GB2332058A

GB2332058A - Electrode with aminoacid polymer coating

Info

Publication number: GB2332058A
Application number: GB9725707A
Authority: GB
Inventors: Pankaj Madganlal Vadgama; Keith Stewart Robert Warriner
Original assignee: Victoria University of Manchester; University of Manchester
Current assignee: Victoria University of Manchester; University of Manchester
Priority date: 1997-12-05
Filing date: 1997-12-05
Publication date: 1999-06-09
Also published as: GB2332058A9; GB9725707D0

Abstract

Sensor devices comprising a working electrode carrying a coating of an aminoacid polymer with a layer superimposed upon it to maintain it in place. The layer is preferably of diamond-like carbon ("DLC"). The electrodes are especially applicable to the analysis of liquid media to detect their ethanol content by electrolytic analysis. The preferred combination is poly-lysine on platinum, with a DLC coating 0.01 to 5 um (preferably 0.5 to 2.0 microns) thick, and the preferred procedure is by pulsed amperometric detection ("PAD"). The DLC coating stabilises the coating of poly aminoacid against being dislodged by acidic media during use. The device is especially applicable to monitoring and measurement of fermentation media, with reduced interference by any sugars present. The combination of the poly aminoacid and DLC gives a sensor which is stable enough to be heat-sterilised and have good pH independence and selectivity to minimise interference by other components, e.g. sugars.

Description

SENSOR DEVICES AND METHODS FOR USING THEM.

2332058 This invention relates to sensor devices and methods for their use, and more particularly to improved sensor devices useful for electrolytic analytical methods for the detection and determination of ethanol.

It is known to make and use a variety of electrolytic sensor devices incorporating one or more electrodes to produce a signal output from which specific analytes can be detected and measured. These electrodes can act in several ways, for example by detecting such conditions as oxidation, reduction, acidity/alkalinity (pH), electrical potential and current flow. The species which can be detected in this way include glucose, ethanol, and many other compounds.

The detection and measurement of ethanol is of great commercial importance, as the wine and brewing industries are very extensive and taxes and duties are payable to governments on the basis of measurement of the ethanol content of fermentation products. Consequently, there is a great demand for reliable devices for monitoring the progress and efficiency of alcoholic fermentation processes by measuring the content of ethanol in them, and also, in many instances, other properties of the fermentation media, for example the reducing sugar (for example glucose) content as it is fermented into ethanol. This monitoring is also desirable for other process or waste liquors or effluents.

Among the proposed sensor devices which have been proposed for carrying out such monitoring and measurement, many have contained enzymes - which act on the substrate chemical being evaluated and generates a different chemical which can be determined, thus providing means for determining the substrate chemical indirectly. Especially, glucose oxidase has been used because it catalyses oxidation of glucose to gluconic acid -- producing hydrogen peroxide via oxygen reduction. The hydrogen peroxide is very readily and conveniently determined electrolytically.

2 Existing sensor devices are not entirely reliable or durable for the more demanding industrial uses, for example continuous monitoring of the whole fermentation cycle, and suffer from the disadvantage of being unable to survive the heat sterilisation steps which are so important in the fermentation industries. These often involve temperatures as high as 140 degrees C (as in steam sterilisation) and enzymes are de-activated at such temperatures.

Therefore, current methods still rely on removing samples periodically from the fermentation process and determining the ethanol content usually by specific density or chromatographic techniques. Clearly, this is inconvenient (as it does not give continuous measurement and may even contaminate the process media) and is slow.

Therefore there is a need for an enzyme-free electrochemical sensor for ethanol which can withstand repeated steam sterilisation or rigorous chemical sterilisation, and thus can be part of the "clean in place" systems used industrially. 20 Ethanol is relatively inactive electrochemically, and electro- oxidation typically requires strongly alkaline solutions, but by using platinum electrodes it is possible to detect ethanol in neutral or acidic solutions. Even so, the response signals are pH-dependent and are also weak, so they are susceptible to masking by background (capacitative) currents or "noise," and a practical sensor is not possible to produce. We have now found that these problems can be overcome and improved performance can be obtained for the detection of ethanol by using a platinum electrode coated with a polyaminoacid (aminoacid polymer).

