Sensor for components of a liquid mixture

This invention relates to equipment and methods for detecting the presence of, measuring the amount of, and/or monitoring the level of, one or more selected components in a liquid mixture, employing an electrode sensing system. We have discovered that a class of mediating compounds has extremely useful properties for mediating the transfer of charge between enzyme-catalyzed reactions and electrode surfaces (15) in electrode sensing systems. Specifically, the specification discloses as electrode sensor mediators, organometallic compounds composed of at least two organic rings, each of which has at least two double bonds in a conjugated relationship; a metal atom is in electron-sharing contact with those rings. An enzyme capable of catalyzing a reaction at a rate representative of the selected compound concentration is in contact with an assay mixture, and the mediator compound transfers charge between the enzyme and the conductive surface of the electrode at a rate representative of the enzyme catalyzed reaction rate.

BACKGROUND 
This invention relates to equipment and methods for detecting the presence 
of, measuring the amount of, and/or monitoring the level of, one or more 
selected components in a liquid mixture. 
While use may be made of this invention in chemical industry, especially 
where complex mixtures are encountered (e.g. in food chemistry or 
biochemical engineering) it is of particular value in biological 
investigation and control techniques. More particularly, it lends itself 
to animal or human medicine, and in particular to in vivo measuring or 
monitoring of components in body fluids. 
For convenience, the invention will be described, inter alia, with 
reference to one particular in vivo measurement, the determination of 
glucose in a diabetic human subject by the use of equipment which, while 
usable on a specific or occasional basis also lends itself to temporary or 
permanent implantation. While the provision of sensors for components in 
biological fluids is one object of the invention, other and broader 
objects are not hereby excluded. 
In vivo glucose sensors have already been proposed. One proposal is based 
on direct oxidation of glucose at a catalytic platinum electrode (see 
Hormone and Metabolic Research, Supplement Series No. 8, pp 10-12 (1979)) 
but suffers from the drawback of being non-specific and of being easily 
poisoned by interfering substances. Another proposal, for a procedure more 
specific to glucose, involves the use of glucose oxidase on an oxygen 
electrode (Adv. Exp. Med. Biol, 50 pp 189-197 (1974) but is not very 
responsive to the high glucose concentrations. Other systems using glucose 
oxidase have been proposed but not fully investigated for in vivo methods, 
see e.g. J. Solid-Phase Biochem. 4 pp 253-262 (1979)). 
A fall in the level of oxygen tension resulting from poor tissue perfusion 
is a particular problem for detecting glucose in blood taken from 
subcutaneous tissue of diabetics. 
The inventors have carried out in vitro studies of enzyme-catalyzed 
reactions using a mediator (e.g. phenazine methosulfate or phenazine 
ethosulfate) in solution to transfer the electrons arising from the 
enzyme, during its action, directly to the electrode, as described in 
Biotechnology Letters 3 pp 187-192 (1981). 
Generally, it is desirable to find a mediator which meets the particularly 
stringent demands of quantitative electrochemical assaying. For example, 
the mediator must rapidly transfer electrons between the enzyme and the 
electrode at a rate representative of the rate of the enzyme-catalysed 
reaction rate. The mediator should be sensitive to potential differences 
of the enzyme; however, the mediator's response should be relatively 
insensitive to the presence of interfering substances. Ideally, the 
mediator should be capable of effecting electron transfer for a broad 
range of enzymes and under a broad range of conditions such as temperature 
and pH. The mediator should not be toxic to cells or carcinogenic. 
It is specifically desirable to find an alternative amperometric detection 
method, based on glucose oxidase, which is not dependent on oxygen as the 
mediator of electron transfer. Previously described electron acceptors for 
glucose oxidase include hexacyanoferrate (III), and a range of organic 
dyes; the former is not readily entrapped at an electrode; the latter, 
though widely used in spectrophotometric measurements, have a number of 
disadvantages for electromechanical use including ready autoxidation, 
instability in the reduced forms and pH-dependent redox potentials. 
SUMMARY OF THE INVENTION 
We have discovered that a class of mediating compounds has extremely useful 
properties for mediating enzyme-catalysed reactions in electrode sensing 
systems. In one aspect, the invention features, as electrode sensor 
mediators, organometallic compounds known as metallocenes, and more 
specifically (bis)polyhaptometallocenes which include two organic ring 
structures, each with conjugated unsaturation, and a metal atom sandwiched 
between the rings, so that the metal atom is in electron-sharing contact 
with each atom in the ring. In another aspect the mediator is composed of 
at least two organic rings, each of which has at least two double bonds in 
a conjugated relationship; a metal atom is in electron-sharing contact 
with those rings. The mediators are broadly useful in electrode sensor 
systems having two conductors insulated from each other, each of which is 
in contact, via a conductive surface, with a mixture of compounds that 
includes the selected compound to be sensed. An enzyme capable of 
catalyzing a reaction at a rate representative of the selected compound 
concentration is in contact with the mixture, and the mediator compound 
transfers electrons between the enzyme and the conductive surface of one 
of the conductors at a rate representative of the enzyme catalyzed 
reaction rate. 
We have discovered that ferrocene-type compounds are particularly useful 
mediators. Other compounds that are envisaged as mediators include 
ruthocene-type compounds and dibenzene chromium. Insoluble compounds, 
particularly ferrocenes, are most preferable. 
Ferrocene, has, as its fundamental structure, an iron atom held 
"sandwiched" by bonds between two cyclo-pentadienyl rings. It is an 
electroactive organometallic compound, acting as a pH-independent 
reversible one-electron donor. Various derivatives are available (e.g. 
with various substituents on the ring structure, possibly in polymer form) 
differing in redox potential, aqueous solubility and binding constant to 
enzymes. 
For instance, the redox potential of the parent compound is +422 mV vs NHE. 
By introducing functional groups on to the ring system, E'o can be varied 
between +300 and +650 mV. Moreover, the water-solubility of the 
carboxyl-substituted ferrocenes is greater than that of the parent 
compound. Further description will be found in Kuwana T., 1977, ACS 
Symposium Series, 38: 154. 
Among specific mediator compounds of this type are ferrocene itself, 
1,1'-ferrocene dicarboxylic acid, dimethyl ferrocene, and polyvinyl 
ferrocene, e.g. of average molecular weight of about 16000. 
Other derivatives, having substitution of one or both cyclopentadienyl 
rings and/or by polymerisation that we have studied include those listed 
in table 1 below. 
The unique structure and properties of ferrocene (Fecp.sub.2) and its 
derivatives have resulted in a considerable amount of theoretical and 
experimental studies. First synthesised in 1951, ferrocene itself was the 
earliest example of the now well-known metallocene compounds. 
Whilst ferrocenes had been found to be of limited value in 
spectrophotometric assays as a result of their poor solubility in aqueous 
solution and low extinction coefficients, we have found them to be more 
suited to a bio-electrochemical system. Ferrocene-type compounds have 
advantages over other mediators used with enzyme/substrate reactions for 
charge-transfer purposes. Specifically, ferrocenes have: 
(a) a wide range of redox potentials accessible through substitution of the 
cyclopentadieneyl rings, which can be functionalised; 
(b) electrochemically reversible one-electron redox properties; 
(c) pH-independent redox potential and slow autoxidation of the reduced 
form; 
(d) the absence of any known problems of toxicity or carcinogenicity from 
ferrocene compounds; 
(e) the capability of redox reaction at a potential sufficiently low to 
avoid excessive interference from competing higher redox-potential 
reactions competing with the enzyme-catalyzed reaction being sensed; 
(f) satisfactory oxygen insensitivity to avoid excessive interference from 
oxygen; 
(g) the ability to be concentrated at the electrode surface by covalent 
attachment or by surface adsorption; 
(h) the ability to control redox potential over a range by controlling 
substitution on the ferrocene ring; 
(i) the ability to control water solubility by controlling substitution on 
ferrocene ring--for example relatively insoluble compounds (e.g. 
