This invention relates to sensor devices such as are used in the determination of a component or components which may be present in a fluid sample, such as a physiological fluid (e.g. blood) or other fluids of biological origin (e.g. fruit), process fluids or effluents.

Many forms of sensor have been proposed, and commonly these rely on some form of membrane to control the extent to which the components present in a sample under examination can gain access to an electrode, at which they can then be detected and determined. Especially, it is well known to make sensors using membranes to separate the media being analysed from the active electrode itself. The main function of the membrane is to separate, as far as possible, those components which are desirable (i.e. can take part in the reactions at the electrode on which the desired determina ion depends) from interferents (i.e. compounds which may be present but are undesirable because they either interfere with the progress of the desired determination reactions or take part in reactions of their own which compete with those of the component sought and distort or overwhelm the signals which are to be measured. The forms of construction have much in common with each other, and mainly differ in the nature of the membrane or media within it or combined with it in some way.

Some forms of sensor rely on the components used to make the membrane, while others rely on the mode of fabrication of the membrane, selec ing its physical properties (for example its porosity) or treatments given to it, as these factors can control its effectiveness and selectivity in use.

Other forms of sensors incorporate an enzyme, which converts one substrate compound or analyte into another which may then be more easily measured. Especially, it is known to use oxidase enzymes, which generate hydrogen peroxide — a substance which can be measured very conveniently and very accurately by electrolytic methods, especially amperometrically. An example is

European Patent No. 216577 (of ICI PIC) which specifies an enzyme electrode sensor with membrane barrier of low (>5%) porosity.

Materials which have been proposed for the fabrication of membranes for sensors include polyvinyl chloride (PVC), but only in plasticised form, as described in European Patent Application No. 92302131.5, where the plasticiser performs a necessary part of the func ion of the membrane.

Such known sensors, utilising various membrane materials, are very valuable in analytical techniques and the analysis of biological fluids (e.g. blood) for the presence of substances considered critical for medical reasons — e.g. glucose and other materials which may be oxidisable or reducable, or have toxic properties.

We have now found that the selectivity of such a membrane- enclosed sensor can be significantly and surprisingly modified and improved by making the membrane of polyvinyl chloride (PVC) itself, in un-plasticised form.

Thus according to our invention we provide an improved sensor device comprising means for detecting components present in fluid samples and providing an output representative of the content of said component comprising a detecting means and a membrane barrier between the detecting means and the sample to be analysed, characterised in that the membrane barrier is composed of polyvinyl chloride (PVC) itself, in un-plasticised form. The detecting means is most conveniently of one of an electrochemical nature, but other types (e.g. spectrophotometric or optical detecting systems) may be used if desired. The detecting means will usually comprise an electrode system and a liquid or gel phase electrolyte-containing medium. In most applications the electrolyte will be aqueous (i.e. aqueous or aqueous-based) but the use of non-aqueous electrolyte media (for example organic-based media) is not excluded.

According to our invention we also provide a method for determining a component in a fluid sample, which comprises contacting, the sample with a sensor device as defined above.

Especially, in the devices and method of our invention, the

sensor device comprises a detect ing means in contact with an electrolyte medium and both associated with a membrane of polyvinyl chloride (PVC) itself , in un-plast icised form , which provides an interface for contact with a sample to be analysed and interposed between the active electrode (anode) of the cel l used as detector . Alternative forms of construction may have (a) both the electrodes of the detecting means (cel l ) enclosed within the PVC membrane (so that both are separated from the sample) or (b) only the anode of the detecting means (cell) enclosed within the PVC membrane (so that the PVC membrane i s between the electrodes and the cathode is not separated from the sample) . Of these, the former is very convenient and compact , but the latter is more simple and is made practicable by the fact that PVC in un-plasticised form is less insulating in its properties , and H ⁺ ions can pass through it.

The sensor device of our invent ion can have a single membrane or , if desired , mult iple layers of membrane material . When multiple layers of membrane are used, these may be the same or different . The preference for the position to be occupied by the un-plasticised PVC membrane differs to some degree according to the particular use to which the sensor is being applied and what substrate compound is to be detected by the sensor .