Thus according to our invention we provide an improved sensor device, useful in electrolytic analysis procedures, which comprises a working electrode carrying a coating of an aminoacid polymer upon which is superimposed a layer of material to maintain it in place.

- 3 The material to maintain the aminoacid polymer layer in place is preferably, and most advantageously, diamond-like carbon, though other means for retaining it in place may be used if desired.

Thus according to our invention we provide an improved sensor device, useful in electrolytic analysis procedures, which comprises a working electrode carrying a coating of an aminoacid polymer upon which is superimposed a layer of diamond-like carbon.

If desired alternatives to the diamond-like carbon may be used, for example a microporous or semi-permeable membrane, but we strongly prefer the diamond-like carbon because it is much superior and allows the assembly to be smaller, simpler and more effective.

According to our invention we also provide a method for electrolytic analysis, especially for the detection of ethanol in a liquid medium, which comprises contacting the said liquid medium with a sensor device as defined above.

This method of analysis is especially applicable to the monitoring, measurement and assessment of media in which ethanol is being formed or produced -- e.g. fermentation media, in which ethanol is formed by alcoholic fermentation of sugars -- and for monitoring and controlling the progress of fermentation processes. Thus, it is especially useful for analysis of the media used in the course of making alcoholic beverages, as well as the "finished" alcoholic products, e.g. alcoholic beverages for which fermentation is not being continued before sale, storage or later treatment. It is also applicable to other alcoholic media, e.g.

distilled or fortifies spirits, whether intended for use as beverages or not, and for the study of waste materials which may contain or give rise to ethanol.

The preferred electrode material is platinum because it has good reactivity towards ethanol and the aminoacid polymer adheres well to this metal, but the electrode may be made of any material with an electrochemically active 4 surface, for example any of those used in electrochemical analysis where the surface is electrochemically polarised, provided the polymer can adhere or can be sufficiently well retained in place while in use, as for example with a semipermeable membrane. The electrode may be in any conventional form, for example sheet or wire, or as a coating deposited upon a substrate.

The aminoacid polymer may be a polymeric form of any aminoacid. Such compounds are well known in the art and may be obtained commercially or can be made by conventional methods for making or synthesising polypeptides and the like, for example by the techniques of automated synthesis.

If desired, the polymer may be a copolymer derived from more than one aminoacid, but it is usually more convenient to use a polymer of only one aminoacid, and the complication of using a copolymer is not necessary.

The polymer may be derived from various aminoacids, and the term "aminoacid" is used in its conventional sense to refer to aliphatic carboxylic (or poly-carboxylic) acids having one or more amino-groups as substituent on the aliphatic chain - optionally with other substituent groups. Preferably, the amino acid contains a secondary amino group as substituent, and especially preferred amino acids are diamino mono-carboxylic acid, for example lysine (diamino- caproic acid) or ornithine (diamino-valeric acid) -- both of which are alpha-omega di-amino mono-carboxylic acids. In practice, we find that lysine is particularly preferred as being the most satisfactory one. The aminoacid may be natural or synthetic, but it is usually more convenient and suitable to use a naturally-occurring form, e.g. L-lysine.

The aminoacid polymer may be applied to the platinum metal support electrode as a thin layer, using any conventional coating technique. The preferred method is to use an solution of the aminoacid polymer and apply this to the electrode by dip-coating. The solution is preferably and most conveniently an aqueous one, but if desired a - 5 solution in a non-aqueous solvent (or a mixture of solvents, with or without water present) may be used provided the aminoacid polymer does not precipitate out from solution prematurely.