1-1'-dimetheylferrocene) are selected where the mediator is to be 
concentrated at the electrode, and soluble compounds (e.g. COOH 
substituted ferrocene) are selected where it is desirable to have the 
mediator diffuse throughout the solution. 
Thus, ferrocene meets the stringent demands placed on the mediator. For 
example, ferrocene compounds readily shuttle electrons between the enzyme 
and the conductive electrode surface at a rate that is high enough to 
render potentially conflicting reactions insignificant. Moreover, the 
response covers as large a region as possible to enhance the precision of 
the concentration reading. The ferrocene compound can be concentrated at 
the electrode surface in sufficient amounts to enhance electron transfer. 
Where the ferrocene compound is covalently bound to the electrode and/or 
the enzyme, the bonding does not interfere with the mediating function. 
The ferrocene compound selected should be relatively insoluble in water 
for most application. It should be stable and non-responsive to 
interfering substances such as oxygen or pH. Most importantly the rate of 
electron transfer with ferrocene must be dependent on the rate of the 
enzyme-catalyzed reaction. That is, the ferrocene effects electron 
transfer during the period of catalytic ativity at a rate representative 
of that activity. 
Satisfactory performance in the above-listed areas is obtained with 
ferrocene-type compounds in an extraordinarily broad range of sensor 
systems. For example, ferrocene can mediate electron transfer for a broad 
range of enzymes. 
The preferred enzymes are non-oxygen-specific flavo-protein enzymes or 
quinoproteins Glucose oxidase and glucose dehydrogenase are particularly 
preferred enzymes. 
Glucose oxidase (.beta.-D-glucose:oxygen oxidoreductase, of enzyme 
classification EC 1.1.3.4) is a well known type of enzyme. Bacterial 
glucose dehydrogenase is of more recent discovery, and is believed to be a 
quinoprotein with a polycyclicquinone prosthetic group (PQQ). Reference is 
made to Duine et al TIBS (October 1981) 278-280 and Arch, MicrobiOl (1982) 
131.27-31. 
Use of such a bacterial glucose dehydrogenase in the present invention has 
certain advantages over the use of a glucose oxidase. The major advantage 
is that it can give an oxygen-insensitive glucose sensor, since the enzyme 
does not use oxygen as an electron acceptor. A suitable enzyme can be 
purified (as described in more detail below) either by conventional 
chromatographic techniques or by two-phase aqueous partition from a range 
of micro-organisms. A preferred micro-organism is Acinetobacter 
calcoaceticus but various Gluconobacter species (e.g. Gluconobacter 
oxidans) or Pseudomonas species (e.g. Pseudomonas fluorescens, Pseudomonas 
aeruginosa) can also be used. 
DESCRIPTION OF THE PREFERRED EMBODIMENT 
The above referenced U.S. patent application, as well as the corresponding 
European Patent application No. 82305597, [published May 11, 1983 as No. 
EP0078636], discloses a sensor electrode composed of electrically 
conductive material and comprising at least at an external surface thereof 
the combination of an enzyme and a mediator compound which transfers 
electrons to the electrode when the enzyme is catalytically active. 
The purpose of such an electrode is to detect the presence of, measure the 
amount of and/or monitor the level of one or more selected components 
capable of undertaking a reaction catalysed by the said enzyme. 
Examples of electrode configurations, mediators and uses are given in the 
above-described U.S. and E.P. patent applications which are hereby 
incorporated by reference. 
The following U.S. patent application filed May 7, 1984 and owned by 
Genetics International, Inc. are hereby incorporated by reference: 
Assay Techniques Utilizing Specific Binding Agents (U.S. Ser. No. 607,695) 
Assay Systems Using More Than One Enzyme (U.S. Ser. No. 607,607, filed by 
Davis, Hill, and Cass); 
Assay Systems Utilizing More Than One Enzyme (U.S. Ser. No. 607,698, filed 
by Davis and Hill); 
Analytical Equipment and Sensor Electrodes Therefor (U.S. Ser. No. 607,599) 
U.S. Ser. No. 607,695 has been abandoned in favor of a continuation 
application, U.S. Ser. No. 039,531, filed Apr. 16, 1987. U.S. Ser. No. 
607,599 has been abandoned in favor of a continuation-in-part application, 
U.S. Ser. No. 002,120, filed Jan. 12, 1987. U.S. Ser. No. 607,607 is now 
abandoned. 
1,1-dimethylferrocene is a particularly preferred mediator. The selected 
component to be sensed is preferably the substrate for the 
enzyme-catalyzed reaction. Also, preferably, the enzyme and/or the 
mediator are confined at the conductive surface of one of the conductors. 
Finally, in preferred systems, the mediator transfers electrons from the 
enzyme to the electrode surface. The properties of a range of ferrocene 
derivatives, together with those of the parent compound are given in the 
table below; 
TABLE 1 
______________________________________ 
Ferrocene derivative 
Eo Solubility 
E 
______________________________________ 
1,1'-dimethyl- 100 I,D -- 
acetic acid 124 S 370 
hydroxyethyl- 161 S -- 
ferrocene 165 I,D 335 
1,1'bis(hydroxymethyl)- 
224 S 385 
monocarboxylic acid 
275 S 420 
1,1'-dicarboxylic acid 
385 S -- 
chloro- 345 I,D -- 
methyl trimethylamino- 
400 S -- 
______________________________________ 
S indicates water solubility; I,D means respectively insoluble and 
detergent solubilised in 3% Tween-20. E.degree. is in mV vs a standard 
calomel electrode, and E is measured in cm.sup.-1 M.sup.-1. 
The E.degree. values of various ferrocenes in phosphate buffer at pH 7.0 
given in the above table, span a range of potentials, E.degree.=100 to 400 
mV vs SCE. The trend in E.degree. values is in agreement with that 
expected on the basis of substituent effects. In general electron-donating 
groups stabilize the positive charge and hence promote oxidation more so 
than electron withdrawing groups. 
In one particularly preferred embodiment, the electrode is designed to 
determine glucose in vivo. The enzyme is therefore preferably a glucose 
oxidase, or possibly a glucose dehydrogenase, for example a bacterial 
glucose dehydrogenase. 
The electrically conductive material of the electrode itself can be a 
metal, particularly silver, or carbon either as a pre-formed rod or as an 
electrode shape made up from a paste of carbon particles or a carbon 
fibre. Surface condition of the electrode is usually important. If metal, 
the surface can be roughened where it contacts the active materials 
(enzyme and/or mediator). If solid carbon, the surface can be "oxidised" 
i.e. previously heat-treated in an oven with oxygen access. 
Of the two types of enzyme listed for the exemplary assay of glucose, the 
dehydrogenase is preferred, and of the mediators the ferrocene-type 
compounds are preferred. 
Certain combinations of the above materials, and certain configurations of 
electrode, are preferable in practice. 
Optionally, enzyme immobilisation materials, or polymeric electrode 
admixtures e.g. TEFLON, or long-chain alkyl derivatives of mediators of 
increased molecular weight and thus decreased mobility, can be 
incorporated. 
In a particularly valuable form of the invention, however, the electrode 
comprises a carbon core, a layer of ferrocene or a ferrocene derivative at 
a surface thereof and a layer of glucose oxidase or glucose dehydrogenase 
at the surface of the ferrocene layer. The enzyme layer is peferably 
immobilised at the surface of the underlying mediator, retained in a 
self-sustaining gel layer thereupon and/or has a retention layer thereover 
permeable to the glucose molecules. 
The carbon core can itself be solid or a stiff paste of particles. 
