Mediators suitable for the electrochemical regeneration of NADH, NADPH or analogs thereof

Disclosed is an improved electrode suitable for the electrochemical regeneration of the co-enzymes NADH and NADPH. The electrode has imparted on its surface a mediator function which is a 3-phenylimino-3H-phenothiazine or a 3-phenylimino-3H-phenoxazine. Also disclosed is a method of improving the performance of a biochemical fuel cell which operates with a dehydrogenase as a catalyst and a co-enzyme as the energy-transferring redox couple which involves using the improved electrode in the fuel cell.

BACKGROUND OF THE INVENTION 
Analytical methods that combine the selectivity of enzymes with the 
sensitivity of amperometric detection are of interest to the diagnostic 
industry. The reduction of the nicotinamide co-enzymes (NAD and NADP) is 
particularly important because they are produced in reactions catalyzed by 
dehydrogenases. Dehydrogenase catalyzed reactions according to the 
equation: 
##STR1## 
play an important role in biological cells and analytical reactions. 
Several hundred different dehydrogenases are known which selectively 
catalyze the conversion of different substrates into products. When the 
substrate, e.g. glucose, is oxidized, the enzymes NAD.sup.+ and/or 
NADP.sup.+ are reduced to NADH and NADPH respectively. These co-enzymes 
are a necessary element in the reaction due to their ability to act with 
the dehydrogenase enzyme to form an energy-transferring redox couple. The 
pyridine linked dehydrogenases transfer reversibly two reducing 
equivalents from the substrate to the oxidized form of the pyridine 
nucleotide; one of which appears in the reduced pyridine nucleotide as a 
hydrogen atom, and the other as an electron. The other hydrogen atom 
removed from the substrate appears as free H.sup.+ in the medium. 
The co-enzymes NAD.sup.+ and NADP.sup.+ are expensive chemicals making 
their regeneration and reoxidation to their original state imperative if 
they are to be economically used in low cost, disposable, analytical 
devices. 
NADH is oxidized directly at different base electrode materials only with 
high overvoltages on the order of 1 volt. However, a decrease in this 
overvoltage can be obtained by the adsorption of functionalities on the 
electrode surface which mediate the electron transfer from NADH to the 
electrode. Such mediators are typically selected from materials which may 
be reoxidized electrochemically without excessive overvoltages rendering 
them useful as an auxiliary system for electrochemical regeneration. 
Various mediator compounds suitable for this purpose are known. In U.S. 
Pat. No. 4,490,464 there are mentioned, by way of background, mediators 
such as phenazine methosulfate (PMS); phenazine ethosulphate (PES); 
thionine and 1,2-benzoquinone. This patent goes on to describe electrodes 
which are modified to catalyze the oxidation of NADH, NADPH or analogs 
thereof by imparting to the electrode surface as mediator a condensed 
aromatic ring system comprising at least three and preferably four or more 
condensed aromatic rings with or without heteroatoms. More particularly, 
this patent describes the electron exchange with the co-enzyme or analog 
thereof by structural elements comprising one of either alkyl phenazinium 
ions, phenazinium ions, phenazinones, phenoxazinium ions, phenoxazinones, 
phenothiazinium ions or phenothiazinones. 
In J. Electroanal. Chem. 287, 61-80 (1990) there is disclosed 
3-.beta.-naphthoyltoluidine blue O (I): 
##STR2## 
which is perhaps the most effective of the known phenothiazinium mediators 
generically disclosed in the '464 patent. A variety of the mediators 
disclosed in this patent are compared in J. Electroanal. Chem. 292, 
115-138 (1990). 
The phenoxazinium and phenothiazinium ions disclosed in the '464 patent are 
positively charged species such as (I) above and are readily 
distinguishable from the mediator compounds of the present invention. The 
phenoxaziones and phenothiazinones claimed in the '464 patent are 
3H-phenothiazines (II) and 3H-phenoxazines (III): 
##STR3## 
in which the 3-position is derivatized with a carbonyl oxygen group. They 
bear a structural resemblance to the compounds of the present invention in 
that the oxygen atoms in (II) and (III) are replaced by nitrogen atom 
bearing substituted phenyl rings. In reality, however, these compounds are 
quite different and there is no suggestion in the prior art that replacing 
the carbonyl oxygen of compounds (II) and (III) with a phenyl-substituted 
nitrogen atom would afford effective mediators. 