The aminoacid polymer for use in the present invention can be defined in terms of its degree of polymerisation and/or its molecular weight -- as these two features are inter-dependent. This choice may also depend upon the particular aminoacid from which the polymer is derived. The molecular weight of the polymer can vary considerably, but in general should be at least 5000 and may be much higher, e.g. 100,000 or more. In practice, we have found a polylysine of molecular weight approximately 70, 000 to be very suitable.

is The layer of aminoacid polymer may be of a thickness in the range 10 to 100 microns (um) but thicknesses which are greater or less than this may be used if desired. We find that the thickness does not need to be very great, and may be limited to the amount of aminoacid polymer that can be easily deposited on the electrode, and may be as thin as a mono-layer.

The choice of aminoacid polymer, its molecular weight and the thickness of the layer used in any particular instance depends upon such factors as the specific aminoacid polymer used, the electrode surface and the readiness with which the polymer adheres to it, the particular conditions of its use and the solutions or materials in contact with it. The optimum should allow a sufficient current/potential to be measured with reasonable ease and convenience. These choices can be determined readily by simple trial.

Once applied, the aminoacid polymer can be stabilised in place by drying and fixing it on the metal surface, for example by drying and heating, e. g. to a temperature of about 50 to 100 degrees C. The time of heating may vary according to the temperature used and the degree of fixation found most effective.

a 6 The aminoacid polymer is used because it adheres well to the substrate metal, especially to platinum, but it tends to be softened and loosened from the platinum metal electrode base by prolonged contact with liquids, especially acid solutions (as would occur in using the coated electrode in fermentation liquors or effluents), so it is rendered more durable by application of a coat of diamond-like carbon over it.

Surprisingly, the application of diamond-like carbon as a coating, applied over the aminoacid polymer, has an unexpectedly beneficial effect on the durability of the aminoacid polymer layer and the reliability of the sensor in use, and is better than any other material we know. Unexpectedly, it is very effective in providing protection for the aminoacid polymer layer, preventing its softening and detachment from the underlying metal electrode, without any unduly adverse effect on the electrolytic measurements. This allows effective use over long periods.

The diamond-like carbon coating not only gives stability to the aminoacid polymer layer but also provides a degree of selectivity in favour of ethanol against various potential interferents such as glucose. We are not sure what the mechanism for this is, but it may be associated with molecular size. Interference by sugars such as glucose in the detection and measurement of ethanol is a key problem in fermentations, so this selectivity effect of a diamondlike carbon over a poly aminoacid layer and the reduction of interference provides a great advantage for our invention.

The combination of the poly aminoacid and diamond-like carbon results in a very desirable combination of properties --- including pH independence, stable adherence of the coating, permeability, heat-stability (which can allow the sensor device to sterilised by heat, and selectivity which can minimise interference by other components (e.g. sugars) in the detection and determination of ethanol. These properties make it eminently suitable for use in making 7 continuous measurement of ethanol content in the progress of fermentation processes.

Diamond-like carbon is already well-known in itself and described in the art, and commonly referred to as 11DLC11.

DLC is a form of amorphous carbon or a hydrocarbon polymer with properties approaching those of diamond rather than those of other hydrocarbon polymers. Various names have been used for it, for example "diamond-like hydrocarbon" (DLHC) and "diamond-like carbon" (DLC), but the term 11DLC11 appears to be the most common. It possesses properties attributable to a tetrahedral molecular structure of the carbon atoms in it, similar to that of diamond but with some hydrogen atoms attached. It has been described in the art as being a designation for "dense amorphous hydrocarbon polymers with properties that differ markedly from those of other hydrocarbon polymers, but which in many respects resemble diamond" [J.C. Angus, EMRS Symposia Proc., 17, 179 (1987)].

The formation and application of the diamond-like carbon (DLC) to the membrane material as coatings or films for the purposes of the present invention may be carried out by methods known in the art. It is usually formed by decomposition of carbon- containing compounds in gaseous or vaporised form (particularly hydrocarbon gases) induced by radiation or electrical fields.