Normally, it will present a smooth surface for the ferrocene or ferrocene 
derivative, which may be adhered thereto in a number of ways, for example, 
(a) For a monomeric ferrocene or ferrocene derivative, by deposition from a 
solution in a readily evaporatable liquid e.g. an organic solvent such as 
toluene. 
(b) For a ferrocene polymeric derivative, deposition from a readily 
evaporable organic solvent for the polymer such as chloroform. J. Polymer 
Sci. 1976, 14 2433 describes preparation of a polyvinyl ferrocene of 
average molecular weight about 16000 which can be deposited in this way. 
(c) For a polymerisable ferrocene-type monomer, by electrochemically 
induced polymerisation in situ, e.g. by dissolving vinyl ferrocene in an 
organic electrolyte containing tertiary butyl ammonium perchlorate in 
concentration about 1M and depositing at a potential of -700 mV vinyl 
ferrocene radicals as a polymer in situ. 
The enzyme to be coated on to the ferrocene or ferrocene derivative can be 
the glucose oxidase or the bacterial glucose dehydrogenase. The glucose 
oxidase can be immobilised to the underlying surface e.g. by the 
carbo-diimide material DCC (1-cyclohexyl-3(2-morpholino ethyl) 
carbo-diimide metho-p-toluene sulphonate) which gives a thin strongly 
bound layer, a good linear response to low glucose concentrations, and 
oxygen insensitivity (because of the competition from the ferrocene with 
oxygen for electrons transferred to the enzyme redox centre from the 
substrate). Using DCC immobilisation of glucose oxidase on ferrocene also 
extends the top end of the linear range of the sensor from about 2 mM to 
40 mM. 
Other methods of immobilisation, or other forms of protection e.g. 
incorporated into a self-supporting gelatine layer, are also possible. 
The bacterial glucose dehydrogenase can also be immobilised at the mediator 
surface, but may be merely deposited from an evaporatable solution, or 
held in a gelatin layer. 
Optionally, but preferably when being used on live blood, a protective 
membrane surrounds both the enzyme and the mediator layers, permeable to 
water and glucose molecules. This can be a film of dialysis membrane, 
resiliently held e.g. by an elastic O-ring. It can however also with 
advantage be a layer of cellulose acetate, e.g. as formed by dipping the 
electrode into a cellulose acetate solution in acetone or polyurethane. 
Membranes may be applied by dip, spray or spin coating techniques. It will 
be apparent that while the invention has primary relevance to a sensor 
electrode, especially such an electrode specific for glucose, it also 
relates to the combination of such an electrode and temporary or permanent 
implantation means, e.g. a needle-like probe. Also, such an electrode, 
connected or connectable, with signal or control equipment, more 
especially with an insulin administration means, constitutes an aspect of 
the invention. Moreover, a method of monitoring a diabetic subject 
involving the use of a temporarily or permanently implanted electrode as 
described above is also within the scope of the invention. 
The electrodes according to the invention permit the manufacture of an 
improved macro-sensor for use in hospital analytical glucose-sensing 
instruments of the existing type. The advantages compared to known 
instruments would be that the increased linear range together with very 
low oxygen sensitivity would allow omission of the dilution step involved 
in blood analysis in current instruments. Moreover, as described in more 
detail below, the response times of such electrodes are short (24-36 
seconds for 95% of steady state depending on complexity of solution). 
The electrodes of the invention, on the macro-scale could be incorporated 
into simple, cheap electronic digital read-out instruments for doctors 
surgeries or diabetic home-testing kits. 
Use of a small version of the macro-sensor would be possible in a device 
which automatically takes a blood sample from the finger, brings it into 
contact with the sensor, amplifies the signal and gives a digital readout. 
Use of a micro-version of the sensor in a watch type device for monitoring 
glucose interstitial fluid in the skin could also be envisaged. It would 
be worn on the wrist and would have a disposable sensor cartridge in the 
back with one more more separate, fine, needle-type sensors. Each would 
feed into the electronics which if several sensors were used would 
cross-refer the current inputs to ensure reliability. 
Connection of such devices to external insulin delivery systems could act 
as a feedback control loop for an insulin pump. Indeed, such a device 
could be housed in the cannula used to feed insulin into the body from a 
pump and again serve as a sensor for the feedback loop. Other uses such as 
a hypoglycaemia alarm, or digital read-out monitor, are also possible. 
The enzymes that can be used with ferrocene-mediated systems include: 
flavo-proteins that are capable of using a variety of electron acceptors, 
including oxygen; and NADPH-or NADH-linked enzymes such as lipoamide 
dehydrogenase and glutathione reductase; dehydrogenase enzymes, termed 
quinoproteins, that contain the above-mentioned polycyclicquinone 
prosethtic group (PQQ). 
A listing of flavoproteins that generate H.sub.2 O.sub.2 appears in Clark 
et al. Biotechnol. Bioeng. Symp. 3: 377 (1972). Particularly preferred 
flavoproteins are: lactate oxidase, pyruvate oxidase, xanthine oxidase, 
sarcosine oxidase, lipoamide dehydrogenase, glutathione reductase, 
aldehyde oxidase, glucose oxidase, glycollate oxidase, L-amino oxidase, 
galactose oxidase. Ferrocenes are also suitable mediators for methanol 
oxidase and carbon monoxide oxido reductase. 
Suitable quinoproteins include glucose dehydrogenase, alcohol 
dehydrogenase, methanol dehydrogenase. A list of PQQ quinoproteins appears 
in Quine et al. J. TIBS 6: 728 (1981). 
Finally, heam-containing enzymes can be used in ferrocene-mediated 
electrode systems. Such enzymes include: horseradish peroxidase, yeast 
cytochrome C peroxidase, lactate dehydrogenase (i.e. yeast cytochome B2), 
and horse heart cytochrome C peroxidase. 
The compatibility of an enzyme such as those listed above with ferrocene 
can be demonstrated using dc cyclic voltammograms in which current at a 
working electrode is measured over voltage sweeps. 
The current measured includes a Faradaic component which results from 
electron transfer to and from an electro-active species in the solution. 
If the rate of electron transfer between the electro-active species is 
sufficiently fast, the Faradiac current is controlled by the rate of 
diffusion of the electro-active species. The enzyme-catalyzed reaction 
causes a perturbation in the voltammogram that depends on the reaction 
rate, compared with the time required for the voltage sweep. 
Thus, the suitability of a particular mediator for transfer between a 
particular enzyme and an electrode can be assessed as described below in 
examples 12-31. 
The preferred enzymes are the flavo-protein enzymes which are not 
oxygen-specific and the quino-protein enzymes, and, in particular, enzymes 
catalyzing glucose reactions such as glucose oxidase and glucose 
dehydrogenase. 
As discussed above, in the preferred sensor system the compound selected to 
be measured is the substrate for the enzyme, and the enzyme and mediator 
are confined at the electrode surface. The electrode is exposed to a 
mixture containing the selected compound, and the enzyme becomes 
catalytically active, generating a current representative of the 
compound's concentration. 
Other configurations are possible, however, in which the rate of the enzyme 
catalyzed reaction is a surrogate for the concentration of another 
compound that is not the enzyme substrate.

EXAMPLE 1 
Purification of Quinoprotein Glucose Dehydrogenase (GDH) from Acinetobacter 
calcoaceticus 
(a) Growth of Organisms 
Strain NCTC 7844 was grown on sodium succinate (20 gl.sup.-1) in batch 
culture at pH 8.5 and 20.degree. C. Cells were harvested after 20 hours 
A.sub.600 =6.0) using a Sharpless centrifuge, and stored frozen. 
(b) Purification of Glucose Dehydrogenase 
The method is based on the method of J A Duine et al (Arch Microbiol, 1982 
vide supra) but with modifications as follows. 
1. 100 g of cells were thawed, resuspended in 3 300 ml. of 56 mM Tris/39 mM 
glycine and treated for 20 minutes at room temperature with 60 mg. 
lyxozyme. 