The compounds, whose utility as mediators is taught herein, are disclosed 
in U.S. Pat. No. 4,710,570 which describes the "leuko" or reduced form of 
these dyes to be "suitable as dye-forming agents in pressure sensitive, 
thermographic, photothermographic and photographic imaging systems." 
U.S. Pat. No. 5,264,092 discloses the mediators of the '464 patent 
covalently attached to polymers which are useful for the electrochemical 
regeneration of NADH. This patent discloses a variety of polymeric 
backbones to which the mediators are attached. Polymer/mediator modified 
electrodes are also disclosed. Certain of the mediators of the present 
invention also perform well when immobilized on polymers. 
SUMMARY OF THE INVENTION 
The present invention involves an electrode suitable for the 
electrochemical regeneration of the coenzymes dihydronicotinamide adenine 
dinucleotide (NADH), dihydronicotinamide adenine dinucleotide phosphate 
(NADPH) or analogs thereof, said electrode having imparted on its surface 
a mediator function comprising one or more mediator compounds selected 
from the group consisting of substituted or unsubstituted 
3-phenylimino-3H-phenothiazines and 3-phenylimino-3H-phenoxizines.

DESCRIPTION OF THE INVENTION 
This invention is predicated on the discovery that 
3-phenylimino-3H-phenothiazines and 3-phenylimino-3H-phenoxizine compounds 
are useful mediators for the electrochemical regeneration (oxidation) of 
NADH. The mediators of the present invention can be represented by general 
formulae (IV) and (V). 
##STR4## 
It is evident that R1 and R2 in formulae IV and V can represent a variety 
of substituent groups without departing from the scope of the present 
invention. Such substituent groups are limited only by the ability of one 
of ordinary skill in the art to prepare stable compounds which have 
electrochemical properties necessary for the requisite electron transport. 
For example, in the above formulae substituents R1 and R2 may be the same 
or different, and selected from the group consisting of hydrogen, lower 
alkyl, aryl, halo, haloalkyl, carboxy, carboxyalkyl, alkoxycarbonyl, 
aryloxycarbonyl, aromatic and aliphatic keto, alkoxy, aryloxy, nitro, 
dialkylamino, aminoalkyl, sulfo, dihydroxyboron (--B(OH).sub.2) and the 
like. It is also intended that aliphatic and aromatic groups incorporated 
into R1 and R2 can themselves bear a variety of substituent groups. 
The substituents R1 and R2 may serve to modulate the reduction-oxidation 
(redox) potential of the mediator, to vary solubility or to function as a 
handle for covalent attachment of the mediator to a polymer or solid 
support. 
Compounds (IV) and (V) can be represented by a single formula (A) in which 
the symbol X is used to represent oxygen and sulfur: 
##STR5## 
Nicotinamide adenine dinucleotide (oxidized form, NAD.sup.+ ; reduced form, 
NADH) is the cofactor providing chemical redox function for many 
dehydrogenase enzymes. This cofactor is reduced during the course of the 
enzymatic reaction as the substrate molecule is oxidized. Amperometric 
biosensors seeking to use these enzymes as a means to measure substrate 
concentration correlate this concentration with the current generated as 
the cofactor is electrochemically re-oxidized. The NADH can be 
electrochemically re-oxidized on graphite, pyrolytic carbon, glassy 
carbon, platinum, gold, or a composite made from these materials, 
electrodes without a mediator, but this reaction occurs with several 
difficulties including a large overpotential and electrode fouling. 
The present invention describes the first use of 
3-phenylimino-3H-phenothiazines (IV) and 3-phenylimino-3H-phenoxizines (V) 
in the electrochemical regeneration of NADH and NADPH coenzymes or their 
derivatives and accordingly, encompasses a wide variety of phenothiazine 
and phenoxazine derivatives. The present mediators can also be used for 
the electrochemical regeneration of NADH and NADPH derivatives. 
Derivatives of NADH and NADPH include modified cofactors such as in the 
case where the coenzyme is attached to a polymer as described by 
Dolabdjian, et al in Enzyme Engineering Vol. 4, G. B. Brown and G. 