Thus, it may be prepared from hydrocarbon precursor gases (e.g. propane, butane or acetylene) by glow-discharge deposition, by laser-induced chemical vapour decomposition, by a dual-ion beam technique, or by introduction of the hydrocarbon gases directly into a saddle-field source. A saddle-field source is a source of ions produced by a collision between gas atoms excited by thermionic emission, and this method is preferred because it allows heatsensitive materials to be coated by a beam that is uncharged -- so facilitating the coating of insulating or nonconductive materials.

8 Its properties can vary according to the particular raw materials used and its mode of formation. It can also be made in other ways, for example by sputtering solid carbon, as an alternative to dissociating hydrocarbon gases.

Further description of DLC, including its constitution, nature and properties, and the variations in its form which can be made, and modes for its preparation, are to be found for example in the following published references (among others):- (a) "Diamond-Like Carbon Applied to Bio-Engineering Materials;" A.C. Evans, J. Franks and P.J. Revell, of Ion Tech Ltd., 2 Park Street, Teddington, TWll OLT, United Kingdom; Medical Device Technology, May 1991, pages 26 to 29.

(b) "Preparation and Properties of Diamondlike Carbon Films;" J. Franks; J.Vac.Sci.Technol. Vol.A, No.3, May/June 1989, pages 2307-2310; (c) "Biocompatibility of Diamond-like Carbon Coating;" L.A. Thomson, F.C. Law, J. Franks and N. Rushton; Biomaterials, Vol.12, January 1991 (pages 37-40); (d) "Categorization of Dense Hydrocarbon Films;" J.C. Angus; E.M.R.S. Symposium Proc., 1987, Vol. 17, page 179; Amorphous Hydrogenated Carbon Films, XVII, June 2-5 1987, Edited by P.Koide & P. Oelhafen.

"Properties of Ion Beam Produced Diamondlike Carbon Films;" M.J. Mirtech; E.M.R.S. Symposium Proc., 1987, Vol. 17, page 377; (f) "Diamond-like Carbon - Properties and Applications;" J. Franks, K. Enke and A. Richardt; Metals & Materials (the Journal of the Institute of Metals); and U.S. Patent No. 4490229; M.J.Mirtich, J.S.Sorey & B.A.Banks.

The convenient source of the carbon is a hydrocarbon gas or vapour, especially one which is readily decomposed by an electric field or discharge. A very convenient source gas is acetylene, though others may be used if desired.

(e) 9 Individual hydrocarbons (or mixtures thereof) may be used, and diluent gases may be added if desired. The decomposition/deposition procedure may be carried out at pressures at atmospheric or above or below atmospheric, as found most suitable for particular instances.

The DLC coating may be made of a thickness which may be varied according to the particular requirements desired for the performance of the sensor and the system to be analysed. The thickness of the MC coating or deposit may be in the range 0. 01 to 5 um, but thicker or thinner coatings may be used if desired. A convenient thickness is in the range 0.5 to 5.0 microns, and preferably in the range 0.5 to 2.0 microns, is usually most suitable. As the thickness need not be very great, a typical and convenient coating deposit is one approximately 0.1 um thick, but this is not necessarily the optimum for all purposes. The thickness in any particular case will depend upon such factors as the nature (physical and chemical) of the material upon which the DLC is deposited, and its porosity or permeability, and the particular characteristics appropriate to the intended use of the sensor.

The coating is conveniently carried out at a rate which allows the deposit to adhere to the membrane material and form a coating of the desired thickness - preferably also evenly coated so as to cover substantially all the surface without leaving any areas too thinly covered or even uncovered.

When using acetylene as a source, for example, the deposition may be carried out at a rate of up to 0.5 um per hour, though higher or lower rates may be used if desired.

The mode of electrolytic analysis used to carry out the method of our invention is preferably amperometric analysis, which is well known and used in the art. In this, we find that problems arise because oxidised products formed from the ethanol, during the electrolytic oxidation which occurs during amperometric analysis, tend to adhere to the - 10 electrode (metal) surface and thereby impede further functioning of the electrode. This passivation effect can be overcome, however, by using the known technique of pulsed amperometric detection ("PAD").