2. Triton X-100 extracts were combined and treated with 0.01 mgml.sup.-1 of 
deoxyribonuclease I for 15 minutes at room temperature. The resulting 
suspension was then centrifuged at 48000 xg for 25 minutes at 4.degree. C. 
The supernatant from this centrifugation was then treated with ammonium 
sulphate. The yellow protein precipitating between 55 and 70% ammonium 
sulphate was resuspended in 36 mM Tris/39 mM glycine containing 1% Triton 
X-100 and dialysed against that buffer at 4.degree. C. for 5 hours. 
3. Active fractions from the CM Sepharose C1-6B Column were combined and 
concentrated using Millipore CX-30 immersible ultrafilters. 
EXAMPLE 2 
Purification of Quinoprotein Glucose Dehydrogenase from Acinetobacter 
calcoaceticus (alternative method) 
(a) Growth of Organisms 
The method of Example 1 was repeated. 
(b) Purification of GDH 
The method is based on the partitioning of proteins between two liquid 
phases. The steps were: 
1. Cells were thawed and resuspended at 3 ml/g wet weight in 50 mM sodium 
phosphate, pH 7.0. They were then pre-cooled on ice and passed once 
through a Stansted pressure cell (made by Stansted Fluid Power Ltd., 
Stansted, Essex, UK) at 25000 psi. This provides the cell-free extract. 
2. The cell-free extract was the mixed for 15 minutes at room temperature 
with 50% (w/v) polyethyleneglycol 1000, 50% (w/v) sodium phosphate, pH 7.0 
and distilled water in the proportions of 2:4:3:1 respectively. This 
mixture was centrifuged at 5000 rpm for 5 minutes to break the emulsion. 
3. The lower layer was aspirated off and desalted immediately, by either 
diafiltration using an Amicon hollow-fibre ultrafiltration cartridge of 
10000 mwt cut off, or by passage through a Sephadex G50 (medium grade) gel 
filtration column. 
4. The resulting solution was concentrated using an Amicon PM10 membrane in 
a nitrogen pressure cell. 
EXAMPLE 3 
Interaction between Ferrocene and Glucose Oxidase 
DC cyclic voltammetry was used to investigate the homogeneous kinetics of 
the reaction between ferrocene and the glucose oxidase enzyme under 
substrate excess conditions. A two compartment electromechemical cell of 
1.0 ml volume fitted with a Luggin capillary was used. The cell contained 
a 4.0 mm gold disc working electrode, a platinum gauze counter-electrode 
and a saturated calomel electrode as a reference. A series of voltamograms 
for ferrocene was recorded at scan rates of 1-100 mVs.sup.-1 in 50 mM 
potassium phosphate buffer, pH 7.0. The data shows that the mediator acted 
as a reversible, one-electron acceptor. 
Addition of 50 mM glucose has no discernable effect on the electrochemistry 
of the mediator (500 .mu.m). Upon addition of glucose oxidase (10 .mu.m), 
however, an enhanced anodic current was observed in the voltamogram at 
oxidising potentials with respect ot the mediator. This indicated 
catalytic regeneration of the reduced form of the mediator by glucose 
oxidase. Quantitative kinetic data was obtained for this reaction using an 
established procedure (Nicholson, R. S. and Shain, J., 1964, Anal. Chem., 
36, 707). The meditor gave a second order rate constant for the reaction 
between ferricinium ion and reduced glucose oxidase of K=10.sup.4 m.sup.-1 
s.sup.-1. This ability of the ferricinium ion to act as a rapid oxidant 
for glucose oxidase facilitates the efficient coupling of the enzymic 
oxidation of glucose. 
EXAMPLE 4 
The procedure of Example 3 was repeated using 1,1'-ferrocene dicarboxylic 
acid instead of ferrocene. The value of Eo' was determined to be +420 mV, 
and the second order rate constant of the ferricinium ion and reduced 
glucose oxidase was again 10.sup.4 m.sup.-1 S.sup.-1, thus confirming the 
conclusions drawn from Example 3. 
EXAMPLE 5 
Glucose Oxidase 1,1-Dimethyl Ferrocene 
Mini electrode for in vivo glucose sensing in skin 
A graphite rod 13 (FIG. 1) with an oxidised surface, 30 mm long.times.0.9 
mm diameter is glued with epoxy resin into a nylon tube 14-25 mm long, 0.9 
mm inside diameter, 1.3 mm outside diameter. The end 15 of the electrode 
is dipped into a solution of dimethyl ferrocene, (10 mg/ml) in toluene, 
and the solvent is then allowed to evaporate. 
The end 15 of the electrode is placed into a solution of water soluble DCC 
(25 mg/ml) in acetate buffer, pH 4.5 for 1 hour. It is then rinsed, in 
buffer only, for 5 minutes and thereafter placed in a solution of glucose 
oxidase (10 mg/ml) in acetate buffer, pH 5.5, for 11/2 hours before again 
rinsing in buffer. The tip of the electrode 15, with the layers of 
dimethyl ferrocene and immobilised enzyme is then dipped into a solution 
of cellulose acetate dissolved in acetone and N,N'-dimethyl formamide and 
put into ice water for several minutes, to give a protected and stable 
electrode. 
This electrode was connected to a potentiostat, together with a suitable 
counter electrode and calomel reference electrode and placed in a solution 
containing glucose. The potential of the working electrode is kept at +100 
mV to 300 mV relative to the calomel electrode, i.e. as low as possible to 
avoid oxidation of potentially intefering substances. A current is 
produced which is proportional to the glucose concentration. The time for 
95% of response is less than 1 minute and the electrode gives a near 
linear response over the range 0-32 mM glucose, as shown in FIG. 2. Slow 
loss of activity of ferrocene (due to slow loss of ferricinium ion) can be 
minimised by keeping the electrode at a potential between 0 and -100 mV 
vs. a standard calomel electrode when not in use. 
FIG. 3 shows in section an electrode structure in which an electrode 
(references as in FIG. 1) of much smaller size is held within a hypodermic 
needle 16 plugged at its point 17 but with side windows 18 for passage of 
blood or other body fluid. The small size of such an electrode and its 
linear response over a large range of glucose concentrations makes it 
possible to use the electrode for in vivo glucose determination on both 
severely diabetic and normal individuals. 
EXAMPLE 6 
Glucose Oxidase/Ferrocene 
In vitro sensor 
A carbon rod 19 (FIG. 4) Ultra carbon, grade U5, 6 mm.times.15 mm with a 
metal connector 20 secured in one end was sealed in glass tubing 21 
(borosilicate, 6 mm i.d..times.mm) with an epoxy resin (araldite). (not 
shown). The exposed surface at 22 was polished with emery paper and washed 
with distilled water. The entire rod was heated in an oven for 40 h at 
200.degree. C. to give an oxidised surface at 22. 
15 .mu.l of ferrocene (20 mg/ml in toluene) was pipetted onto the oxidised 
surface and allowed to dry completely. The rod was then placed in 1 ml of 
water-soluble DCC (25 mg/ml in 0.1M acetate buffer, pH 4.5) for 80 min at 
room temperature. The rod was then washed in 0.2M carbonate buffer, pH 9.5 
and placed in a glucose oxidase solution (Sigma type X, 12.5 mg/ml) for 
11/2 hours at room temperature. It was finally washed with water with a pH 
7 buffer containing 0.2 g/l glucose) and stored at 4.degree. C. 
The characteristics of the above electrode were determined in a 
nitrogen-saturated buffer solution (0.2M sodium phosphate, pH 7.3) and are 
shown in FIG. 5. The curve is linear from 2 to 25 mM glucose and reaches 
saturated current at 100 mM in glucose. 
