either or bot~ layer3.-
In most in~t~ces it will b~ preferred that
~he layered electrode half cell o~ the invention be
combined on the same ~ubstrate wit~ a similar half
cell which is coated with a salt bridge layer, which
l~yer in the pre~ence o~ analyte serves as an ionic
bridge between the two half Cell3- The primary pur-
17
~L2070~718
pose o~ this second half cell is to be a source o~ a
constant signal, i~e., a constant potential, based
upon a fixed concentration o electrolyte.
A PVA/salt bridge layer is printed on the
reference electrode side of the sensor, abov~ the PVC
_ layer, using a 165 me~h screen. ~pically, the PVA
: (polyvinyl ~lcohol) ~ormulation consist~ of appro~i-
~ately 37 weight percent PVA: 0.17 weight percent
Triton~-x 100 nonionic sur~a~tant: 0.75 weight per-
cent Foama~ter DP-122 NS. Where the analy~e is
an electrolyte, for example, potas6ium, the PVA layer
i8 doped with a ~tandard amount of that electrolyte,
for example, RCl.
The above-described PVA layer is dried ~or
10 minute8 at room temperature and cured for 30
minute~ at 35C~
The topmost layer of the sen~or i~ an ovex
coat layer, typically of sili~one ~ubber, which is
printed with a 1~5 mesh screen over the ~eference
~ide of the sensor. It is dried for 10 minutes at
- room temperature and cured for 1 hour at 50~C in a
humidi~ied C02 atmosphere. ~hi8 topmost layer
covers the entirs chip e~c pt for a ~mall area w~ich
is le~t open to provide electrical contact between
analyte solution contained in the receiving æone
(described below) and the PVA/salt layer. The
primary purpo~e of the small open area is to allow
electrical contact between the analyte ~olution and
the ~alt bridge while mini~izing contamination and
concentration chanyes within the ~alt bridge layer
arising ~rom suGh contact. Within th time ~rame of
mOBt mea~urements ~usually less than five minutes),
no ~ignificant migration of analyte into the 3alt
bridge layer will take place and thu~ signal drift
rom this source i8 ~ ~or all practical purpose~,
eliminated.
, .
~,
L2 C)~0~
19
In the operation of sensors made from the
electrode half ~211s of the invention, the liquid
electrolytic analyte is placed in on area overlapping
the two half cells atop an appropriate insulating
layerO The analyte must overlap the half cells suf-
ficiently to establish ioni~ contact with both half
cells. ~hi8 area between the hal~ cells constitutes
a receiving ~one for the analyte. Though it is not
re~uired, thi~ zone may be given a dish-shaped or
other type of configuration to de~ine the zone and
hold the liquid analyte in place more effectively.
In addition, the zone can be provided with an inert
adsorbent material to hold the analyte more tightly
within ~he rec iving zone.
~IELDS O~ USE
The device of this invention i~ useful in
~easuring co~centrations of a large number of ana-
lytes of cl~nical interest in biological samples.
(By analyte i~ meant a ubstance whose concentration
20 -iæ de~ired to be determined~) The biological ~ample
c~n be a biological fluids such a~ whole blood, biood
serum, blood plA~ ~, saliva, cerebro~pi~al Eluid, or
urin~ or it can be a cell or tis~ue e~tract. The
analyte i~ o~ten an electrolyte (e.g., H~, ~a~, Ca2+,
~5 K , C1 , ~H4, HCO3, etc.), ga~ (e.g., C02~ 2)~
or a metabolite ~e.g., glucose, blood urea nitrogen,
triglyceride , phenylal~nine, tyrosine, ereatinine,
etc.) present in one of the~e biological fluids.
- Other analyte~ include protein8, drugs~ hormones,
30 vit~ ins~ enzymes, enzyme sub6trates, antibodie~,
polysaccharides, bacteria, prstozoa, fungi, ~iruses,
cell and ti~ue antigens and other blood cell or
blood fluid substanca~.
Electrolytes are measured potentiometrically
35 by the device o~ this invention. By potentiometric
19
~ 07(~27
~ o
measurement is meant that the potential di~erence
between the half cells is measured at substantially
zero curr~nt 10w (less than 1 x 10 12 and preferably
less khan about 1 x 10 15 amperes)O
~ n ion-selective membrane is used in the
device to select the ion for the ultimate potentio-
me~ric measurement. In the case of the electrolytes,
an ionophore is used to select the desired ion f~r
measurement~ Table 1 above li3t~ typical ionophores
and the analyte for which they are selective.
