JPS6017345A - Electrode sensor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention detects the presence of one or more selected components in a liquid mixture, measures υ thereof, monitors its content, or detects the presence of one or more selected components in a liquid mixture. It relates to a device for detecting the presence and measuring its quantity.

The invention is of particular value in the chemical industry, especially where complex mixtures are encountered (eg food chemistry or biochemical technology), but in particular in biological research and control technology. Furthermore, it is particularly useful for measuring or monitoring components in animal or human medicine, especially body fluids, in vivo.

For convenience, the invention will be described for measuring glucose in diabetic patients by use of a device that lends itself to temporary or permanent infusion, although it may be used on a specific basis, particularly with respect to one measurement in vivo. Although it is one object of the present invention to provide a sensor for components in biological fluids, other broader objects are also included.

In vivo glucose sensors have already been proposed. One proposal is based on the direct oxidation of glucose at a catalytic platinum electrode.
esearch, Supplement 5erie
s AR, pp 10-12 (1979)) is not unique and has the disadvantage of being easily poisoned by interfering substances. Suggestions for more unspecific manipulations of glucose include the use of glucose oxidase at the electrode (AdV, EXp, IKeci, B.
iol, 50 pp 1.89-107 (1,974
3) does not show a significant response to high glucose concentrations.

Other systems using glucose oxidase have been proposed, but in vivo methods (e.g. J, 5o11d-Ph
ase B soil 00h6m, 4 pp258-262 (
1,979)) and has not been sufficiently studied.

European Patent Application No. 82805597 of the applicant describes a sensor comprising an electrically conductive material and having on at least its outer surface an enzyme in combination and a mesoieter compound which transports electrons to an electrode when the enzyme is catalytically active. Electrodes are described and claimed.

The purpose of such electrodes is to detect the presence, evaluate and/or monitor the amount of one or more selected components capable of carrying out the enzyme-catalyzed reaction. Examples of electrode structures, mesoiators and uses are described in the B-Rotspa patent application.

The present invention shows that one particular mesoieter compound exemplified in the European patent application has general utility as a mesoieter for a wide range of enzymes.

The inventors of the present invention
RS 8 pp 187-192 (1981), in vitro studies of enzyme catalyzed reactions using mesoieters in solution to transport electrons generated from the enzyme directly to the electrode during enzyme production. It was carried out in

Numerous attempts have been undertaken to find a satisfactory mesoieter for enzyme catalyzed electrode systems. Some of the documents disclosing such an attempt are: 11 U.S. Patent No. 8,88;
No. 8.038, No. 4.224.125, No. 4,
144, No. 1.48 and No. 4.888.1.66
There is a number.

Decreased oxygen tension levels are associated with poor tissue perfusion.
This is a particular problem in detecting glucose in blood taken from the subcutaneous tissue of diabetic patients.

It would be particularly desirable to find other amperometric detection methods based on glucose oxidase that do not rely on oxygen as the mesoiator of electron transport. The electron acceptor of glucose oxidase mentioned above is hexacyanoferate (Ill
) (2) and a range of organic dyes (8);
The former are not easily confined with electrodes, and the latter are used extensively in spectrophotometric measurements, but for electrochemical use they require facile autoxidation, instability in the reduced form and pH dependence. It has many drawbacks including redox potential.

More generally, it is desirable to find mesoieters that meet the particularly strong demands of quantitative electrochemical analysis.

The inventors have discovered that a group of mesoieta compounds have extremely useful properties for mediating enzyme-catalyzed reactions in electrode sensing systems.

In particular, the present invention is characterized in that an organometallic compound composed of at least two organic rings, each of which has at least 2 double bonds in a conjugated relationship, is used as an electrode sensor mesoieter; The metal atom is in an electron-sharing state in contact with such an organic ring. These mesoiators are extremely useful in electrode sensor systems having two mutually insulated conductors, each of which is in contact via a conductive surface with a mixture containing the selected compound to be detected. . Selected compound? At a typical reaction rate of 7'41 degrees, the enzyme capable of catalyzing the reaction is contacted with the mixture, and the mesoieta compound between the enzyme and the conductive surface of the conductor increases the rate of the enzyme-catalyzed reaction. Move electrons at a typical speed of

In the present invention, mezoieta is mainly a compound of ferrocentib, but mesoieta also includes a compound of lutenocentib, which is particularly insoluble,
The enzyme is anoxic specific flavoprotein enzyme or chinoprotein. Glucose oxidase and glucose dehydrogenase are particularly suitable enzymes.
4 BD-glucose:oxygen oxidoreductase) is a well-known type of enzyme. Bacterial glucose dehydrogenase has recently been discovered and is a quinoprotein with a polycyclic quinone prosthetic group (PQQ).
(TlB5. (Mon. 1981) 278-28)
Arch, Microbiol (1982
') 181, 27-81).

Such bacterial glucose dehydrogenase (bacterial glucose dehydrogenase) in the present invention
The use of drogenase) has some advantages over the use of glucose oxidase. The main advantage is that oxygen-insensitive glucose sensors can be obtained with this glucose dehydrogenase. The reason for this is that enzymes do not use oxygen as an electron acceptor. The appropriate enzymes are purified by conventional chromatographic techniques (as described in detail below) or by two-phase aqueous partitioning methods from a microbial range.

can be manufactured. A suitable microorganism is Acinetobacter.
Although the genus Calcoaceticus may also be used, any number of Gluconobacter species (eg, Gluconobacter oxidase) or Pseudomonas species (eg, Pseudomonas aeruginosa, Fluorescence) can also be used.

As mentioned above, a particularly preferred form of mesoieta compound is ferrocene or ferrocene derivatives.

Ferrocene has as its basic structure an iron atom held "sandwiched" between two pentadiethyl rings by a dative bond. It is a pH-independent, reversible electroactive organic compound that acts as an electron donor. 411 (metallic compounds) with different redox potentials, water solubility, and binding constants to glucose oxidase or bacterial glucose dehydrogenase (e.g., derivatives with various substituents in the ring structure, or polymeric forms). ) are available.

For example, the reduction potential of the parent compound is +42 with respect to NHK.
2 mV. By introducing a functional group into the annular beam, E' can be varied between +800 and +650 mV (7). Furthermore, the water solubility of carbogylic storage ferro7ine is greater than that of the parent compound. For details, see ACS Symposium Series, 88, l 5.
4j (Uwana T., 1977).

Among the particular mesoieta compounds of this type are fuxocene itself, 1,11-ferrocenedicarboxylic acid, dimethylferrocene and polyvinylferrocene, for example having an average molecular weight of about 16,000.

The demands placed on mesoieta are particularly urgent. Preferably, the mesoiator readily transports electrons between the enzyme and the conductive electrode at a rate high enough to eliminate potentially incompatible reactions. Therefore, the response speed is quick. In general, the response should increase the accuracy of the concentration reading over as large an area as possible. The mesoieta is 'I!' in sufficient concentration to achieve intermittent transport. j Should be concentrated at the extreme surface. If the mesoieter is covalently bonded to the electrode and/or the enzyme, this bond should not interfere with the mediating action. Therefore, mesoieta should be relatively insoluble for most applications. Mesoieters are stable and non-responsive to interfering substances such as oxygen or pH.