Manecke, eds., Plenum Press, New York, 1978, Pp. 399-400 or covalently 
attached to the dehydrogenase enzyme as described by M. Persson, et al in 
Biotechnology 9, Pp. 280-284 (1991) or synthetic analogs bearing other 
substituents so long as they function as the cofactor for the 
dehydrogenase enzyme. These references are incorporated herein by 
reference. In a preferred embodiment, the mediator is covalently bonded to 
Gantrez, i.e. poly(methylvinyl ether-co-maleic anhydride). Gantrez can be 
represented by the formula 
##STR6## 
where n is preferably from about 120 to about 500. 
The mediator compounds useful in this invention are depicted by general 
formulae IV and V. The structures of the compounds synthesized and tested 
are numbered with Arabic numerals and appear in the first column of Table 
1. Columns 2-7 of Table 1 summarize results of mediator evaluations as 
described in Examples III and IV. 
3 TABLE 1 
Mediator Structures and Activities NADH Titration on GRE Glucose 
Titration on Sensor Compound E.sub.ox.sup.o Potential Slope E.sub.ox.sup. 
o Potential Slope 
##STR7## 
55 mv 155 mv 0.0061 
##STR8## 
108 mv 210 mv 0.0072 
##STR9## 
79 mv36 mv 160 mv200 mv 0.00420.0125 
##STR10## 
62 mv101 mv 162 mv200 mv 0.00770.0109 
##STR11## 
71 mv92 mv 171 mv200 mv 0.00660.0079 
##STR12## 
143 mv130 mv 
##STR13## 
72 mv74 mv 160 mv200 mv 0.00390.0132 
##STR14## 
136 mv 250 mv 0.0042 
##STR15## 
141 mv 250 mv 0.0056 
##STR16## 
24 mv 100 mv 0.0059 125 mv 0.0145 
##STR17## 
57 mv 150 mv 0.0061 160 mv 0.0192 
##STR18## 
92 mv 200 mv 0.0094 
##STR19## 
71 mv 200 mv 0.0072 150 mv 0.0185 
##STR20## 
-20 mv 100 mv 0.0067 
##STR21## 
189 mv &-120 mv -15 mv 300 mv 0.0016 
##STR22## 
86 mv 400 mv 0.0142 
##STR23## 
138 mv 100 mv200 mv300 mv 0.01560.02030.0205 
##STR24## 
27 mv 200 mv 0.0090 -14 mv 100 mv300 mv 0.08480.0962 
##STR25## 
##STR26## 
-116 mv 100 mv 0.079 
##STR27## 
Among those phenothiazines and phenoxazines which have been prepared and 
found to have suitable properties as NADH mediators are 
3-(4'-chloro-phenylimino)-3H-phenothiazine, 
3-(4'-diethylamino-phenylimino)-3H-phenothiazine, 
3-(4'-ethyl-phenylimino)-3H-phenothiazine, 
3-(4'-trifluoromethyl-phenylimino)-3H-phenothiazine, 
3-(4'-methoxycarbonyl-phenylimino)-3H-phenothiazine, 
3-(4'-nitro-phenylimino-3H-phenothiazine, 
3-(4'-methoxy-phenylimino)-3H-phenothiazine, 
7-acetyl-3-(4'-methoxycarbonylphenylimino)-3H-phenothiazine, 
7-trifluoromethyl-3-(4'-methoxycarbonyl-phenylimino)-3H-phenothiazine, 
3-(4'-.omega.-carboxy-n-butyl-phenylimino)-3H-phenothiazine, 
3-(4'-aminomethyl-phenylimino)-3H-phenothiazine, 
3-(4'-(2"-(5"-(p-aminophenyl)-1,3,4-oxadiazoyl)phenylimino)-3H-phenothiazi 
ne, 3-(4'-.beta.-aminoethyl-phenylimino)-3H-phenothiazine, 
6-(4'-ethylphenyl)amino-3-(4'-ethylphenylimino)-3H-phenothiazine, 
6-(4'-[2-(2-ethanoloxy)ethoxy]-ethoxyphenyl)amino-3-(4'-[2-(2-ethanoloxy)e 
thoxy]ethoxyphenylimino)-3H-phenothiazine, 
3-(4'-[2-(2-ethanoloxy)ethoxy]ethoxy-phenylimino)-3H-phenothiazine, 
3-(4'-phenylimino)-3H-phenothiazineboronic acid, 
3-(3',5'-dicarboxy-phenylimino)-3H-phenothiazine, 
3-(4'-carboxyphenylimino)-3H-phenothiazine, 
3-(3',5'-dicarboxy-phenylimino)-3H-phenoxazine, 
3-(2',5'-phenylimino)-3H-phenothiazinedisulfonic acid, and 
3-(3'-phenylimino)-3H-phenothiazinesulfonic acid. 