Metabolites, enzymes and enzyme ~ubstra~es
are usually mea~ured by devices which couple the
selectivity inherent in enzyme-sub~trate reactions
with that of the ion-selective ~ensor. The ul~imate
detection i8 ~ potentiometric measurement of the
resultant ion (~uch as a blood urea nitrogen ~BUN~
moaaurement thro~lgh the urease-coupled generation of
NH4). Table 3 below lists ~ome of the analy~es
which can be measured through enzyme-coupled sensors
and al30 gives ~he specie~ w~ich is measurea.
TABLE 3
~ZYME-COUPLED MEASUREME~T DEVIC~S
Species
An~lyte Substrate/Enzyme Monitorea
BU~ Urea6e ~H4~
Creatinine Creatininase NH4+
Lactic acid . Lactic dehydrogenase N~D+
Triglyceride3 Lipase H~
Cholin~stera~e Acetylcholine ~
The device o~ this invention can also be
used in the mea~urement of analytes in immunochemical-
coupled a89ay8 For example, an antigen can be mea
2~70;2 7
21~ured by u~ing a urease-labelled antibody to the
antigen in the detector. ~he detector is sen3itive
to the amount of ~H4 generated by the urease, which
in turn is related to the a~ount of antigen in the
S biological fluid. Thus, variou~ antigens or anti-
bodies of clinical intere6t can be measured through
this type o~ device. Other en~yme labels, such as
aecarboxylase, or hydrolase, or electroactive label~,
such as porphyrin~O quinones, ~errocene, or ~AD ,
can be used in the immunochemical coupled device.
~he ~electivity in t~ese devices i8 due pri~arily to
the specific bin~ of antigen and antibody.
Be~ide~ the previously discussed applica-
tions in clinical or diagnostic c~emi~try, the device
of thi~ invention can also be used in the detection
of analy~es of intere~t in, for example, chemîcal
procedures, manufacturing proce~se~, chemical hazard
detection (dosimeters), and microbial growth pro-
cesses.
n~T~rT.~n DESC~IP~IO~ OF TH~ DRAWI~G
Figurs 1 ~s a schematic overhead YieW of a
typical ~ensor which incorporate two of the half
cells of the invention as the indicator and reference.
sides of the sensor. Visible in this view of fhe
device a~e ceramic substrate 1 over which are printed
con~uctor leads 3 and 3' which underlie indicator
half cell 4 and reference half cell 4', both of which
are multilayered ~tructures as described with respect
to Figure 2 below. In operation, conductor lead~ 3
and 3' are connected elactxically to an electrometer-
amplifier.
Pigure 2 is a schematic sectional view of
the analytical electrode o Figure 1 taken along the
vertical section de~ined by lines A-A.
21
lZ~:~7~
Two-layered half cell~ are ~hown which are
printed upon a ~- - ceramic substrate 1 made ~rom
-96% by weight aluma. The layered electrode half cell
on the left 4 i8 the indicator hal~ cell an~ the
~ layered ele~trode half cell on the right 4' is the
reference half ~ell.
The lowermost layer of the indicator or
sensing half cell 4 con~ s o a patterned area o
silver metal 3 which ~erves a3 an electrical conduc-
tor to which leads of an electrometer are attached.
~ikewi~e, the lowermost layer of the reference half
cell 4' has a functio~ally identical silver metal
j layer 3'- ~o insulat~ the two condu~tive layers 3
j 15 and ~' when the device is ~ontacted with analyte 15,
a layer of ~ielectric Sinsulating) material S is
interposed between the sides of the silver layers 3
and 3'. A dispe~sed carbon layer 7 and 7' is posi-
tioned upon the conductor layers 3 and 3' in such
20 ~nner that the layers are phy~ically shielded from
any direct contact with the analyte 15~ A5 shown
here, the ovexlying carbon layer~ 7 and 7' each over-
lap ~lightly ~he in~ulating layer 5 to effect com-
plete shielding o~ ~he conduct~rs 3 and 3'.
Upon t~e carbon layers are positioned iono- .
phoxic layers 9 and 9i in suc~ manner that the carbon
layer i8 also physically shielded from any direct
contac~ with analyte 15.
¦ . - Up to this point the structures of half
~, ~o cells 4 and 4' are functionally the same and ~truc-
turally similar except for po3sible difference~ in
geometry.
~ owever, the ionopXoric layer 9' of refer-
ence hal~ c211 4 ~ i8 overlaid by salt bridge layer 11
and covered by a protective layer 13 which physically
~hields referenee half cell 4' from direct contact
I
22
~2~37C~Z'7
.~ ,
23
with analyte 15. The salt bridge layer 11 is sub-
stantially but in~ ~letely ~overed by impermeable
1 ayer 13 in such -nner that the exposure of the salt
bridge layer 11 to the liquid analyte 15 is quite
restricted relative to the total surface of the layer
Layer 11 comprises a constant composition of
eleetrolyte to which layer 9' is sensitive and selec~
tive. Protective layer 13 ensures that the electro-
lyte composition is not altered during the measure
ment te~g. ~ 2 minutes) and hence func~ions to ~ain-
tain a constant E.M.F. during mea~uremen~ o~ ana
lyte. The expo~ed edge of layer 11 en~ures contact
of the ~eference half cell with the analyte, while ~t
the ~ame time retarding ionic migratio~ into the bulk
of layer 11.