Most importantly, however, the rate of electron transport should depend on the rate of the enzyme-catalyzed reaction. That is, the mesoiator should affect electron transport during its activity at a rate that indicates catalytic activity.

Satisfactory performance in the areas listed above is obtained with very broad ferrocentive compounds of the sensor system. For example, ferrocene can mediate electron transport for a wide variety of enzymes.

1,1-Dimethylferrocene is a particularly preferred mesoieter. Preferably, the selected compound to be detected is a substrate for the enzyme-catalyzed compound. It is also preferred to confine the enzyme and/or mesoietor to the electrically conductive surface of one of the conductors. Finally, in a preferred system, a mesoiator transports electrons from the enzyme to the electrode surface.

The properties of the ferrocene derivative along with the properties of the parent compound are shown below.

1.1/-dimethyl-100I+D-acetic acid 124
S 870 Hydroxyethyl-1613- Ferrocene 165 1. D 885], ]l-bis(hydroxymethyl)-2243885
Monocarboxylic acid 275 S, 4201゜11-dicarboxylic acid 885 S -chloro-845, D methyltrimethylamino-4003 In Table 1, S is soluble in water, ■, D is insoluble in water, and 8% detergent (Twe 'en -20). Eo is mv for standard force four-mel electrode (!jOE)
where E is a measured value expressed in cm M-.

E of various ferrocenes from the above table in phosphate buffered solution, pH 7. The values span a range of potentials, Eo = 10
0 to 400 mv/saE. The trend in EO values is consistent with what would be expected based on the influence of substituents.

Generally, electron donating groups stabilize the positive charge and therefore promote oxidation more than electron accepting groups.

- In one preferred specific example, the electrode is designed to measure glucose in vivo. The enzyme is therefore preferably glucose oxidase or preferably glucose dehydrogenase. An example is bacterial glucose dehydrogenase.

The electrically conductive material of the electrode itself can be metal, especially silver, or carbon, such as an electrode molding composed of a preformed rond or paste of carbon particles or carbon fibers. The surface conditions of TLi poles are generally important.

In the case of metals, the surface in contact with the active substance (enzyme and/or mesoieta) can be roughened. In the case of solid carbon, the surface can be "oxidized",
That is, the surface can be heat-treated in advance in a furnace having an oxygen atmosphere.

Of the two enzymes shown for typical assay results for glucose, dehydrogenase is preferred, and for mesoieta, ferrocentib compounds are preferred.

Certain combinations of the above substances and certain arrangements of electrodes are indeed preferred.

Enzyme immobilization materials, polymeric electrode mixtures such as Teflon, or long chain alkyl derivatives of mesoieters with reduced mobility due to increased molecular weight can optionally be incorporated.

However, in a particularly important form of the invention, the electrodes are
It consists of a carbon core, a layer of ferrocene or a ferrocene derivative on its surface, and a layer of glucose oxidase or glucose dehydrogenase on the surface of the ferrocene core. This layer of enzyme is preferably maintained in a self-supporting gel layer on the mesoieter, immobilized on the underlying surface of the mesoieter, and/or having a retention layer thereon which is permeable to glucose molecules. preferable.

The carbon core itself can be solid carbon or a hard paste of carbon particles or carbon fibers. Generally, this carbon core has a flat surface for ferrocene or ferrocene derivatives. Set up an it point. Such ferrocene or ferrocene derivatives can be prepared in various ways, for example: (a) in the case of monomeric ferrocene or ferrocene derivatives, by removal from solution in a readily evaporable liquid, for example an organic solvent such as toluene; (b) In the case of polymeric ferrocene derivatives, the easily evaporated properties of polymers such as chloroform? 11 by precipitation from an organic solvent (J, Dolyme Sc.
Sat., 1976, 10-2488 discloses a method for producing polyvinyl fercene having an average molecular weight of about 16,000, which can be precipitated in this way. in case of,
For example, vinyl ferrocene is dissolved in an organic electrolyte containing tert-butylammonium perchlorate at a concentration of about LM, and vinyl ferrocene radicals are precipitated as a polymer at a potential of -700 mV, thereby performing electrochemical polymerization in situ. (d) covalent modification of carbon electrodes, e.g. by carbodiimide cross-linking of ferrocene or ferrocene derivatives onto carbon;
By n); it can be deposited on the carbon core.

Ferrocene or ferrocene iTl [The enzyme to be coated on the body can be glucose oxidase or bacterial glucose dehydrogenase. Glucose oxidase is characterized by, for example, a tightly bound thin layer, a good proportional response to low glucose concentrations, and an insensitivity to oxygen (because oxygen competes with those from ferrocene for electrons transferred from the substrate to the enzyme redox center). The carbodiimide material DOG (1-Schiff-tetrahexyl-8(2-morpholinoethyl)carbo-diimidometh-p-)luenesulfonate) can be immobilized on the underlying surface. Also, using DOG immobilization of glucose oxidase on ferrocene extends the top end of the linear range of the sensor from about 2 mV to 40 mV.

Other methods of immobilization or other forms of protection are also possible, such as incorporating a self-supporting gelatin layer.

Bacterial glucose dehydrogenase can also be immobilized on the mesoieter surface, which can simply precipitate out of an evaporable solution or be retained in a gelatin layer.

Although it is optional, live blood (1ive bloo)
&'), it is preferred to surround both the enzyme and the mesoieter layer with a protective membrane that is permeable to water and glucose molecules. This can be, for example, a dialysis film held elastically by an elastic O-ring. However, it is also advantageous for such a protective film to be a layer of cellulose acetate or a layer of polyurethane, such as can be formed by immersing the electrode in a solution of cellulose acetate in acetone. The membrane can be applied by dipping, spraying or spinning, coating methods.

The present invention relates to a sensor electrode, in particular a special sensor electrode for glucose, but also to the combination of such an electrode with a temporary or permanent injection means, such as a needle probe. In other respects, such electrodes also constitute the invention, as they are or can be coupled to a signaling device or a control device, and more particularly to insulin management means. Additionally, methods of monitoring diabetic patients involving the use of temporary or permanently injected electrodes as described above are also within the scope of the present invention.

The electrode according to the invention makes it possible to produce rigid macro-sensors used in existing types of hospital gluten sensing devices. The advantage compared to known devices is the very low sensitivity to oxygen and the enhanced proportional range, which makes it possible to eliminate the dilution step associated with blood analysis in conventional devices. Furthermore,
As discussed in more detail below, the response time of such electrodes is short (24-36 seconds for 95% steady state depending on the complexity of the solution).

The electrodes according to the invention can also be integrated on a macroscopic scale into simple and inexpensive electronic digital displays for surgeons or diabetic home test kits.

A slight variation of the macro-sensor can be used in a device that automatically collects blood from a finger, contacts the blood with a sensor, amplifies the signal, and provides a digital display. It is also conceivable to use microscopically modified sensors in monitoring-type devices for monitoring glucose-mediated fluids in the skin. This sensor is worn on the wrist and has one or more separate thin serrated sensors and a disposable sensor cartridge at the rear. Each sensor can be powered using electronics technology that ensures reliability by referring to the current input when using several sensors.

When such a device is connected to an external insulin delivery system, it can act as a control loop for the insulin pump. In fact, such a device is housed in the cannula used to deliver insulin into the body from the pump and again serves as a sensor for the feedback loop. Other uses include, for example, a hypoglycemia alarm or digital display monitor.