In separate tests with an air-saturated buffer at 8 mM glucose the current 
was measured as being at least 95% of that produced in the 
nitrogen-saturated buffer. 
Response time was also measured, being the time taken to achieve 95% of 
maximum current for the given concentration. With the nitrogen-saturated 
buffer an electrode as described above had a response time of 24 seconds 
at 2 mM glucose and 60 seconds at 6 mM glucose. With the same buffer, such 
an electrode modified by a cellulose acetate membrane coating (produced as 
in Example 7) gave response times of 36 seconds (2 mM) and 72 seconds (6 
mM). With blood, this modified electrode gave response time of 36 seconds 
(blood with a known 2 mM glucose content) and 72 seconds (blood at a known 
6 mM glucose content). 
Electrodes as above were stored in 20 mM NaPO.sub.4, pH7 for 4 weeks at 
4.degree. C. as a stability test and thereafter re-examined as above. The 
results were within 10% and usually with 5% of results with a freshly made 
electrode. 
EXAMPLE 7 
Glucose Dehydrogenase/Ferrocene 
A stiff carbon paste was made up from 1.6 g of Durco activated charcoal and 
2.5 ml of liquid paraffin. A pasteur pipette of 6 mm internal diameter was 
blocked 2 mm from its wide end by a silver disc to which a connecting wire 
was soldered. The space between the disc and the end of the pipette was 
filled with the carbon paste, and the surface of the paste was polished 
with paper until smooth and even. 
A single 20 microliter drop of a toluene solution of ferrocene (20 mg/l) 
was placed on the smooth surface and allow to spread and evaporate to 
leave a film of the ferrocene. 
A further drop of 25 microliters of bacterial glucose dehydrogenase 
solution as obtained in Example 1, containing between 1 and 10 mg of 
protein per ml, was placed on this ferrocene surface and allowed to 
spread. 
A cover of dialysis membrane was secured over the so-coated end of the 
electrode by a tight-fitting O-ring. 
EXAMPLE 8 
Glucose Dehydrogenase/Ferrocene 
The procedure of Example 7 was repeated but using as electrode the same 
carbon paste packed into the space defined between the end of a length of 
nylon tubing and a stainless steel hypodermic needle shaft inserted 
therein terminating 2 mm. short of the tubing end, so as to define a small 
electrode body. The electrode was further fabricated using only 5 
microliters of the ferrocene solution and 1 microliter of the enzyme 
solution. 
EXAMPLE 9 
Glucose Dehydrogenase/Ferrocene 
The procedure of Example 8 was repeated using as electrode a solid carbon 
rod (Ultracarbon grade U5 6 mm diameter) within a Pyrex glass tube 3 cm 
long and 6 mm internal diameter and connected to a stainless steel 
hypodermic shaft, giving a construction similar to that shown in FIG. 4. 
The end of the carbon rod was polished smooth with emery cloth and 
aluminium oxide powder prior to the application of the ferrocene solution. 
EXAMPLE 10 
Glucose Dehydrogenase/Ferrocene 
A gelaton-entrapped glucose dehydrogenase was prepared by mixing at 
37.degree. C., 25 mg gelatin, 0.5 ml of the glucose dehydrogenase solution 
as described in Example 9 and 2.5 microliters of TEMED. After complete 
dissolving of the gelatin 200 microliters of the solution was spread over 
an area of 2 cm.sup.2 and allowed to dry under a stream of cold air. 
A disc of 0.25 cm.sup.2 area was then used instead of the drop of enzyme 
solution in Example 8. 
EXAMPLE 11 
Glucose Dehydrogenase/Ferrocene 
Example 10 was repeated using a disc of the gel of 1 mm.sup.2 area and 
applying it instead of the drops of enzyme solution in the construction of 
example 10. 
The results obtained from the electrodes described in Examples 7-11 are all 
similar, and show a very specific electrode of low oxygen sensitivity. By 
way of example, the electrode of Example 10 was calibrated and gave the 
results shown in FIG. 6. 
EXAMPLES 12-24 
dc cyclic voltammetry was used to demonstrate the ability of a ferrocene 
compound (usually ferrocene monocarboxylic acid) to generate and enhance 
anodic current in the presence of each of the following enzymes, together 
with their respective substrates: 
TABLE 2 
______________________________________ 
Enzyme Substrate 
______________________________________ 
Flavo-proteins 
Pyruvate Oxidase [EC 1.2.3.3] 
Pyruvate 
L-Amino Acid Oxidase [EC 1.4.3.2] 
L-Amino Acids 
Aldehyde Oxidase Aldehydes 
Xanthine Oxidase [EC 1.1.3.22] 
Xanthines 
Glucose Oxidase Glucose 
Glycolate Oxidase [EC 1.1.3.1] 
Glycolate 
Sarcosine Oxidase [EC 1.5.3.1] 
Sarcosine 
Lactate Oxidase Lactate 
Glutathione Reductase [EC 1.8.1.4] 
NAD(P)H 
Lipoamide Dehydrogenase [EC 1.6.3.4] 
NADH 
PQQ Enzymes 
Glucose Dehydrogenase Glucose 
Methanol Dehydrogenase 
Methanol and 
other Alkanols 
Methylamine Dehydrogenase 
Methylamine 
Haem-Containing Enzymes 
Lactate Dehydrogenase Lactate 
(Yeast Cytochrome B2) 
Horse-Radish Peroxidase 
Hydrogen Peroxide 
Yeast Cytochrome C Peroxidase 
Hydrogen Peroxide 
Cupro-protein Enzymes 
Galactose Oxidase [EC 1.1.3.1] 
Galactose 
Metalloflavoproteins 
Carbon Monoxide Carbon monoxide 
Oxidoreductase 
______________________________________ 
In each case, the enzyme/mediator system gave an enhanced anodic current, 
indiative of the enzyme-catalysed reaction. Second order homogeneous rate 
constants calculated from the date thus obtained indicated that the 
ferrocene compound effectively mediated the enzyme-electrode electron 
transfer in a manner suitable for construction of an electrode assay 
system. 
A representative protocol for the cyclic voltammetry is as follows: 
EXAMPLE 25 
(a) Electrochemical Instrumentation 
dc cyclic voltammetry, which is a controlled potential electrochemical 
method, is based upon the maintenance within cell 30 of the potential of 
the working electrode (WE) with respect to a reference electrode (RE) by 
making a current pass between the working and counter electrode (CE). FIG. 
7 shows the circuit that was used which incorporates two operational 
amplifiers. These were built into an Oxford Electrodes potentiostat. 
Current-potential curves were recorded with a Bryans X-Y 26000 A3 chart 
recorder. Applied potential=V in; current=V out/R. 
A 380Z micro-computer (Research Machines Ltd), interfaced to a potentiostat 
via digital-to-analogue and analogue-to-digital converters, was used for 
the potential step methods. The potentiostat incorporates a multiplexer 
which facilitates both switching and monitoring of more than one working 
electrode. 
(b) Cells and Electrodes 
dc cyclic voltammetry experiments were performed using a two compartment 
cell, with a working volume of ca. 1 ml, of the configuration shown in 
FIG. 8, i.e. where the two cells are placed in communication by Luggin 
capillary 31 FIG. 9. 
In addition to a 4 mm diameter working electrode 32 made of gold, (platinum 
and pyrolytic graphite were also tried successfully), the cells contained 
a 1 cm.sup.2 platinum gauze counter electrode 33 and a saturated calomel 
electrode, 34 type K401 (supplied by V. A. Howe Radiometer Electrodes) 
accurate in the range -10.degree. C. to 60.degree. C., as reference. All 
potentials are referred to the saturated calomel electrode (SCE), which is 
+241 mV at 20.degree. C. versus the normal hydrogen electrode (NHE). 
Working electrodes were polished before each experiment using an 
alumina-water paste on cotton wool and then washed with deionised water. 