The invention will be better understood by
reference to the following examples in which the
fabrication and operation of the half cells of the
~! invention ar2 described in detail.
~l 20 EXAMPLES
~i EX~MPLE 1
A pota~sium ~nRor chip was ~abricated using
the pro~edure de~cribed hereîn~hove. In particular,
the sensor. consisted of a 1" x ~l- x 0~025' (2.5 x
5.0 ~ 0.06 cm) alumina chip upon which were printed
two terminated silver conductor patterns. The entire
uncoated area of the sub~trate chip wa~ co~ered with
a layer of glass to shield the side~ of the conductor
~ - pattern from analyteO Correspondinglyj the ~ides of
the disper~ed carbon layer wexe shielded by the over-
lying ionophoric layer. In the reference electrode
half cell, the ionophoric layer wa~ shielded from the
2nalyt~ by the overlying salt bridge layer. ~owever,
the salt bridge layer was covsred with a layer of
~ilastic~ polymer film except or one side of ~he
23
`` ~2~70~'~
24
ion-~elective membrane which was left expos~d to
provide for limited ionic contact wi~ the analyteg
The e ,o-itions of the carbon, ionophoric
and salt bridge layers are given in Table 4 which
follows:
TABLE 4
CODPOS ition of Electrode Functional L~yers
Carbon Di~persion Layer % wt.
10 Vinyl chloride~vinyl ac~ate copol~mer
~utyl cellosolve acetate
Carbon
lonophoric Layer
Poly(vinyl chloride) 11.3
~ioctyl adipate (DOA) 26.9
Cyclohex~ne 6G.7
Valino~y~in 1.1
- 20 100.
Salt Bridge Layer
Poly(vinyl alcohol) 37.4
Water 60,1
25 KCl 1.3
Tri on~X-100 0.1
. FoamasterTM DP-122~S 1.1
., . .. .. . .. . . .. . '~ oo . O
3~
~XAMPLE 2
~ he sen~or chip of Example 1 was then used
to determine ~he potential difference between ~wo
aqueous analytes, one con~i~ting o~ 1 mMol of KCl and
140 ~Mol o~ ~aCl and the other consisting o~ 10 mMol
of RCl and 140 mMole~ of ~aCl.
24
2~
In this te~t, th~ sensor wa~ first expo~ed
to the weaker KCl analyte solution and then, af~er 60
~econds, the weak electrolyte was rapidly washed of
and replaced with the more concPntrated KCl analyte
solution and the cycle was repeated two times, during
which the potentiometric voltage wa~ con~inuously
recorded. A plot of t~e correla~ion of poten~ial
(mVolts) as a function o~ time reveals that a full
re~ponse to each 10 fold change in ~+ concentration
took pla5e in le~ than 10 ~econds with a slope (or
sen~itivity) of at least 56 mV~ As used herein~ the
term ~ull re~ponse refers to attainment of at least
99% of steady state. Sensitivity is determined by
the relationship mY/pK . The response curve for
thi8 electrode (~ignal voltage V8~ K~ concentra-
tion) i5 given in Figure 3.
In addition, upon observing the response or
10 minutes, it was ound that the drift was below
1 mV/min. Furthermore, upon observing the individual
response of the sen~ors it wa~ fou~d that the refer-
ence hal~ cell ~as, as it s~onl~ be, essen~ially
nonresponsive to ~he analyte. That i~, it gave an
es3entially con6tant signal during that extended
p~riod.
EXAMPLES 3-10
A series of eight s~n~or chip~ was fabri-
~ated in the above-de~crib~d -nner in which the ion-
~elective membrane contained 3~ by weight valinomycin
and the amount of DOA p~a3ticizer wa6-varied from
3~ zero to gO% by weight, the weight ratio of ionophore
to PVC binder in ea~h formulation being con~tant.
Using the s~me ~ -containing analyte~ as in Exam-
ple 1, the sensitivity of eac~ sensor wa~ determined
by ~easuring the 810pe of its response curv~. The
results were a8 follows:
"`" ~Z~ '7
26
TABLE 5
Efect of Plastici~er Level in the
Ionop~oric Layer Upon Sensitivity
Example Amount of Slope ~mV/p~)
No.Pla~ticizer, % w~. mV
3 0 ~oisy, unstable
4 25 Noisy, unstable
~oisy, uns~able
6 50 52
7 60 ~8
8 70 56
9 80 57
53
The above data show ~hat for thi~ particular
polymer ~ystem (0.65 I.V. PVC), in exce~s of 40% by
weight plasticizer was necessary to obtain suitable
ion carrier mobility through the ionophoric layer.