Enzymes that can be used with the ferrocene-mediated system include: flapin protein, which can use a variety of electron acceptors, including oxygen: NADPH- or N
ADH-binding enzymes, such as ribamide dehydrogenase and glutathione reductase; there is a dehydrogenase called gituprotiin, which contains the polycyclic quinone prosthetic group (PQQ) mentioned above.

The above-mentioned flavoprotein that generates H2O2 is described in Biotechnol, Bioeng, S by C1ark et al.
ymp, 8+877 (1972). Particularly suitable flavi' protein 91 lactic acid "v#'-J
・7': l f >, t #, "dase, riboamide dedrogenase, glu J'J-one reductase, carbon monooxide reductase, gluco oxidase, glycolate oxidase, L-amino oxidase, galactose oxidase 0 Suitable chinoproteins include glucose dehydrogenase, alcohol dehydrogenase, and methanol dehydrogenase. PQQ chinoproteins are specified in Quine et al. J, TlB56.

278 (1981).

Finally, heme-containing enzymes can be used in ferrocene-mediated electrode systems. Such enzymes include horseradish peroxidase, horseradish λ-cytochrome, cytochrome peroxidase, yeast cytochrome C peroxidase, and lactate dehydrogenase or yeast cytochrome B2.

The compatibility of enzymes such as those mentioned above with 7-erocene can be determined by measuring the current at the working electrode over a voltage sweep or by
, can be expressed using a cyclic voltam Durham.

The current measured is due to electron transport to electroactive species in solution and T'! Contains an induced current component caused by electron transport in active species. If the rate of electron transfer between electroactive species is sufficiently fast, the conduction current is controlled by the diffusion rate of the electroactive species. Enzyme-catalyzed reactions produce perturbations in the voltam durum that depend on the reaction rate compared to the time required for the voltage sweep. Therefore, the suitability of a particular mesoieter for transfer between a particular enzyme and an electrode can be evaluated as described below in Examples 12-28.

Enzymes such as glucose oxidase and glucose dehydrogenase.

As mentioned above, in the preferred seven-nax system, the selected compound to be measured is a substrate for the enzyme, and the enzyme and mesoieter are confined to the electrode surface. When this electrode is exposed to a mixture containing the selected compound, the enzyme becomes catalytically active and produces a current that is representative of the concentration of the compound.

However, other arrangements are possible in which the enzyme-catalyzed reaction rate is substituted for the concentration of other compounds that are not enzyme-catalyzed.

The invention will now be explained by way of example.

Example I Purification of Kinoprotein Dehydrogenase (G D H') from Acinetobacter - Chalcoaceticus (&) Growth of the Organism
1J: I) Sodium succinate (20 l/t) at H8,5 and 20'C
Seta. 1illll Ilf'l After a total of 20 hours (A
6oo = 6.0) was collected using a shoe press centrifuge and stored frozen.

This method is the method of Shu Nie Duyin et al.
ch Hle: rob soil o1. [82vj-de 5
upra), but the following modifications were made.

1 Thaw 1009(7) cells, 8800d(1')
Lysozyme (56 mM) was suspended in 789 mM glycerin and treated with 60 μl of lysozyme for 20 minutes at room temperature.

2 Mix Triton X-100 extract and add 0.01”■
15 at room temperature using deoxyribocreatic acid of f
Processed for minutes. The resulting suspension was then incubated at 4°C for 4 hours.
Centrifuged at 8000x9 for 25 minutes. The supernatant obtained by this centrifugation was then treated with ammonium sulfate. Yellow protein precipitated between 55% and 70% ammonium sulfate containing 1% Triton X-100 86
mM) Lys/suspended in 89 mW Chrysin, 4
Dialyzed against buffer for 5 hours at °C.

Active moiety from 80M Sepharose 0l-6B.

Stability of operation at normal body temperature, wide range of proportional response, lack of significant interference by other metabolites and oxygen independence make it a novel current for in vitro use and continuous monitoring of glucose in vivo. It is worth developing a titrating enzyme electrode. The columns were assembled and concentrated using a Millibore CX-80 immersion ultrafilter.

Example 2 Purification of Quinobacterium glucose dehydrogenase from Acinetobacter calcoaceticus (other methods) (a) Growth of the organism The method of Example 1 was followed.

(b) Purification of CDI (This method is based on the partitioning of the protein between two liquid phases. This step involves: l Thawing the cells and adding 50 ml of phosphoric acid solution at pH 7.0) The mixture was suspended in a wet overlay of 8I/9. Such cells were then precooled on ice and incubated at 1758 9
/cJ (25000pSi・) with standard pressure cell (5ta
nsted Fluid, manufactured by Power, 5tan
sted, Es5ex, UK).

This resulted in a cell-free extract.

2 This cell-free extract was then mixed with 50 wt% polyethylene glycol 1000.50 wt% sodium phosphate, pH 7 and distilled water in a ratio of 2:4:8:I, respectively, for 15 minutes at room temperature. . 5000 ml of this mixture
The emulsion was broken by centrifugation at rpm for 5 minutes.

8. Aspirate the bottom layer and desalt by diafiltration using a 10,000 mwt cut Am1con hollow fiber ultrafiltration cartridge cartridge or by passing through a Sephadex (intermediate) gel filtration column. did.

4 The resulting solution was concentrated in a nitrogen pressurized cell using an Amifun PMIO membrane run.

Example 8 Interaction between Ferrocene and Glucose Oxidase DC cyclic portammetry was used to study the homogeneous reaction kinetics between ferrocene and glucose oxidase under substrate excess conditions. An electromechanical two-chamber cell of 1,0 ml volume fitted with a Luggin tubule was used. The cell is equipped with a 4-0 mm gold disk working electrode, a platinum mesh counter electrode, and a saturated calomel electrode as a reference electrode. A series of portamographs for ferrocene were performed in 5 (1 m% calcium phosphate buffer pH 7).
, 0 at a scanning rate of 1-100 mVs-1. This data indicates that the mesoieta is a reversible one-electron acceptor E. ■-+ 165' Indicates that it acted as MV SO'E.

With the addition of 50 mM glucose, mesoieta (5
00μm) 1! There was no appreciable effect on gas chemistry. However, upon addition of glucose oxidase (10 μ m ), an increase in anodic current for mesoieta was observed in the portamogram at the oxidation potential. This indicates catalytic regeneration of the reduced form of mesoieta by glucose oxidase. Establishment method (N1chols)
on, R,S.

and 5hain, J., 1964 Anla1.
Quantitative kinetic data were obtained for this reaction by using a method (Ohem, , 86°7117). Regarding this mesoieta, K about the reaction between ferridinium ion and reduced glucose oxidase.
A second-order reaction rate constant of =1.0' m-13-1 was obtained. Such ability of the ferridinium ion to act as a rapid oxidant for glucose oxidase facilitates efficient coupling of the enzymatic oxidation of glucose.

Example 4 The process of Example 8 was carried out using 1,1/-7 erocene dicarboxylic acid in place of 7 erocene. The value of E0/ was measured and found to be +420 mV, and the second-order reaction rate constant between ferridinium ion and reduced glucose oxidase was also 10 m s.

This confirmed the conclusion drawn from Example 8.