The method of practicing the invention is further illustrated by the 
following examples. 
EXAMPLE I (Synthesis of Mediators) 
In general, the synthesis of the compounds useful as mediators in the 
present invention involves the oxidative coupling of an aniline with a 
phenothiazine or a phenoxazine. The synthesis of the unsubstituted 
3-phenylimino-3H-phenothiazine was reported in Liebigs Ann. Chemn. 322, 39 
(1902) whereas the synthesis of two other analogs is reported in U.S. Pat. 
No. 4,710,570. Both of these references are incorporated herein by 
reference. The general synthetic scheme for preparing these compounds is 
presented in Scheme I in which compound 18 from Table I is exemplified: 
##STR28## 
The synthesis of specific compound from Table I (compounds 4, 12 and 20) 
was carried out as follows: 
3-(4'-trifluoromethylphenylimino)-3H-phenothiazine (4) 
Phenothiazine (2.0 g, 10 mmole) and 4-aminobenzotrifluoride (1.77 g, 11 
mmole) were dissolved in methanol (MeOH) (100 mL) with warming to 
45.degree. C., treated with a solution of I.sub.2 (5.0 g, 19.4 mmole) in 
MeOH (80 mL) in one portion and allowed to stir for 3 h at 45.degree. C. 
The reaction mixture was filtered and the collected solid was washed with 
MeOH (100 mL). The solid was dissolved in CHCl.sub.3 (60 mL) containing 
triethylamine (NEt.sub.3) (6 mL) then this solution was diluted with 
hexane (200 mL) and chilled in an ice bath for 2 h. The purple solid that 
separated was collected by filtration and redissolved in CHCl.sub.3 
/NEt.sub.3 (10:1, v:v) (50 mL) and chromatographed on silica gel (250 g) 
using CHCl.sub.3 /acetone (96:4, v:v) development. The purple product band 
was collected and evaporated to dryness in vacuo to afford 4 (0.26 g, 
7.3%) with mp=207.degree.-8.degree. C. 
Anal. Calcd. for C.sub.19 H.sub.11 N.sub.2 F.sub.3 S: C, 63.84; H, 3.38; N, 
7.84. Found: C, 63.69; H, 3.26; N, 7.84. 
3-(3',5'-Dicarboxyphenylimino)-3H-phenothiazine (18) 
Phenothiazine (1.0 g, 5.0 mmol) and 5-aminoisophthalic acid (0.9 g, 5.0 
mmol) were dissolved in MeOH (200 mL), cooled to 10.degree. C. and treated 
with 1 M aqueous AgNO.sub.3 (30 mL, 30 mmol). After stirring at 10.degree. 
C. for 30 minutes H.sub.2 O (40 mL) was added and stirring continued for 
an additional 5 minutes. The solid product was then collected by 
filtration and washed with H.sub.2 O (50 mL). The solid was dissolved in 
MeOH (500 mL) containing concentrated aqueous NH.sub.4 OH (10 mL), stirred 
for 20 minutes then filtered to remove Ag. The filtrate was concentrated 
to ca. half its original volume under reduced pressure and refrigerated 
overnight at 5.degree. C. The mixture was then filtered through Celite 
(Celite Corp.) and concentrated in vacuo until solid began to separate. 
The resulting solution (ca. 100 mL) was again filtered through Celite, 
diluted with ethyl acetate (900 mL) and allowed to stand for 15 minutes. 
The red solid that separated was collected by filtration, washed with 
ethyl acetate and dried to give 18(1.55 g, 82.5%). 