In PAD analysis the waveform of the electrical potential applied is cycled continuously between positive and negative voltages, and the current flow is measured at a predetermined point in the cycle for a specified duration.

The average current generated during this specified measurement period is then plotted to give a response curve of current against time. This procedure has the advantage of cleaning the electrode (removing the troublesome oxidation products) and also giving more rapid and stable responses -- so making the resulting measurements easier to make and more reliable to use, for example in being more free from noise and interference. The voltages applied to the electrode in this procedure can be varied over a considerable range, for example between +0.7 and -0. 6 volts, and the measurements taken during made during the "specified duration" part of the cycle can be at a voltage appropriate for the measurement itself - conveniently at about 400 mV. Likewise, the duration of the measurement and the parts of the cycle may vary, but usually can be taken so that the whole cycle takes about 1 to 2 seconds. These voltages mentioned here are those made with reference to a standard silver/silver chloride (Ag/AgCl) electrode.

Description of the Pulsed Amperometric Detection ("PAD") techniques are to be found for example in the following published references (among others):- (a) J. Moracova, J. Stanek, J. Capkova & R. Lowcka; Rostlinna Vyroba, (1997), Vol. 43, 289-292. (b) J.I. Yu, W.G. Huang &: D. B. Hibbert; Electroanalysis, (1997), Vol. 19, 544-548. (C) D.J. Tarnowski & C. Korzeniewski; Anal.Chim.Acta. (1996), Vol. 332, 111-121. (d) W.K. Herber & R.S.R. Robinett; J.Chromatography A, (1994), Vol. 676, 287-295.

The pH of the sample liquid contacted with the sensor devices of this invention for examination can have a considerable effect on the magnitude of the response signal when the aminoacid polymer is not coated with DLC. Applying the coating of aminoacid polymer (e.g. poly lysine) overcomes this dependency upon pH and the DLC coating secures the aminoacid polymer layer and gives it some selectivity. the combination thus enables the sensor to be stabilisOd and have a much lower pH dependency that bare uncoated electrodes.

The principal advantage of our sensors is that they can be used in conditions of variable pH and be subjected to heat without their performance being destroyed. This enables them to be used for long periods of time and through repeated cycles of use/cleaning/sterilising/re-use, as is commonly done in commercial fermentation processes, especially in batch processes in which steam sterilisation is used between batches to avoid any contamination of the fermentation media.

Without a coating (especially of DLC), the aminoacid polymer layer tends to become loose and detached, wholly or partly, from the electrode surface when in contact with liquid media (e.g. solutions, samples, electrolytes, etc.) and so tends to be dislodged and fall off the electrode.

Applying a DLC coating effectively stops this very undesirable effect of loosening or detachment, and does so without any adverse effect of the electrode performance, so that the electrode performance can be improved.

The sensor devices and electrodes of our invention need not necessarily be made with a single electrode element, but can if desired be made in the form of a multiplicity of separate electrode elements. Such separate elements can be made small (as in a so-called micro-array or micro-electrode array), and may be connected electrically in any convenient way so that the signal output from them can be measured. For such arrays, the coating of DLC is especially useful, as it provides a much more viable, convenient and reliable way than a restraining membrane (e.g. of polycarbonate) for coating the aminoacid polymer to keep it in place.

An advantage of using multiple small electrode elements is that the surface of an array of them can be made to have various surface formations and configurations which can assist the construction and use of the device in practice.

Also, such arrays can allow use of higher current densities, which can decrease background noise. For example, the surface can be made in a pitted form (i.e. a form in which the surface has a multiplicity of recesses or "pits" which are small enough to hold the aminoacid polymer and any coating such as DLC). These recesses. or "pits" can then, if desired, be provided with means over or around their entrances (especially "guard electrodes" - conveniently in the form of small rings or conducting regions to which an appropriate voltage potential can be applied) to affect or control the entry of charged solutes into the recesses and so to the underlying electrodes.