Example 5 Glucose oxidase 1, 1-dimethylferrocene Small electrode for in vivo skin glucose sensing Graphite blob with oxidized surface 13 as shown in FIG.
(length 80 mm x diameter Q, 9 mm) was glued into a nylon tube 14 (length 25 my + 1, inner diameter 0.9 mm, outer diameter 1.3 mm) using epoxy resin. 'The end of the sleeping electrode 15 was immersed in a toluene solution of dimethylferrocene (1.0 my/ml), and then the solvent was evaporated. Electrode 15
The ends were placed in a solution of aqueous DOO (25 mg/ml) in acetate buffer, pH 4,5 for 1 h.

It was then washed for 5 minutes with a broth, and then washed with a solution of glucose oxidase (10 μ/-) in acetate buffer, pH 5.
Washed again in buffer at 1% time intervals within 5 days.

The tip of the electrode 15 with a layer of dimethylferrocene and a layer of immobilized enzyme was prepared by mixing cellulose acetate with acetone and NN/N/N.
- A stable electrode was obtained by immersing it in a bath solution containing 111 yen of dimethylformamide and keeping it in ice water for several minutes.

This electrode was connected to a potentiostat along with a suitable counter electrode and a calomel reference electrode. The potential of the working electrode was maintained between +100 mV and 80 (1 mV) relative to the calomel electrode, i.e. as low as possible to prevent oxidation of potentially interfering substances. A current proportional to the glucose concentration occurred. 95% response. In less than 11·1 min, the electrode showed a nearly proportional response over the range 0 to 82 mM as shown in Figure 8. A slow loss of ferrocene activity (based on a slow loss of ferrodinium ions) could be minimized by maintaining the electrode at a constant potential, 0. to -100 mV/standard calomel electrode, when not in use.

FIG. 4 shows a cross section of an example electrode. In this electrode, as shown in Fig. 2, an extremely small electrode is used.
The needle is kept in the hypodermic needle 16 with a stopper at position 17 but with side openings 8 to allow passage of blood or other body fluids. It takes % 1 of the bucket! Due to its small size and its proportional response over a wide range of glucose concentrations, it is possible to use this electrode for in vivo glucose measurements in severe diabetics and normal humans.

Example 6 As shown in FIG. 5, a carbon rod 19 (Ultra carbon, grade U5.6
5 arn) to the glass tube 21 (borosilicate glass,
It was fixed to one end of the tube (inner diameter 6 mm) and sealed with Evogishi resin (aramid resin). The surface exposed in step 1 at the 22nd place 1i゛7 was polished with diamond sand paper and washed with distilled water. The bar was heated in an oven at 200'C for 410 hours to obtain an oxidized surface at position 22.

15μ of ferrocene (20m9/i in toluene) was pipetted onto the oxidized surface and allowed to dry thoroughly. The rod was then injected with 1 ml of water-soluble DOG (0,1
The sample was placed in M. acetate buffer (25μ/I, pH 4,5) for 80 minutes at room temperature. The rods were then washed with 0.2 M carbonate buffer p) (9,5) and then with glucose oxidase solution (Sigma type X, 12.51n9/r).
The mixture was kept for 2 hours at room temperature for 2 hours. Finally add this to water,
The cells were washed with a buffer solution of I)) I7 containing 0.22/] glucose and stored at 4"C.

The characteristics of the above electrode were determined using a nitrogen saturated buffer solution ((1,2M Na
PO, pH 7, 8) and is shown in Figure 6. In Figure 6, the curve shows glucose between 2 and 25
It was linear up to mM and saturation current was reached at 100 mM glucose.

In another test with air-saturated buffer at 8 mM glucose, current was measured as at least 95% of the current generated in nitrogen-saturated buffer.

Additionally, response time was measured. Note that this time is the time required to reach 95% of the maximum current at a predetermined concentration. When using a nitrogen saturated buffer solution, the electrode as described above
The response time was 24 seconds with mM glucose and 60 seconds with 6 mM glucose. When using a similar buffer, such an electrode modified by coating with a cellulose acetate membrane (prepared as in Example 7) lasted for 86 seconds (2 m
M) and showed a response time of 72 seconds (6mM).

In the case of blood, this reforming electrode has a
The response times of blood with a glucose content of mM) are shown.

As a stability test for the above electrode, NaPo at pH 7,
Stored for 4 weeks at 4'C in 20 mM and then retested as described above, the reported results were within 10% of freshly made electrodes, typically 5%.

Example 7 Glufus dehydrogenase/fethene hard carbon paste was prepared from 1.67 Du, rco activated carbon and 2.5 liquid paraffin. 6 Intrapharyngeal%
A Bathstool pipette with a wire was plugged two angles from the wide end of the pipette with a silver disc to which the connecting wire was brazed. The space between the disc and the end of the pipette was filled with carbon paste, and the paste surface was polished with paper until it was smooth and coplanar.

- of ferrocene toluene solution (20+++s+/l)
A 20 μt drop was placed on such a smooth surface, spread and evaporated leaving a film of ferrocene.

Furthermore, a 25 μl drop of a solution of bacterial glucose dehydrogenase as obtained in Example 1, containing 1 to 10 tng of protein per drop, is dropped onto the ferrocene surface;
I expanded this.

The dialysis membrane cover was secured over the coated electrode end with an interference fit O-ring.

Example 8 Glucose dehydrogenase/ferrocene The procedure of Example 7 was repeated. However, the same carbon paste was used and filled into the space defined between the end of the nylon tube and the stainless steel hypodermic needle shaft inserted two spaces short of the end of the tube to define a small electrode body. It was used as an electrode.

This electrode was further assembled using only 5 μt of ferrocene solution and 1 μt of enzyme solution.

Example 9 Glucose dehydrogenase/ferrocene The procedure of Example 8 was repeated. However, as an electrode, a solid carbon rod (Ultra grade U5, diameter (3rr, m) was placed inside a Pyrex glass tube (length 3cm, inner diameter 6) and contacted with a stainless steel hypodermic injection shaft, as shown in Figure 5. The inscription was similar to the structure 1if shown.The end of the carbon body was polished and smoothed with emery cloth and aluminum oxide powder.
Thereafter, a 7Erocene solution was applied.

Example 10 Glucose dehydrogenase/ferrocene 25 μl of gelatin, Q, 5 tnl of glucose dehydrogenase and 2.5 Itl of TEMED as described in Example 9
Gelatin containing glucose dehydrogenase was prepared by mixing at °C. After fully dissolving the gelatin, 200 μl of the solution was spread over an area of 2 CM and dried under a stream of cold air.

Then instead of the drops of enzyme solution in Example 8, 0.25cr
A disk with an area of 1 was used.

Example 11 Glucose dehydrogenase/ferrocene The procedure of Example 10 was repeated. However]-rnn? A disk coated with gel having an area of 100 ml was used, and the gel was used in place of the drops of enzyme solution in Example 10.

The results obtained using the electrodes described in Examples 7-11 are similar, with a very specific 1! It turned out to be extreme. To illustrate, the electrode of Example 10 was tested with the results shown in FIG.

Examples 12-24 D, C, cyclic portammetry was used to demonstrate the ability of ferrocene compounds (usually ferrocene monocarboxylic acids) to produce and enhance anodic current in the presence of the following enzymes and their respective substrates.