3-(3',5'-Dicarboxyphenylimino) 73H-phenoxazine (20) 
Phenoxazine (0.92 g, 5 mmole) was dissolved in tetrahydrofuran (THF) (10 
mL) and stirred at ambient temperature with a solution of 
5-aminoisophthalic acid (0.88 g, 4.86 mmole) in H.sub.2 O (10 mL) 
containing concentrated aqueous NH.sub.4 OH (1 mL). The mixture was 
rapidly treated with a solution of 1.0 M aqueous AgNO.sub.2 solution (13.5 
mL) and stirred for 2 h, during which time a solid separated. The solid 
was collected by filtration and extracted three times with THF (100 mL 
each). The combined extracts were evaporated to dryness in vacuo to yield 
a dark solid (0.8 g) which was taken up in CHCl.sub.3 /MeOH (1:1, v:v) 
(100 mL), with warming, and chromatographed on silica gel (300 g) using 
CHCl.sub.3 /MeOH (2:1, v:v) development. The dark orange product band was 
collected and evaporated to dryness in vacuo to afford 20(0.30 g, 16.7%). 
Using the procedures outlined above, one skilled in this art could 
synthesize a wide variety of substituted analogs of these compounds. 
EXAMPLE II (Synthesis of Polymer-Bound Mediators) 
The synthesis of a polymeric mediator, designated herein as P-3, is 
representative of the method used to prepare other polymer bound mediators 
within the scope of the present invention. This synthesis is illustrated 
in Scheme 2 and the description which follows it. P-3 may be described as 
"40% 13 on Gantrez" because 40% of the anhydride groups on the polymer 
have been reacted with the amino moiety of 13. 
##STR29## 
40% 13 on Gantrez (P-3) 
A solution of GANTREZ AN-119 (ISP Technologies, Inc., Wayne, N.J.) (0.156 
g, 1.0 mmol anhydride) in anhydrous N,N-dimethylformamide (DMF) (10.0 mL), 
maintained under an inert gas atmosphere at ambient temperature, was 
treated at once with a solution of 13 (0.1326 g, 0.4 mmole) and anhydrous 
triethylamine 0.56 mL, 4.0 mmole) in anhydrous DMF (5.0 mL). Additional 
DMF (3.0 mL) was used to rinse in traces of the 13 solution then the 
reaction mixture was allowed to stir for 1 hour. The mixture was diluted 
with H.sub.2 O (25 mL), stirred for 15 minutes then blended into 
additional H.sub.2 O (175 mL). The resulting solution was acidified with 
conc. HCl (3.0 ML) whereupon the product separated as a solid. The mixture 
was centrifuged for 20 min. at 10,400 X g and the supernatant discarded. 
The pellet was resuspended in 0.1 M HCl (20 mL) with sonication and 
centrifuged for 20 min. at 33,400 X g. The supernatant was discarded and 
the pellet resuspended in 100% ethanol (20 mL) with sonication, then 
centrifuged for 30 min. at 33,400 X g. The supernatant was discarded and 
the pellet was dried in vacuo to afford P-3 (0.26 g) as a black powder. 
Polymeric mediators P-1 (20% 11 on Gantrez) and P-2 (20% 13 on Gantrez) are 
prepared analogously to P-3 except that the 0.4 mmol of 13 is replaced 
with 0.2 mmol of 11 or 0.2 mmol of 13 respectively. 
EXAMPLE III (Evaluation of Mediators on Graphite Electrodes 
Graphite rod electrodes (3 mm in diameter from Johnson Matthey Electronics, 
Ward Hill, Mass.) were prepared by polishing the electrode's surface first 
using a fine grit sandpaper then a suspension of .gtoreq.1 micron alumina 
particles. A 1-2 mM solution of the mediator in methanol was prepared and 
the electrode soaked in this solution for 2 minutes. The electrodes were 
then rinsed with water and soaked for a short time in 0.25 M phosphate 
buffer (pH=7.0). A current vs. voltage profile was first run to determine 
the cathodic and anodic peak positions vs. a Ag/AgCl reference electrode. 
Currents were then measured in pH=7.0 solutions containing NADH in 
concentrations from 20 to 200 .mu.M, using a potential that was typically 
100 mv more positive than the oxidative peak potential (E.degree..sub.ox) 
obtained in the cyclic voltammetry experiment above (actual potentials 
used are listed in column #3 of Table 1), and the slope of the line 
obtained from a least squares fit of the current vs. NADH concentration 
data gave the relative sensitivity of each mediator in .mu.A/.mu.M NADH. 