Calibration of our sensor device for use can be carried out in conventional manner, preferably by immersion of the device in samples of the medium which is to be monitored or examined (e.g. fermentation liquor). An isotonic or other buf f er may be used, but it is pref erable to use one which has an ionic strength similar to to that of the media in which the device is to be used 0 ----- - ill-

Claims

WHAT WE CLAIM IS:-

1. Sensor device, useful for electrolytic analysis, which comprises a working electrode superimposed a coating of an aminoacid polymer upon which is a layer of material to maintain it in place.

2. Sensor device as claimed in Claim 1 wherein the material to maintain the aminoacid polymer layer in place is diamond-like carbon PIDLC11).

3. Sensor device as claimed in Claim 1 or Claim 2 wherein the electrode material is platinum.

4. Sensor device as claimed in any of Claims 1 to 3 wherein the aminoacid polymer is derived from an amino acid containing a secondary amino group as substituent, preferably an di-amino mono-carboxylic acid, for example lysine or ornithine and especially L-lysine.

5. Sensor device as claimed in any of Claims 1 to 4 wherein the aminoacid polymer is on the support electrode (especially platinum) as a thin layer, preferably formed by dip-coating with a solution of the aminoacid polymer.

6. Sensor device as claimed in any of Claims 1 to 5 wherein the aminoacid polymer, after application to the electrode, is stabilised in place, for example by drying and heating, e.g. to a temperature of about 50 to 100 degrees C.

7. Sensor device as claimed in any of Claims 1 to 6 wherein the aminoacid polymer has a molecular weight of at least 5000 and especially 100,000 or more.

8. Sensor device as claimed in any of Claims 1 to 7 wherein the DLC coating is of a thickness in the range 0.01 to 5 um, for example in the range 0.5 to 5.0 microns, and preferably in the range 0.5 to 2.0 microns.

9. Sensor device as claimed in any of Claims 1 to 8 wherein the layer of aminoacid polymer is of a thickness in the range 10 to 100 microns (um).

10. Sensor device containing a layer of an aminoacid polymer substantially as described.

11. method for electrolytic analysis of a liquid medium which comprises contacting the said liquid medium with a Ak- sensor device as claimed in any of Claims 1 to 10.

12. Method of electrolytic analysis which comprises using a working electrode carrying a coating of an aminoacid polymer upon which is superimposed a layer of material to maintain it in place, as defined in any of Claims 1 to 10.

12. Method as claimed in Claim 11 or Claim 12 wherein the material superimposed on the layer of aminoacid polymer to maintain it in place is diamond-like carbon ("DLCII).

13. Method of electrolytic analysis as claimed in any of Claims 10 to 12 wherein the analyte sought is ethanol.

14. Method as claimed in Claim 13 as applied to monitoring, measurement and assessment of media in which ethanol is being formed or produced, for example fermentation media, or for monitoring and controlling the progress of fermentation processes.

15. Method as claimed in any of Claims 10 to 14 wherein the mode of analysis used is amperometric analysis.

16. Method of analysis as claimed in Claim 15 wherein the analysis is carried out by pulsed amperometric detection ("PAD").

17. Method as claimed in any of Claims 10 to 16 wherein the sensor devices and electrodes of our invention are made using a multiplicity of separate electrode elements, for example as a multi-electrode array or micro-array.

18. Method as claimed in any of Claims 10 17 wherein the electrode surface is made in a pitted form (i.e. a form in which the surface has a multiplicity of recesses or "pits") small enough to hold the aminoacid polymer, especially with means over or around the entrances of such recesses or "pits" to affect or control the entry of charged solutes into the recesses and so to the underlying electrodes, especially "guard electrodes" - conveniently in the form of small rings or conducting regions to which an appropriate voltage potential can be applied.

19. Method of electrolytic analysis using a working electrode coated with an aminoacid polymer and a material to maintain it in place, substantially as described.