Table 2 Pyruvate oxidase Pyruvate L-amino acid oxidase L-amino acid aldehyde oxidase Aldehydoxanthine oxidase
) Hriboamide dehydrogenase N, A, D
HPQQ#glucose dehydrogenase glucose methanol dehydrogenase methanol i5 and other alkanols methylamine dehydrogenase methylamine heme-containing enzyme lactate dehydrogenase lactate (yeast cytochrome B2) horseradish peroxidase hydrogen peroxide in each case, enzyme /Mesoieta threads produce increased anodic current indicative of enzyme-catalyzed reactions. The second-order uniform rate constant calculated from the data thus obtained showed that the ferrocene compound effectively mediated the electron transport of the enzyme electrode, making it suitable for creating an electrode assay system.

Typical records for cyclic portammetry are as follows.

Example 25 a) Electrochemical measurement potential control
) Electrochemical method or O, cyclic portammetry is used in the cell 80 facing the working electrode (WE)! It is based on maintaining the potential of the working electrode (WE) with respect to the reference electrode by passing a current between -M (G E ). No. 8 @ shows the circuit used, incorporating two operational amplifiers. These amplifiers are manufactured by Oxford Electrodes (Oxford Ele
ctrodes) was incorporated. The current-potential curve was calculated using Bryans X-Y
Recordings were made using a 26000A3 chart recorder. Applied potential −vin+current −■. It was set as ut/R.

An 880X Microcs computer (Research Machines) connected to a potentiostat via digital-to-analog and analog-to-digital converters was used for the potential step method. The Botensio stat incorporates a multiplexer to facilitate switching and monitoring of one or more working electrodes.

b) Cell and electrode, O, cyclic portammetry experiment, 9th
The experiment was carried out using the arrangement shown in the figure, that is, a two-chamber cell in which two cells are connected by a Luzin tubule 81 in FIG. 9.

In addition to a gold working electrode 82 with a diameter of 4 fins (white plum and pyrolytic graphite could also be used satisfactorily), the cell was 1 cm”
Platinum mesh counter electrode 38 and accurate saturated calomel electrode 84 type 4 in the range of -10°C and 0°C as a reference.
01. All potentials are standard hydrogen electrode (NHK)
) was referenced to a saturated calomel electrode (SOE) of +241 mV at 20°C.

The working electrode was polished on cotton wool with alumina-water paste before each experiment and then rinsed with deionized water. A particle size of approximately 0.8 μm was obtained with BDH.

c) Temperature-controlled electrochemical experiments were performed under thermostatic control by using a Churchill chiller thermocirculator connected to a water bath in which the electrochemical cell was placed.

d) Spectrophotometry All light spectra were measured by placing the sample and standard solution into a small quartz cuvette with an appropriate optical path of 1 cm.
O recorded on a Unicam S P 8200 spectrophotometer e) Purification of water When possible, all solutions were
pore) Milli-RO4 and LSI 1ll
It was prepared using water purified by a series of reverse osmosis ion exchange and carbon p-filtration using a combined i-Q system.

Ultrapolar filtration and diafiltration of proteins,
This was done using an appropriately sized Amlcon cell equipped with an appropriate Diaflo membrane.

Pharmacia for protein purification
The test was carried out using FPLq manufactured by Co., Ltd. This is single wavelength UV
- two p-500s controlled by a gradient programmer GP-250 operated with a monitor (λ-260nm) and an automatic fraction collector FRAO-100;
Equipped with a pump. The analytical ion exchange column and preliminary ion exchange column were also from Pharmacia.

D, 0. Cyclic portammetry experiments were performed in argon saturated solution as follows. First, the reversible electrochemical properties of 7-erocene monocarboxylic acid (200 μM) in a suitable electrolyte were recorded using portum monograms at various scanning speeds (1 to 100 mV) over a potential range of 0 to 400 mV. It was established by Substrate was then added to the cell, typically to a final concentration of 10 mM and always above the Michaelisis-Menten constant for the enzyme. Record a set of Portummoregrams -
The effect of substrate on the electrochemical properties of C mesoieta was evaluated. Enzyme was then added to a final concentration ranging from 10 to 100μ16. If the anodic current is increased and the dependence of the anodic current effect on the scan rate indicates a catalytic reaction, repeat the experiment with the addition of substrate as the final component to confirm that the reaction is dependent on the presence of the substrate. I confirmed it.

Under the conditions used, the substrate did not interfere with the electrochemical action of ferrocene. θ~400 m relative to SCE
Over the V range, the substrate or enzyme did not exhibit any direct electrochemical effects.

h) Material 1 Flavoprotein, pyruvate oxidase (EOl, 2
．． 3.3), xanthine oxidase (E C1,2,
8,2), sarcosine oxidase (E O1,5,3
,1), riboamide dehydrogenase (E C1,6,
3,4) and glutathione reductase (E O1,
6,4,2) to Boehringer
), and these were stored and stored at -20°C. Each concentration of flavoprotein was expressed as the amount of catalytically active flavin.

Carbon monoxide oxide reductase by Dr. J.

Biochemistry Depa written by C01by
rtment.

It was isolated from Pseudomonas sp.-7carboxydoborans by 5underland Polytechrric and supplied at a concentration of 8.6 f1 in phosphate buffer containing 50% ethanediol as a stabilizer. Before use, the enzyme was diluted with 20 mM Tris-HGl-(1)H7.
, 5) at 4°C and purified by FPLO using an analytical MOno-Q column. The enzyme was then injected into 20 mW of Tris-HCII) H7,5 (buffer A).
) was loaded onto the column at a concentration of 1, Omgmr'' and eluted using a linear ionic strength gradient with buffer (A+1., OMKO7). As such, it eluted as one major peak at an ionic strength equal to 85% Buffer B.

Kino-protein alcohol dehydrogenase (E(3)
Purification of 1,1,99,8) was described above. Lactate dehydrogenase (EOl, 1.1.27) and isocitrate dehydrogenase (EOl, 1.1°42) were obtained from Boehringer.

Sodium lactate, sodium isocitrate, sarcosine, sodium pyruvate, xanthine.

Cholesterol, potassium oxalate, coli, phosphorus and chloride were recovered. - Carbon oxide was obtained from BOG. Ferrosense/
Fluorochem carboxylic acid
I got more.

1) Electrolyte 100 mM Tris-HC7 1p for all experiments
H7,o was used. However, the one containing oxalate oxidase using 1100 In succinic desalting buffer pH 3.0 and the one containing 100 m containing 14 mM NH, O7
The one with alcohol dehydrogenase using M silt-NaOH was excluded.

The electrochemical cell shown in Figure 9 incorporating four pyrolytic graphite disc working electrodes was used in all experiments. However, in such experiments for lipoamide dehydrogenase and glutathione reductase, a four-fin disc metal electrode was used. In an experiment in which cytochrome C was investigated using carbon monoxide oxyside reductase, bis(41,4,7-pyridyl)-1,2-
An ethene-modified gold electrode was used.