Alumina with a particle size ca. 0.3 .mu.m, was supplied by BDH. 
(c) Temperature Control 
Electrochemical experiments were performed under thermostatic control by 
using a Churchill chiller thermocircular connected to a water bath into 
which the electrochemical cell was placed. 
(d) Spectrophotometric Measurements 
All optical spectra were recorded with a Pye-Unicam SP8 200 
spectrophotometer with the sample and reference solutions in matched 
quartz micro-cuvettes of path length 1 cm. 
(e) Water Purification 
Where possible, all solutions were prepared with water purified by a 
sequence of reverse osmosis, ion exchange and carbon filtration using a 
combined Milli-RO4 and Milli-Q system supplied by Millipore Ltd. 
(f) Ultrafiltration and Diafiltration 
Ultrafiltration and diafiltration of proteins were performed by using the 
appropriately sized Amicon cell with a suitable Diaflo membrane. 
(g) Fast Protein Liquid Chromatography 
Protein purifications were performed using an FPLC system supplied by 
Pharmacia. This encorporated two P-500 pumps controlled by a gradient 
programmer GP-250 operated in conjunction with a single wavelength 
UV-monitor (.lambda.=260 nm) and an automatic fraction collector FRAC-100. 
Analytical and preparative ion-exchange columns were also supplied by 
Pharmacia. 
dc cyclic voltammetry experiments are performed in argon-saturated 
solutions using the following protocol. Firstly, the reversible 
electrochemistry of ferrocene monocarboxylic acid (200 uM) in a suitable 
electrolyte is established by recording voltammograms at different scan 
rates (.nu.=1-100 mVs.sup.-1) over the potential range 0-400 mV. Substrate 
is then added to the cell, typically to a final concentration of 10 mM and 
always in excess of the Michaelis-Menten constant for the enzyme. A set of 
voltammograms are recorded to assess the effect of the substrate upon the 
electrochemistry of the mediator. Enzyme is then added to final 
concentrations in the range 10-100 uM. If an enhanced anodic current is 
obtained, and the dependence of the current function upon the scan rate 
was indicative of a catalytic reaction, the experiment is repeated adding 
the substrate as the final component to insure that the reaction was 
dependent upon the presence of substrate. 
Under the conditions that were used, none of the substrates interferred 
with the electrochemistry of the ferrocene. Over the range 0-400 mV vs 
SCE, none of the substrates or enzymes exhibited any direct 
electrochemistry. 
(h) Materials 
The flavo-proteins pyruvate oxidase (EC 1.2.3.3), xanthine oxidase (EC 
1.1.3.22), sarcosine oxidase (EC 1.5.3.1), lipoamide dehydrogenase (EC 
1.8.1.4) and glutathione reductase (EC 1.6.4.2) were supplied by 
Boehringer and stored at -20 C. The respective concentrations of the 
flavo-proteins are expressed in terms of the amount of 
catalytically-active flavin. 
Carbon monoxide oxido-reductase was isolated from Pseudomonas 
thermocarboxydovorans by Dr. J. Colby, Biochemistry Department, Sunderland 
Polytechnic and supplied at a concentration of 8.6 mg ml.sup.-1, in 
phosphate buffer containing 50% ethanediol as a stabilizer. Before use, 
the enzyme was dialysed against 20 mM Tris-HCl (pH 7.5) at 4.degree. C., 
and purified by FPLC using an analytical Mono-Q column. The enzyme was 
loaded on to the column at a concentration of 1.0 mg ml.sup.-1 in 20 mM 
Tris-HCl pH 7.5 (buffer A) and eluted with a linear ionic strength 
gradient using buffer B (A +1.0M KCl). Carbon monoxide oxido-reductase 
eluted as one major peak at an ionic strength equivalent to 35% buffer B, 
as shown in FIG. 9. 
Purification of the quino-protein alcohol dehydrogenase (EC 1.1.99.8) is 
described above. Lactate dehydrogenase (EC 1.1.1.27) and isocitrate 
dehydrogenase (EC 1.1.1.42) were supplied by Boehringer. 
Sodium lactate, sodium isocitrate, sarcosine, sodium pyruvate, xanthine, 
cholesterol, potassium, oxalate, choline, reduced nicotinamide adenine 
dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide 
phosphate (NADPH) were supplied by Boehringer. Carbon monoxide was 
supplied by BOC. Ferrocene monocarboxylic acid was supplied by Fluorochem. 
(i) Electrolytes 
All experiments used 100 mM Tris-HCl buffer pH 7.0, except those involving 
oxalate oxidase which used 100 mM succinate buffer pH 3.0, and those with 
alcohol dehydrogenase which used 100 mM borax:NaOH, pH 10.5 containing 14 
mM NH.sub.4 Cl. 
(j) Electrochemical Experiments 
All experiments used the electrochemical cell of FIG. 8 incorporating a 4 
mm pyrolytic graphite working electrode, except in experiments on 
lipoamide dehydrogenase and glutathione reductase where a 4 mm disc gold 
electrode was used. In experiments where cytochromes c was investigated 
with carbon monoxide oxido-reductase, a bis(4,4'-pyridyl)-1,2-ethene 
modified gold electrode was used. 
EXAMPLES 26-31 
The above described cyclic voltammetry was used to demonstrate the 
electron-transfer capability of a variety of ferrocene compounds listed 
with a glucose/glucose oxidase system: 
TABLE 3 
______________________________________ 
Rates of glucose oxidase oxidation 
measured at pH 7 and 25.degree. C. 
ferrocene derivative 
Eo/mV vs SCE 
##STR1## 
______________________________________ 
1,1'-dimethyl- 100 44 
ferrocene 165 15 
vinyl- 250 18 
carboxy- 275 115 
1,1'-dicarboxy- 
385 15 
trimethylamino- 
400 300 
______________________________________ 
(a) Solution Kinetics 
A variety of ferrocene derivatives, Table 3, with a range of potentials 
(150 to 400 mV vs SCE) were investigated as possible oxidants for glucose 
oxidase using D.C. cyclic voltammetry. FIG. 10 shows at (a) a voltammogram 
of carboxyferrocene which fulfills electrochemical criteria as a 
reversible one-electron couple (.DELTA.E.sub.p =60 mV; i.sub.p 
/.mu..sup.1/2 =constant). The addition of glucose alone in solution has no 
discernable effect upon the voltammogram. However, upon further addition 
of glucose oxidase to the solution (at b) a striking change in the 
voltammogram occurs. Enhancement of the anodic current is characteristic 
of a catalytically-coupled reaction and can be interpreted in terms of the 
following scheme, 
##STR2## 
where R is the ferrocene, O is the ferricinium ion and Z the reduced 
glucose oxidase. The enhanced anodic current results from the reaction 
between the reduced glucose oxidase and the ferricinium ion, the latter 
being generated at oxidizing potentials. The kinetics of the homogeneous 
reaction between glucose oxidase and a number of ferrocene derivatives 
were analyzed by the theory developed by Nicholson and Shain. From the 
data, a pseudo-first order rate constant, independent of scan rate, can be 
derived. The variation of this parameter as a function of the glucose 
oxidase concentration yields the second order rate constant for the 
reaction. The validity of this analysis depends upon two conditions being 
fulfilled; the heterogeneous electrode reaction must be fast compared to 
the catalytically coupled homogeneous reaction and there must be 
sufficient glucose present to ensure that the enzyme is always in the 
reduced form. Both conditions hold true in this study. The data shown in 
Table 3, indicate that the oxidized form of all ferrocene derivatives 
investigated act as a rapid oxidant for the enzyme, with rates of reaction 
comparable to that of the natural electron acceptor, molecular oxygen, 
which has a value of .sup.k s=10.sup.6 M.sup.-1 S.sup.-1. 