EXAMPLES 11-14
Four additional chip sensors were fabricated
in the manner o~ Example 1. In each a di~ferent ~ol-
25 vent ~y3tem for the ionophoric layer wa~ used. Again
using the same potassium-containing analytes as in
Example 1, the ~en~itivity of each of the sen~or~ was
measured by determining the slope of it8 ~e3ponse
,' ____ 9',__, ' curve-- The re~ults wer~-as ~ollows~
~6
~,
7~'7
27
TABLE 6
Effect of Solvent Composition of
Ionophoric Layer on Sensitivity
Example Solvent Slope ~ mV/pK~)
S No. C~ csition mV
11 Cycloh~Y~n~ne caO 57
12 N me~hyl-pyrrolidone ca. 59
13 Isophorone ca. 45
14 Tetrahyarouran ca. 57
The above data show that the solvent medium
u~ed in casting the ionophoric layer can have a 3ig-
nificant effect on ~en~itivity of the sensor~ The
data suggest t~at, for any given binder/ionophore
system, the solvent can be chosen to improve sen
tivity to a s~all but ~ignificant d~gree.
EXAMPLES 15-25
Another ~erie~ of sensox chips was formu-
lated in the m~nner of Eæample 1 in which the thick-
ness of the ionophoric layer was varied to determine
the e4fect of membrane thicknes~ upon sen~itivity of
the ~hip. For still further co~r~rison~ an elec~
tro~e wa~ formulated and tested which contained no
ionop~oric ~embrane at all. .Each o~.these chips was
evaluated a~ in the previous example for sensitivity
by me~surement of the slope of the res~onse curve.
The re~ults given in Table 7 below are the average
; ~ -obta ned -~ro~ tP~ts of-2- or-3 electrode chips-a~- each -
30 thiC~n~ level.
27
7~)2~
28
TABLE 7
Ef~ect of Ionophoric Layer
Thickness on Sensitivity
Example Innsphoric Layer ~lope S~V/pK~)
5 ~o. Thickness (mil~ mV
- 1
16 0.05 ~
17 0.08 . 55
18 0.15 4~
19 ~.2 54
0.4 54
21 0.~ 54
22 0.7 5
23 1~1 54
2~ 2~7 53
2~ ~5 6.0 40
~ he oregoing data ~how that an ionophoric
membrane ~hickness of at least about 0.2 mil is
n~eded ~or this particular matri~/plasticizer sy8-
tem~ It i~ }ikely that the poorer sen~itivity o~ therelatively thin layers was due to unsvenness and pin-
holes w~ich occur in such thin ~ilm~. On the other
hand, it i5 apparent that the sensitivity decreases
. to an unsati~actory level when t~e ionophoric layer
thickness exceeds about our or ~ive mil~. Thi5 lat-
ter ~dverse effect i8 probably due at least in part
to the fact tha~ the signal ~mped~nce i8 reduced
escessively with suc~ high io~lophoLic layer thick
ne3~. An i~op~oric membrane thickness o~ no more
than 3~0 mil i8 preferred.
28
~0~2'7
~9
EXAMPLES 26-32
A still further BerieS o~ sens~r chips was
fabricated in the ~nner of Example 1, bu~ the amount
of ionophore in the ion-selective layex was varied to
det~rmine the e~fect of ionophoxe conoentration on
the ensitivity of the chips. The sen~itivity of
each o~ the~e chip~ was measured as in the previous
examples ~y determining the ~lope of the response
curYe. The results in Table R are the average of 2
or 3 chips of each composition.
TAB~E 8
Ef~ect of Ionophore Concentration
on Sensitivity
~xample Ionophoric Slope (~m~/pK~)
- 15No.Concentration % wt. mV
26 0.1 52
~7 ~.3 58
78 0.5 61
2029 1.0 60
2.0 ~4
31 3.0 61
32 5.0 60
These d~ta ~how that quite good sensi~ivity
can be obtained at even very low ionophore concentra-
tions, bu~ that ma~imum sen~itivities are attaine~ at
an ionophore level of at l~ea~t~abbut~0-.-5%-by-weight~ --
~urthermore, there.seems to be no additional advan-
tage to using lonophore concentrations above 2-3% by
weight since no greater Yensitivity i8 obtained-
Though for this analyte good ~ensitivities were ob-
tained at low ionophore concentr~tions, other more
conc~ntrated ionic analytes will likely require
higher ionophore levels to get a satisfactory
~ensitivity of ~0 mV or higher.
29