Using cyclic portammetry as described above, we demonstrated the electron transport ability of various ferrocene compounds listed in the glucose/glutose oxidase system: 7 erocene 165 15 vinyl-25018 carboxy-275115 1,17-dicarboxy-8851 , 5 to 117 Nalaminol 400 : 300, a) Solution kinetics Potential in a constant range (100 to 400 mV vs. SOE
) in Table 8, D, O
, investigated as a possible oxidant for glucose oxidase using cyclic portammetry. 1st
Figure 1 shows (a) the portamogram of carboxyferrocene that satisfies the electrochemical criterion as a reversible one-electron couple t (δEp−6omv + lp /
When glucose alone was added to the X-constant) 0 solution, there was no appreciable effect on the portamogram.

However, when further glucose oxidase was added to the solution (b), a significant change occurred in the portamogram. The increase in current at the anode is a characteristic of catalytic coupling reactions and can be represented as the following scheme: R is ferrocene, 0 is ferridinium ion,
and 2 indicates reduced glycose oxidase.

The increased anodic current is due to the reaction between reduced glycose oxidase and 7-erysinium ions. still,
The latter ions occur at oxidation potentials. The mechanism of homogeneous reaction between glucose oxidase and a number of ferrocene derivatives was analyzed by the theory derived by Winifelson and Schein. From the data it was possible to derive pseudo-order rate constants independent of scan speed. Changes in such parameters as a function of glucose oxidase concentration resulted in a quadratic reaction rate constant. The effectiveness of this analysis depends on two conditions that must be met; the heterogeneous electrode reaction must be faster than the catalytic coupling homogeneous reaction, and it must ensure that the enzyme is always in the reduced form. There must be as much glucose as possible. These same conditions were valid in this study. The data presented in Table 8 show that all hepta-rocene derivatives in the oxidized form studied have a reaction rate of molecular oxygen, the natural electron acceptor, with a value of ks-10'M-1.s-1. It is shown to act as a rapid oxidant for enzymes with comparable reaction rates.

Although all ferrocene derivatives listed in Table 8 produce efficient 'electrochemical oxidation of glucose via glucose oxidase, other criteria are important when designing enzyme electrodes for practical use.

The solubility of the heptadrocene derivative in reduced form in aqueous medium is:
? l! The oxidized form should be stable at physiological pH;
The apparent potential is low and steep to prevent interference by reducing compounds present in the physiological sample. 1, 1'
-Dimethylferrocene offers the best compromise between the constraints imposed by these factors and was chosen for incorporation into the enzyme electrode.

b) Enzyme electrode digital simulation technology calculates the apparent steady-state current for a regular enzyme electrode using the Michaelis-Menten constant, the membrane permeability, the effective electrode surface area, and the enzyme,
It was shown that it is mainly determined by the load factor (concentration per unit volume). When considering effective enzyme immobilization techniques, covalent attachment to the functionalized electrode surface generally resulted in the best enzyme activity. Moreover, the single layer coating obtained was the most suitable with regard to optimal response properties. Based on this, a batch of 24 standard glucose enzyme polarizers were constructed as shown below, and their performance was evaluated with 611 values.

C) Reagent glucose oxidase (EC1,], 8.4 type 2, from Aspergillus niger) was obtained from Boerinkermann Eimka. Furthermore, this glucose oxidase is 2
It had an activity of 74 IU/m9.

D-glue (AnalaR) from BDH
↓; Ferrocene and its derivatives are subjected to Strem (3h
Obtained from em, Qo. All solutions were mixed with high purity water (Myribore) using an Arista blade (Ar15tar gr).
ade) Prepared from reagents. Supporting electrolyte is HOIO
The glucose solution was stored overnight to equilibrate the α- and β-amomers. The properties of this enzyme are shown in the table below.0 Table 4 Source: Black black mold RMM 8600 C゛-factor 2 FAD co-substrate Oxygen optimum pH 5,6 K[Il glucose 3.0mM d) Biological sample Diabetic patient Heparinized plasma samples were obtained by the Metapolis Department, Guy's Hospital, London, UK and previously analyzed for glucose on a Yellow Springs Instruments glucose analyzer.

e) equipment A two-chamber cell with a working volume of 1 ml is used, and C.

Cyclic portammetry experiments were performed. This cell is a 4th, rn pyrolytic graphite disk actuated
fIll! in addition to a 1 cm" platinum mesh counter electrode and a saturated calomel reference electrode (Baudelon et al., J.
.

Amer, Qhem, Soc, 102, 428
1, 1980).

All potentials were relative to a saturated calomel electrode (SOE). For cyclic volcummetry, an Oxford Electrodes botensiostat was used with a Pryons X-Y26000A3 chart recorder. , electrolytically controlled steady-state amperometric measurements using a cell with a working volume of 100 fnl and separate chambers for the counter and reference electrodes, adapted to accommodate up to seven enzyme electrodes. went. Current hourly curves were recorded on a Bryans Y-tBs-271 recorder. During the experiment, the humidity of the electrochemical cell was controlled within a range of ±0.5°C using a Churchill thermoculator.

f) Configuration of Glucose Enzyme Electrode A single-portion graphite foil manufactured by Union Carbide was used as the base sensor. If this graphite is 4 mm in diameter
Electrodes were formed by cutting into disks and bonding them to glass pellets with epoxy resin. The electrodes were then heated in air at 200°C for 40 hours, cooled and injected with 15 μl of 1,
11-Dimethylferrocene (0.1.M in toluene) was deposited on the electrode surface and then air-dried. Covalent deposition of glucose oxidase onto oxidized graphite surfaces was accomplished by a method similar to that described by Beaudillon. The electrode was water-soluble 1 made by Sigma Chemical Company.
- 1 ml solution of cyclohexyl-8-(2-morpholineethyl)carbodiimidometh-p-toluenesulfonate (0.15 M in 0.1 M acetone, pH -4,
5) for 80 min at 20 °C, washed with water, and then placed in a stirred solution of acetate buffer (o, 1M, pH-5,5) containing glucose oxidase (12,5 μ/ml). The mixture was incubated at 20°C for 90 minutes. After washing, the electrodes were covered with polycarbonate membranes (nucleopores, 0.03 μm) and stored at 4°C in buffer containing 1 mM glucose.

, g) Enzyme Electrode Pretreatment After completion of the assembly and before the start of the experiment, the electrode response was stabilized by continuously operating the electrode in 8 rl 1M glucose for 10 hours under electrolysis potential control at 160 mV. Thereafter, the electrode was found to provide a more stable response during continued operation for an additional 100 hours. In 8 mM buffered glucose solution, the electrode showed an average current reduction of 8%±1 during the above operation period.

All electrodes modified with glucose oxidase were subjected to such preconditioning treatment.

All electrodes showed a linear current response within the range of 0.1-85 mM glucose and were finally saturated at approximately 70 mM glucose. Within the linear region, the electrodes are 60-9
It showed a rapid response time reaching 95% of the steady current in 0 seconds. The reproducibility of the electrode structure report was examined by measuring the steady state current for each electrode in 8 mM glucose.

Each batch of standard electrodes had an average current response of 7.9 μA with a standard deviation of 2.8.