Although all the ferrocene derivatives shown in Table 3 lead to the 
effective electrochemical oxidation of glucose via glucose oxidase, other 
criteria are important in designing a practical enzyme electrode. The 
solubility of the reduced form of the ferrocene derivative in aqueous 
media must be low to aid entrappment within the electrode; the oxidised 
form should be stable at physiological pH; the formal potential should be 
low to obviate interference from reduced compounds present in 
physiological samples. 1,1'-dimethylferrocene provided the best compromise 
between the constraints imposed by these factors and was chosen for 
incorporation into the enzyme electrode. 
(b) Enzyme Electrode 
Digital simulation techniques have shown that the steady state current for 
an amperometric enzyme electrode is determined predominantly by the 
apparent Michaelis-Menten constant, K.sub.M ', the membrane permeability, 
the effective electrode surface area and the enzyme loading factor 
(concentration per unit volume). Considering the available enzyme 
immobilization techniques, covalent attachment to a functionalised 
electrode surface generally gives the most lasting enzyme activity. 
Additionally, the resulting monolayer coverage is the most appropriate for 
optimum response characteristics. On this basis, a batch of twenty-four of 
the prototype glucose enzyme electrodes were constructed as described 
below and their performance evaluated. 
(c) Reagents 
Glucose oxidase (EC 1.1.3.4 type 2, from Aspergillus niger), was supplied 
by Boehringer Mannheim, had an activityof 274 IU/mg. D-glucose (AnalaR) 
was from BDH; ferrocene and its derivatives were from Strem Chem. Co. All 
solutions were prepared from Aristar grade reagents (BDH) in high purity 
water (Millipore); supporting electrolyte was 0.1M K.sub.2 HPO.sub.4 
adjusted to the required pH with HClO.sub.4, glucose solutions were stored 
overnight to allow equilibration of .alpha.- and .beta.-anomers. 
Properties of this enzyme are given in the table below; 
TABLE 4 
______________________________________ 
source; Aspergillus Niger 
RMM; 186,000 
Co-factor 2FAD 
Co-substrate Oxygen 
Optimum pH 5.6 
K.sub.m Glucose 3.0 mM 
______________________________________ 
(d) Biological Samples 
Heparinised plasma samples from human diabetics were supplied by the 
Metabolic Unit, Guy's Hospital, London., and had been previously analysed 
for glucose with a Yellow Springs Instruments, Ohio, glucose analyzer. 
(e) Apparatus 
dc cyclic voltammetry experiments were performed using a two-compartment 
cell that had a working volume of 1 ml. In addition to the 4 mm pyrolytic 
graphite disc working electrode, the cell contained a 1 cm.sup.2 platinum 
gauze counter electrode and a saturated calomel electrode as reference. 
Bourdillon et al., J. Amer. Chem., Soc. 102: 4321 (1980). All potentials 
are referred to the saturated calomel electrode (SCE). For D.C. cyclic 
voltammetry, an Oxford Electrodes potentiostat was used with a Bryans X-Y 
26000 A3 chart recorder. The potentiostatically-controlled steady-state 
current measurements were made using a cell, designed to accommodate up to 
seven enzyme electrodes, with a working volume of 100 ml with separate 
compartments for counter and reference electrodes. Current-time curves 
were recorded with a Bryans Y-t BS-271 recorder. The temperature of the 
electrochemical cells during experiments were controlled to within 
.+-.0.5.degree. C. with a Churchill thermocirculator. 
(f) Construction of the glucose enzyme electrode 
Graphite foil 1 mm thick, supplied by Union Carbide was the base sensor. 
Electrodes were constructed by cutting the graphite into 4 mm diameter 
discs and sealing into glass rods with epoxy resin. The electrodes were 
then heated at 200.degree. C. in air for 40 hours, allowed to cool, 15 ul 
of 1,1'dimethylferrocene (0.1M in toluene) was deposited on to the surface 
of the electrode and air-dried. Covalent attachment of the glucose oxidase 
to the oxidised graphite surface was achieved by a method similar to that 
described by Bourdillon. The electrodes were placed in 1 ml of a solution 
of water-soluble 1-cyclohexyl-3-(2-morpholine ethyl)carbodiimide 
metho-p-toluene sulphonate from Sigma Chem. Co. (0.15M in 0.1M acetate, pH 
4.5), for 80 mins at 20.degree. C., washed with water and then placed in a 
stirred solution of acetate buffer (0.1M, pH 5.5) containing glucose 
oxidase (12.5 mg/ml.) for 90 mins at 20.degree. C. After washing, the 
electrodes were covered with a polycarbonate membrane (Nucleopore, 0.03 
um) and stored in buffer containing 1 mM glucose at 4.degree. C. 
(g) Enzyme electrode pre-treatment 
After fabrication and prior to experiments, the electrode response was 
stabilized by continuous operation of the electrode under potentiostatic 
control at 160 mV in 8 mM glucose over a 10 hours period. Thereafter the 
electrodes were found to give a more stable response during 100 hours 
further operation. In 8 mM buffered glucose, the electrodes gave a mean 
current decrease of 3%.+-.1 over this period. All electrodes which had 
been modified with glucose oxidase had undergone this pre-conditioning 
process. 
All electrodes gave a linear current response in the range 0.1-35 mM 
glucose and finally saturate at approximately 70 mM glucose. In the linear 
region, the electrodes showed a rapid response time reaching 95% of the 
steady state-current in 60-90 secs. The reproducibility of the electrode 
construction protocol was investigated by measuring the steady-state 
current for each electrode in 8 mM glucose. The batch of prototype 
electrodes gave a mean current response of 7.9 uA with a standard 
deviation of 2.8. 
(h) The effect of temperature 
The effect of temperature on the enzyme electrode response was studied in 
the range 10.degree.-50.degree. C., and showed the increase in steady 
state current with increasing temperature, ca. 0.2 uA/.degree.C. All 
electrodes showed similar behaviour. Assuming Arrhenius type behaviour, 
the absence of maxima in the electrode response, is indicative of the 
thermal stability of the immobilized enzyme at temperatures up to 
50.degree. C. This electrode configuration should be suitable for extended 
use at normal body temperature. Similar thermal stability was also found 
with soluble enzyme, the dependence of the second order rate constant upon 
temperature, giving an activation energy for the reaction of 49.6 KJ 
Mol.sup.-1. 
(i) Interfering Substances 
The effects of substances which might interfere with the response of the 
electrodes, either through direct electrode oxidation, reaction with the 
mediator, or, inhibition of the enzyme, were examined. Analyses of 
solutions containing 8 mM glucose, to which metabolites were added to give 
their normal blood concentrations were carried out. Though, L-ascorbate at 
a final concentration of 0.13 mM gave a mean increase in current of ca. 
4.0%, addition of uric acid (0.20 mM), where the transition from hypo to 
hyperglycemia reflects a change in blood glucose of ca. 0.5-30 mM, a 
practical glucose electrode is required to respond linearly in this range 
so as to eliminate the necessity of sample dilution. In this respect, it 
seems that the immobilization protocol for the ferrocene-based electrode 
is important in changing the apparent Michaelis-Menten constant, K.sub.M 
', of glucose oxidase for glucose which results in the high upper limits 
of linearity. If the electrode response is kinetically rather than 
diffusion-controlled, i.e. the steady-state current is independent of 
whether the solution is quiescent or stirred, K.sub.M ' may be calculated. 
These prototype, ferrocene-based electrodes, which were found to be 
kinetically-controlled, gave values of K.sub.M ' in the range, 30-40 mM. 
This compares to a K.sub.M, for glucose, of 3 mM for the non-immobilized 
enzyme. 