These slopes for various mediators are listed in the 4th column of Table 
1; the greater the slope the better the mediator. For comparison, the 
slope obtained for 3-.beta.-naphthoyl-toluidine blue O (I) under these 
conditions is 0.0075. 
EXAMPLE IV (Evaluation of Mediators on Printed Electrodes) 
Experiments involving printed electrodes comprising a printed sensor card 
with a graphite/carbon working electrode and a silver/silver chloride 
reference electrode were carried out. The ink used for the graphite/carbon 
electrode was No. 423SS (from Acheson Colloids Co., Port Huron, Mich.) and 
No. 427SS silver ink (same vendor) blended with 15-25% AgCl for the 
silver/silver chloride reference electrode. Electrode surface area was 
0.03 cm.sup.2. 
Cyclic voltammetry experiments were performed on printed electrodes just as 
on the graphite rod electrodes. FIG. 1 shows the cyclic voltammogram of 
0.6 mM compound 18 in 0.6 mM PIPES buffer at pH=7.0 alone and in the 
presence of 5 mM NADH. The increase in current at a potential of 26 mv in 
the presence of NADH is due to the oxidation of mediator that had been 
reduced by NADH. The mediator is reduced as it oxidizes NADH to NAD.sup.+ 
and is then re-oxidized electrochemically at the electrode. In effect the 
mediator facilitates the electrochemical oxidation of NADH at potentials 
considerably lower than those required to oxidize NADH directly. FIG. 2 
shows a typical cyclic voltammogram of NADH being oxidized directly (no 
mediator) on a printed electrode. A much higher peak potential (443 mv) is 
apparent. 
A biosensor for measuring glucose concentration uses the enzyme glucose 
dehydrogenase to reduce NAD.sup.+ to NADH as it oxidizes glucose to 
gluconolactone. Mediated electrochemical re-oxidation of the NADH to 
NAD.sup.+ generates a current that is proportional to the glucose 
concentration. 
A typical glucose biosensor was fabricated as follows: A solution of 4 mM 
mediator in 100 mM pH=7.0 phosphate buffer containing 25 mM KCl was 
prepared and diluted with an equal volume of a solution composed of 1.96 g 
5% Surfynol (Air Products and Chemicals, Inc., Allentown, Pa.), 0.32 g 
NAD, 0.70 g Glucose Dehydrogenase GDH (Toyobo), 1.44 g 0.5M PIPES buffer 
pH--7.0 and 5.5 mL DI H.sub.2 O. 1.75 .mu.L of the mixture was applied to 
the sensor area and allowed to dry at RT for about 20 min. The electrode 
was assembled in a format having a small capillary gap, treated with a 
solution of aqueous glucose and the current measured at the potential 
noted in the 6th column of Table 1. This was done for samples containing 
glucose concentrations of 0, 50, 100, 200 and 500 mg/dL and the slope of 
the lisine line obtained from a least squares fit of the current vs. 
glucose concentration data gave the relative sensitivity of each mediator 
in .mu.A/mg dL.sup.-1 glucose; the larger the slope the better the 
mediator. These slopes are listed in the last column of Table 1. 
FIG. 3 shows the plot of current vs. glucose concentration for compound 18 
at 100 mv and 300 mv potentials. 
Polymeric mediators were incorporated into complete glucose biosensors as 
described above and the relative sensitivities are listed in the last 
column of Table 2. 
TABLE 2 
______________________________________ 
Activity of Polymeric Mediators 
Titra- 
tion on 
NADH Poten- on Glucose 
Titration 
Sensor 
E.degree..sub.ox 
tial GRE E.degree..sub.ox 
Potential 
Slope 
______________________________________ 
P-1 -26 mv 100 mv 0.0204 
(20% 11 - 300 mv 0.0362 
Gantrez) 
P-2 -62 mv 300 mv 0.0276 
(20% 13 - 
Gantrez) 
P-3 -43 mv 300 mv 0.0217 
(40% 13 - 
Gantrez) 
______________________________________ 
FIG. 4 shows the plot of current vs. glucose concentration for 3 polymeric 
mediators at 300 mv potential.