, h) Effect of temperature The effect of temperature on the enzyme electrode response was studied in the range 10-50 °C, and the increase in steady-state current with increasing temperature was approximately 0.
．． It was shown to be 2 μA/'C. All electrodes showed similar behavior. Assuming Arrhenius-shaped behavior, the lack of a maximum in the electrode response indicates thermostability of the immobilized enzyme at temperatures up to 50°C. This electrode (iq structure) is suitable for long-term use at standard body temperature.We confirmed that similar thermal stability exists for soluble enzymes, and the temperature dependence of its second-order rate constant is 49.6 KJ MOl-1. Listen to the activation energy.

i-) Interfering substance direct charge t+ Q? The influence of substances that interfere with the electrode response by oxidation, reaction with mesoieta, or inhibition of enzymes was investigated.A solution containing 8 mM glucose was analyzed in which a substitute was added to obtain a standard blood concentration.O , I, L-ascorbate at a final concentration of 8\mM is approximately 41.0
%, but the addition of urine m(o, zom74) increases the transition from hypoglycemia to hyperglycemia by approximately 0.5, ~1
When reflecting a blood glucose change of 30 mM, a practical glucose electrode is required to respond linearly within the range mentioned above to eliminate the need for sample dilution.

In this regard, we believe it is important to change the apparent Michaelis-Menten rate KM of glucose oxidase for glucose, where the immobilization report to the electrode is the -1-limit value with high linearity. The axis can be calculated if the electrode response is in a dynamic rather than diffusion-controlled state (14), i.e. the steady current is independent of the stationary or stirring of the solution. Standard ferrocene group ν confirmed to be kinetically controlled fl;II
At the U pole, KM7 values ranged from 30 to 40 mM. This is compared to a KM of 8mM for glucose non-immobilized enzyme.

Specific interfering substances, ie potential interfering substances, are shown in Table 5 below. Although all of the substances listed are substrates for glucose oxidase, the relative rate of the reaction is much lower than that of the primary substrate, glucose. Although it has been experimentally determined that the effect of these substrates on the glucose assay is minimal, it can be seen that the electrode sensor of the present invention can be used in any of the listed substrate assays in the absence of glucose.

β-D-glucose 100 2-deoxy-D-glucose 25 6-methyl-D-glucose 2 D-mannose 1 α-D-glucose 0.6 L-cysteine HGl (0.08mM), reduced curtathione (0.49mM ), sodium formate (7,3
(1mM), D-X-O-ose (8,00mM), α
- Galactose (7,77mM) and α-mannose (7,77mM) did not cause any observable interference with the electrode response to glucose.

However, changing from nitrogen saturated buffer to air saturated buffer resulted in an average decrease in current response of 4.0%. □... Interference from oxygen is not surprising, but current reduction occurs if the base electrode is not positively balanced enough to reoxidize the hydrogen peroxide generated by the enzymatic reaction. Operating the electrode at a sufficiently positive potential to reoxidize hydrogen peroxide will increase interference from L-ascorbate.

j) Effect of pH Since the pH of plasma samples from diabetic patients changes with the addition of heparin or loss of carbon dioxide, the effect of pH on the response of the glucose electrode was investigated over a clinically relevant range. The steady current of the enzyme electrode mentioned above is substantially p
It has nothing to do with H.

This is comparable to the behavior of soluble enzymes, where the second-order rate constants for all ferrocene derivatives listed in Table 3 are between 6 and 6.
It was confirmed that it was independent of pH changes in the pH range of 9. The desirability of such a pH-independent response is likely due to the fact that no proton transfer is involved in the 7-erocene oxidation compared to oxygen-mediated glucose enzyme electrodes.

A device such as the one shown in the example offers various advantages over most enzyme-based sensors currently available. When compared to these sensors before the dilution step, the electrodes of the present invention have comparable or faster response times and operability under anaerobic conditions, −19
1 has great oxygen insensitivity (important in blood samples with variable oxygen concentrations), a wide linear range protecting specificity comparable to the full physiological range, and stability as well as ease of manufacture.

Additionally, included within the mesoietors specified in this invention are thiols of ferrocene or similar sulfur derivatives where the mesoieter can be directly bonded to the gold electrode.

The diol group can be bonded directly or indirectly to one ring of the ferrocene structure, for example by a lower alkyl group having 1 to 6 carbon atoms.

A simple thiol (ferrocene)-8H made as described in J. Ohem, Soc, 692 (1958) can be used. The inventors have determined that alkylthiolne, (ferrocene)-0,H8SH is valuable. Other, more complex thiol thread compounds are also possible, such as 1,2,8-)lithia-(3)-7 ero-7nophane in which the two rings are linked by a chain of sulfur atoms (chain sulfur atoms). mixture of different substances).

Gold electrodes can be fabricated for repeated use by bonding the mesoietaferrocene structure to the conductive metal, for example by immersion in a solution of a galvanizing compound.

Examples of the production of such substances are as follows: 1, 2.
3-trithio-(3)-ferronanophane (J-O
rganOm6jallO Ohem. 1 9 7
1 , 2 7 +241) The literature procedure was followed, but the product was not appreciably reduced by sublimation of the crude mixture. The most obvious (i.e. most odorous) potentially sublimated material was chromatographed on silica using hexane as eluent.
A seed product was obtained.

1. As a result of analysis, it is not sulfur.

2, C: 4 8.7 2, H: 2.8 8, S
: 8 8.0 5, C engineering. H8FeS8 is (3
: 4, 2.9.8, H: 2.8 9, S
;: 3 4.4 2 is required and the yield is 0.
A complex molecule of 0.45 and 0.11, which could not be examined beyond the spectra, showed that it was not a ferrocene fan with one sulfur atom.

Ferrocentiobencune (Ferrocenylthiobutane) 1 Ferrocenylbutyric acid (J.Am.Chem.Soc
1957.

79, 8420) Fo- GO - CH2- (3I(2-0)12
-OH2- OH2000H was prepared by the method described in the literature.

2. Ferrocenylbutyric acid Fo- OH2- OH2- (3H2- OH2-
000H (made with Talemenson's reduction, zinc/mercury and hydrochloric acid as described above) 3Ferrocenylbutanol Fo- OH2- OH, - OH2- (EI(2-
OH20H acid (2) (12 g) was dissolved in ether (distilled from sodium/potassium) and treated with lithium hydride under nitrogen atmosphere. When the reaction was complete, the lithium aluminum hydride was destroyed using ethyl acetate and then water. The organic phase was separated and the aqueous phase was washed with ether (2×20 tnl). Mix the organic phases and dry (M
gSO4')L, after filtration the solvent was removed on a rotary evaporator. The resulting red oil contained two components. Column chromatography on silica (80C
m X 2 Cm) to form ether:hexane l;l
Elution gave the alcohol and ester.

Fo- OH2- OH2- CH2- OH2-
000CH84 Ferrocenylthiobutane (8) (4oom9) was dissolved in pyridine (dried over sodium hydroxide, lQi) and cooled in a water bath. Tosyl chloride (1!7) was added and the solution was stirred until clear and then left at 4°C for 24 hours. The mixture containing solid pyridine hydrochloride was poured onto ice/water to precipitate the Tris salt. This was filtered with a water pump to obtain a yellow solid. This dried part gave the characteristic Tris salt infrared spectrum. residue, (0,65
(still wet) in ether:meth/-le (1
: Sodium hydrosulfide hydrate (X) I20 dissolved in 1)
') Add 1.69 while stirring and bring the mixture to 5°.
It was maintained at C. After 80 minutes, the water bath was removed and the mixture was allowed to warm to room temperature. After 8 hours f, 1. c.

(silica, ethyl ether (Et2o)'hexane 1:1) indicated the reaction was complete.