Particular interfering, or potentially interfering substances are listed in 
table 5 below. All of the listed substances are substrates for glucose 
oxidase, however the relative rates of reaction are much lower than that 
of the primary substrate, glucose. Although experiment showed that the 
effect on glucose assay of these substances was minimal, it is envisaged 
that in the absence of glucose the sensor of the present electrode could 
be employed to assay for any of the listed substrates. 
TABLE 5 
______________________________________ 
Substrate Relative Rate 
______________________________________ 
.beta.-D-glucose 100 
2-deoxy-D-glucose 
25 
6-methyl-D-glucose 
2 
D-mannose 1 
.alpha.-D-glucose 
0.6 
______________________________________ 
L-cysteine HCl (0.08 mM), reduced glutathione (0.49 mM), sodium formate 
(7.35 mM), D-xylose (8.00 mM) .alpha.-galactose (7.77 mM) and 
.alpha.-mannose (7.77 mM) did not cause any observable interference to the 
electrodes response to glucose. 
There was however, a mean decrease in the current response of 4.0% when 
changing from nitrogen-saturated to air-saturated buffer. Whilst 
interference from oxygen is not surprising, the current decrease occurs as 
the base electrode was not poised sufficiently positive to re-oxidise the 
hydrogen peroxide generated by the enzymatic reaction. Operation of the 
electrode at potentials sufficiently positive to re-oxidize the hydrogen 
peroxide also leads to increased interference from L-ascorbate. 
(j) Effect of pH 
Since the pH of diabetic plasma samples may vary either through the 
addition of heparin or loss of carbon dioxide, the effect of pH on the 
response of the glucose electrode was investigated over the clinically 
relevant range. The steady-state current of the above-described enzyme 
electrode is essentially independent of pH. This paralleled the behaviour 
of the soluble enzyme, where the second order rate constants for all 
ferrocene derivatives shown in Table 3 were found to be independent of 
changes in pH in the range pH 6-9. This desirable feature of a non-pH 
dependent response, is presumably due to the fact that, in contrast with 
oxygen-mediated glucose enzyme electrodes, no proton transfer is involved 
in ferrocene oxidation. 
Devices such as shown in the Examples offer advantages over most of the 
enzyme-based sensors currently available. When compared to such sensors 
prior to dilution steps, the present electrode has an equal or faster 
response time, the ability to operate under anaerobic conditions, greater 
oxygen insensitivity (important in blood samples, where oxygen 
concentration is variable), extended linear range covering the complete 
physiological range and comparable specificity. It is additionally 
proposed, in accordance with the present invention that among the 
mediators named figure the thiol or like sulphur derivatives of ferrocene, 
whereby the mediator can link directly to a gold electrode. 
The thiol group can be directly or indirectly attached to one ring of the 
ferrocene structure, e.g. by a lower alkyl group containing 1 to 6 carbon 
atoms. The simple thio(ferrocene)-SH can be used, prepared as in J. Chem 
Soc. 692 (1958) Knox and Pauson. We have also established that of the 
alkyl thiols ferrocenyl thiobutane is valuable i.e. (ferrocene)-C.sub.4 
H.sub.8 -SH. Other more complex thiol-like compounds are possible e.g. 
1,2,3-trithia-(3)-ferrocenophane in which the two rings are linked by a 
chain of sulphur atoms (a mixture of substances with different numbers of 
chain sulphur atoms is possible.) 
The gold electrode can be prepared for repeated use e.g. by dipping into 
solutions of such compounds, so as to link the mediator ferrocene 
structure to the conductive metal. 
Examples of the production of such materials are as follows: 
1,2,3-trithia-(3)-ferrocenophane (J. Organometallic Chem. 1971, 27, 241) 
The literature procedure was followed, but no product was obviously evident 
from the sublimation of the crude mixture. The sublimed material that had 
the most obvious (i.e. smelliest) potential was chromatographed on silica 
(30 cm.times.2 cm colume) with hexane as eluant to give three products. 
1. No sulphur on analysis. 
2. C: 43.72, H: 2.83, S: 33.05, C.sub.10 H.sub.8 FeS.sub.3 requires C: 
42.98, H: 2.89, S: 34.42 Yield) 0.45 g. 
3. 0.11 g A complex molecule, not examined beyond the mass spec. which 
indicated it was not however a ferrocenophane with a simple number of 
sulphur atoms. 
Ferrocene thiopentane (ferrocenyl thiobutane) 
1. Ferrocenyl butyric acid (J. Am. Chem. Soc. 1957, 79, 3420 
F.sub.c --CO--CH.sub.2 --CH.sub.2 --CH.sub.2 --CH.sub.2 COOH 
Prepared by the literature method 
2. Ferrocenyl butyric acid 
F.sub.c --CH.sub.2 --CH.sub.2 --CH.sub.2 --CH.sub.2 --COOH 
(As above prepared by Clemmenson reduction zinc/mercury and hydrochloric 
acid) 
3. Ferrocenyl butanol 
F.sub.c --CH.sub.2 --CH.sub.2 --CH.sub.2 --CH.sub.2 --CH.sub.2 OH 
Acid (2) (12 g) was dissolved in ether (distilled from sodium/potassium) 
and treated with lithium hydride (1.27 g) in a nitrogen atmosphere. When 
reaction was complete the excess lithium aluminium hydride was destroyed 
using ethyl acetate and then water. The organic phases were separated and 
the aqueous phase washed with ether (2.times.20 ml). The organic phases 
were combined and dried (MgSO.sub.4) and after filtration the solvent was 
removed on the rotary evaporator 4. The red oil resulting had two 
components. Column chromatography (30 cm.times.2 cm) on silica eluted with 
1:1 ether:hexane gave the alcohol and an ester. 
EQU F.sub.c --CH.sub.2 --CH.sub.2 --CH.sub.2 --CH.sub.2 --COOCH.sub.3 
4. Ferrocenyl thiobutane 
(3) (400 mg) was dissolved in pyridine (10 ml, dried over sodium hydroxide) 
and cooled in an ice bath. Tosyl chloride (1 g) was added and the solution 
stirred until clear, then left for 24 hours at 4.degree. C. The mixture, 
containing solid pyridine hydrochloride, was tipped into ice-water and the 
tosylate precipated out. This was filtered at the water pump to give a 
yellow solid. A dried portion of this gave the characteristic tosylate 
i.r. spectrum. The remainder (0.65 g, but still damp) was dissolved in 
ether:methanol (1:1) and sodium hydrosulphide.times.H.sub.2 O (1.6 g) was 
added while stirring, the mixture being maintained at 5.degree. C. After 
30 min, the ice bath was removed and the mixture allowed to warm to room 
temperature. After 3 h t.l.c. (silica, 1:1 Et.sub.2):hexane) indicated 
that reaction was complete. The mixture was reduced to dryness on the 
rotary evaporator and then dissolved in the minimum of Et.sub.2 O/hexane 
(1:11) and chromatographed on silica (60-120 mesh, 25 cm 25 cm.times.2 cm 
colume, eluted with Et.sub.2 O/hexane (1.11). The thiol runs very quickly 
and was collected in approximately 150 ml. Yield 200 mg. 
EQU F.sub.c --CH.sub.2 --CH.sub.2 --CH.sub.2 --CH.sub.2 --CH.sub.2 SH 
As used herein, the term "a ferrocene" includes various derivates (e.g. 
with various substituents on the ring structures, possibly in polymer 
form) differing in redox potential and aqueous solubility. Hill U.S. Ser. 
No. 607,695, filed May 7, 1985, referenced above at p. 11, describes (at 
pp. 16 et seq.) specific systems in which there is a chemical link between 
the mediator and a ligand (e.g. a drug to be assayed such as 
phenobarbital, phenytoin, procainamide, or theophylline). Hill also 
describes (p. 53) various ligand assay schemes including those in which 
the ligand is bound to ferrocene and all system components are freely 
diffusing (i.e., in solution).