The governing factor is mainly the fact that un-plas icised PVC is permeable to hydrogen peroxide and some low molecular weight species (for example oxalate) but impermeable to sugars , for example glucose . Thus , when the sensor is to be used for detect ing a species to which the PVC is impermeable , a sensor assembly is best made with an enzyme (with which the desired substrate can interact and generate a species which can pass through the un-plast ici sed PVC) situated outside the un- plast icised PVC membrane , so that the desired species in the sample under examinat ion can make contact with the enzyme — either directly or indirect ly , through another selective membrane layer — and thereby generate a species to which the un- plasticised PVC membrane is permeable . Especial ly , when glucose is the species sought , an oxidase enzyme can be used and the

hydrogen peroxide thus generated can pass through the un- plasticised membrane and be determined at the detector means.

When mul iple membrane layers are used, any membrane layer or layers other than any comprised of un-plasticised PVC may be made of any of the wide variety of materials known in the art . Examples of these include dialysis membranes, and in general are preferably non-diffusion limi ing membranes, at least to the extent that they do not limit diffusion and passage of desired species towards the detecting means. When un-plasticised PVC is used as in inner membrane (i.e. a layer which is not the outermost), then one or more outer layers may be used which are of material which protects the sensor assembly in a mechanical manner (e.g. from mechanical damage) or in a chemical or any other manner considered appropriate for the use to which it is to be applied. Thus for example, there may be used an outer layer comprising a polycarbonate (especially in a porous form).

The active electrode may be any of those known in the art, for example a metal electrode, but especially a platinum anode. This is most conveniently made in combination with a silver/silver chloride counter electrode, as for example in the so-called Clark electrode, which comprises a platinum electrode surrounded by a silver/silver chloride ring.

The PVC (polyvinyl chloride) may be any polymer of vinyl chloride, as for example those made and available commercially, but should be free from any added plasticiser (an ingredient which is often present in some commercial products intended for uses such as moulding). Such "un-plasticised" PVC polymers are readily obtainable in commerce, however, and it is necessary only for the quality and purity of any polymer to be checked, whether by its specification or labelling. The molecular weight of the PVC is relatively non-critical, and most commercial grades will be satisfactory in use. A typical molecular weight is in the range 10,000 to 200,000, but others may be used if desired.

The material (i.e. the un-plas icised polyvinyl chloride) may be made into membranes by any conventional method. Most conveniently, this can be done by solution casting techniques,

using solvents to dissolve the polymer and then spreading the solution on a plate or flat surface and allowing the solvent to evaporate. A convenient solvent is tetrahydrofuran (THF) , but other solvents or mixtures of solvents which are known to be able to dissolve PVC be used if desired.

The thickness of the membranes can be of the order already used conven ionally in the art, but may be varied as found most appropriate having regard for the particular mixed polymer composition being used and the conditions under which it is to be used. Thus a convenient thickness is in the range 10 to 40 ιm, though larger or smaller thicknesses can be used if desired.

There are two principal forms of construction which may be used to secure the advantages of the new membrane material we now propose. In one, the un-plas icised PVC is the outer membrane, and in the other it is the inner membrane.

The forms using an enzyme will have basically the construction sequences:-

(A) OR (B)

Sample Sample

Membrane (e.g. Polycarbonate) Membrane (Un-plasticised PVC)

enzyme enzyme

Inner (Un-plasticised PVC) Inner (e.g. polycarbonate) membrane membrane

Electrode. Electrode.

For these, the components (apart from the un-plasticised PVC membrane) are mainly the conventional ones, and the many variants known in the art may be used.

Un-plasticised PVC is not permeable to glucose, so direct electrochemical detection of glucose cannot be achieved through this material as membrane. However, it is permeable to hydrogen peroxide, so enabling an alternative glucose-permeable membrane

and an oxidase enzyme to be used to produce hydrogen peroxide, which then can selectively permeate the un-plasticised PVC membrane.

This un-plasticised PVC membrane has a particular advantage over plasticised PVC in that it is (unlike the plasticised form) impermeable to paracetamol — so that this compound is excluded from interfering with hydrogen peroxide detection, as well as ascorbate/urate. This selectivity against paracetamol and the like while in favour of hydrogen peroxide is a valuable property which is not easily found and it very useful in clinical and related analytical, diagnostic and monitoring work.