The mixture was reduced and dried on a rotary evaporator, then Et2
0/hexane (1:11) and chromatographed on silica (60-120 mesh, 25 cm
I25 cm x 2 cm column, eluted with Et20/hexane (1:11)). Thiols passed through very quickly and were collected at approximately 150 thiols. Yield is 200 m9
Met.

Fo-0H2-C1 (2-0H8-0H2-0H2SH

[Brief explanation of drawings]

FIG. 1 is a partial vertical sectional view of a glucose sensor electrode according to an example of the present invention. FIG. 2 is a vertical sectional view of a glucose sensor electrode according to another example of the present invention. FIG. , a diagram showing the relationship between the current detected by the electrode shown in FIG. 2, and FIG.
FIG. 5 is a vertical cross-sectional view of the electrode shown in FIG. 2 provided in the needle of a hypodermic syringe; FIG. 5 is a vertical cross-sectional view of a glucose sensor electrode according to still another example of the present invention; FIG. Figure 7 is a diagram similar to Figure 3 for electrodes; Figure 7 is a diagram similar to Figure 8 for electrodes incorporating glucose dehydrogenase; Figure 8 is for use in D, O, cyclic portammetry; Figure 9 is a two-chamber cell for D, O, cyclic portammetry; Figure 10 is a carbon monoxide oxide reactor isolated from Pseudomonas mocarboxydoborans. FIG. 11 is a diagram showing a portamogram of carboxyferrocene. , 13... graphite rod 14... nylon tube 15
... Electrode 16 ... Hypodermic injection needle 18 ... Side window
19... Carbon rod 20... Metal connector 21... Glass tube 80...
・Cell 81...Lusin tubule 82...Working electrode 3
3...Counter electrode. FIG, 11° (GB)■8405262 ■February 29, 1984■United Kingdom (GB)■8405263 0 Inventor Eliot Verne Plotkin 43 Nissey George Street, Bedford MK, United Kingdom Procedures Amendment (Form) % Formula % 1 Indication of the case Patent Application No. 90835 of 1983 2, Name of the invention Electrode Sen Power 3 So 11i Relationship with the case Patent applicant name Name J2 Tix International Incorporated

Claims (1)

[Scope of Claims] An electrode sensor for detecting the presence of at least one selected component of a mixture of 1 components, wherein the mixture and 1! at least two mutually insulated electrically conductive devices in gaseous contact; and a mesoieter compound that transports electrons between the enzyme and one of the electrically conductive surfaces when the enzyme is catalytically active. and the mesoieter is an organometallic compound having at least two organic rings, each of which has at least two double bonds that are funshugated and a metal atom that is in covalent electron contact with each of the rings. An electrode sensor featuring: λ A sensor according to claim 1, wherein the mesoiator is limited to the electrically conductive surface. & The sensor of claim 1, wherein the enzyme is confined to the electrically conductive surface. Mori: The sensor according to claim 1, wherein the selected component is a substrate for the enzyme-catalyzed reaction. 5. The sensor according to claim 1]'cI, wherein the mesoieta compound is a ferrocentib compound. 6 The above ferrocentib compounds are ferrocene, chloroferrocene, methyl-trimethylami7ferrocene,
1.1-dimethylferrocene, 1.1/-dicarboxyferrocene, carboxyferrocene, vinylferrocene, trimethylamino/ferrocene, 1,1/-dimethylferrocene, polyvinylferrocene, ferrocene monocarboxylic acid, hydroxyethylferrocene, acetoferrocene and 1,1/-bishydroxymethylferrocene. 7. The sensor according to claim 1, wherein the enzyme is an oxygen-specific flavoprotein. & The above flavoproteins were combined with methanol oxidase, pyruvate oxidase (EOl, 2°a, S, ), xanthine oxidase (EC31°2, 8.2.),
Sacosine oxidase (EC1,5,8,1,), ribamide dehydrogenase (E O1,6,8,4,),
Glutathione reductase (EOl, 6°4.2.)
, carbon monooxyoxide-reductase, glucose oxidase, glycolate oxidase (EC1
, 1, 8, 1,), L-amino acid oxidase (EC1
, 4, 8, 2,) and lactose oxidase (Ec
1, t, a, t, ). 9. The sensor of claim 1 d, wherein the enzyme is chinoprotein. 10. The sensor according to claim 911, wherein the chinoprotein is selected from the group consisting of glucose dehydrogenase, alcohol dehydrogenase, and methanoldehyde tetragenase. 11. The sensor according to claim 1, wherein the metal atom is iron. 1a The sensor according to claim 1, wherein the metal atom is cobalt. 1 & The sensor according to claim 1, wherein the meso-Ita compound is cobal)cene. 14 Claim 1 in which the metal atom is chromium
Sensor described in section. 15. The sensor according to claim 14, wherein the mesoieta compound is dibenzenechromium. 1a. The sensor according to claim 1, wherein the enzyme catalyzes the reaction of glucose to obtain a glucose sensor. 17. The sensor according to claim 16, wherein the enzyme is glucose oxidase. 1& The sensor according to claim 16, wherein the enzyme is bacterial glucose dehydrogenase. 19. The sensor according to claim 18, wherein the glucose dehydrogenase is isolated from Acinetono (Futa genus Calcoaceticus). 20. In 1, the electrode is made of a material selected from silver, carbon particle paste and solid carbon. 21. The sensor according to claim 1, wherein one of the electrically conductive devices comprises carbon, a layer of a ferrocentib compound on the outer surface as an electron transport mesoieta compound, and a layer of glucose provided on the layer of the mesoieta compound. A sensor according to claim 1 for use in a liquid mixture containing glucose to respond to the presence of glucose, comprising an electrode consisting of an enzyme selected from oxidase and bacterial glucose dehydrogenase. 22. A sensor according to claim 21, wherein the compound of ferrocentib is in the form of a polymer, and the compound of ferrocentib is in the form of a polymer, and the sensor according to claim 24. The sensor according to claim 21, wherein the compound of ferrocentib is bonded to the carbon electrode by a carbodiimide crosslink.
The sensor described in item 2. 25. The sensor according to claim 21, wherein the enzyme is glucose oxidase immobilized on the mesoieta by DOO. 2& The sensor of claim 21, wherein the enzyme is bacterial glucose dehydrogenase deposited onto the mesoieta layer from an evaporable solution. 27. The sensor of claim 21, wherein the enzyme is bacterial glucose dehydrogenase maintained in a gelatin layer on the surface of the mesoieta layer. 28. The sensor according to claim 21, having an outermost protective film that is permeable to water and glucose molecules. 29. The sensor according to claim 29, wherein the protective film is cellulose acetate deposited from a solution of cellulose acetate. 80. The sensor according to claim 1, comprising an injection device suitable for injection into a person n11. 81. The sensor of claim 1, wherein the mesoieta compound transports electrons from the enzyme to the conductive surface. 8z Claim 12, wherein the enzyme is a heme-containing enzyme
Sensor described in section. 83. The sensor of claim 82, wherein the heme-containing enzyme is selected from the group consisting of lactate dehydrogenase, yeast cytochrome C peroxilase, and horseradish peroxidase. 8. The sensor according to claim 1, wherein the enzyme is cuberobrotiin. 8& The sensor according to claim 84, wherein the cuproprotein is galactose oxidase.