Consequently, any other sensor device or system using an oxidase enzyme to generate hydrogen peroxide can, with advantage, incorporate an un-plasticised PVC membrane as part of its construction, especially as an inner membrane near to the active electrode surface.

Another advantage is that sensor devices of the electrolytic (amperometric) type are often used for examination of biological fluids for example blood, urine, and the like. It happens that urine contains a number of interferents which — even though their identity has not yet been established — have the ability to permeate many of the known conventional membrane materials, for example plasticised PVC, and so interfere severely with the accurate estimation of components such as hydrogen peroxide and of compounds which can be used to generate it . The un- plasticised form of PVC does not allow these unidentified components to pass through, is it constitutes an effective barrier to them and facilitates the wider applicability and accuracy of measurement. Thus, for many applications the un- plasticised PVC membrane will be preferable, though it will not always be so in applications in which it is desired to have a phenolic compound permea ing the membrane.

Another use for the un-plasticised PVC membrane is in the construction of a sensor device for the determination of oxalate. It acts in this in two ways, both of which are valuable. One is a form of sensor in which the un-plasticised PVC membrane is used

as an inner membrane beneath a layer or membrane comprising an oxalate oxidase system, so that hydrogen peroxide is generated from the sample and the resulting hydrogen peroxide passes through the un-plasticised PVC membrane; this relies on its action as a membrane selectively permeable to hydrogen peroxide.

Alternatively, the un-plasticised PVC membrane can be used as an outer layer with the means for oxalate detection beneath it; this relies on its unexpected ability to act as a membrane selectively permeable to oxalate. Un-plas icised PVC is the only material that we know of at present which is oxalate-selective, and the mechanism by which it performs in this way is still not clear to us. In this mode or embodiment of our invention, the means for detecting the oxalate which has passed through the un- plasticised PVC membrane may be any of those known and may be either an enzyme one (oxalate oxidase based) or one which detects and measures the oxalate directly by electrolytic action. This may be amperometric, and the pH of the medium around the active electrode may be adjusted as may be found most appropriate by simple trial. Whatever the mechanism by which the permeability to oxalate takes place, the ability of an un-plasticised PVC membrane to act so as to exclude serum and urine interferents from the active electrode region of a sensor is true and highly effective and a valuable feature of this invention. An example of a basic construction sequence utilising this un-plasticised PVC membrane can be represented as follows:-

(1) SAMPLE

(2) ==-=--_===-=---= un-plasticised PVC membrane

(3) ENZYME (Oxidase) (4) ϊBBsssssss inner membrane

(5) ELECTRODE.

In this , oxalate or oxal ic acid from sample (1 ) passes through the un-plasticised PVC membrane (2) and then, by contact with the enzyme (3) , generates hydrogen peroxide and carbon dioxide . The hydrogen peroxide then passes through the inner membrane (4) to the electrode (5) , at which it is determined

electrolytically.

The inner membrane (4) may be of any convenient material — for example a. dialysis membrane or a polycarbonate membrane — intended to perform such actions as regulation of the flow of components to electrode (5) (should this be considered desirable) or to provide mechanical protection and add to the robustness of the sensor assembly and minimise risk of damage to the innermost electrode elements.

Thus according to our invention we also provide, as useful new products, membranes comprising un-plasticised polyvinyl chloride. Especially, we provide these in the form of multi¬ layer membrane products, in which at least one layer is formed of un-plasticised polyvinyl chloride and is combined with one or more layers of other materials of appropriate properties to enhance the properties of the un-plasticised polyvinyl chloride itself. Such materials may be of appropriate permeability to regulate the access of components before or after passage through the un-plasticised polyvinyl chloride, and/or of a physical form or strength which protects the un-plasticised polyvinyl chloride from damage or provides it with any desired degree of stability of shape or positioning in use.

Especially, we provide membranes of un-plasticised polyvinyl chloride incorporating an enzyme, for example an oxidase. In these, the enzyme may be immobilised by the chemical means known in the art, or they may be held between the layers of a multi¬ layer structure.