WO2000054039A1

WO2000054039A1 - Selective polymer material

Info

Publication number: WO2000054039A1
Application number: PCT/GB2000/000874
Authority: WO
Inventors: Elizabeth Anne Howlett Hall
Original assignee: Elizabeth Anne Howlett Hall
Priority date: 1999-03-10
Filing date: 2000-03-10
Publication date: 2000-09-14
Also published as: GB9905442D0; AU2933200A

Abstract

The invention relates to a selective polymer material for use in measuring the concentration of a substance in a solution characterised in that the material is a thermoplastic polymer with an acrylate backbone and a plurality of pendant lipophilic plasticising groups, for example C3-7alkyl groups, to provide the polymer with a Tg of -10 °C or less and in that it contains a moiety selective for the substance to be measured chemically bonded to the poolymer or physically entrapped therein. The polymer material can be formed into membranes for use in selective electrodes or optodes.

Description

SELECTIVE POLYMER MATERIAL

The present invention relates to a selective polymer material suitable for use in measuring the concentration of a substance in solution The invention also relates to selective membranes made of the polymer material and to electrodes and optodes incorporating such membranes

There is a general requirement in the field of analytical and clinical chemistry to measure the concentration of a particular ion in a solution which may contain that ion together with other ions. For example, in the field of clinical chemistry there is a requirement for analyzers capable of measuπng ions such as Na⁺, K⁺ , Ca^" or H⁺ (pH) in solutions such as whole blood, serum and plasma Potentiometπc measurements can be used for this purpose employing ion-selective electrodes, ι e electrodes which respond preferentially or exclusively to one particular ion in a solution

Potentiometπc measurements determine the difference in electrical potential between two electrodes in contact with a liquid, the electrodes and the liquid forming an electrochemical cell. If one of the electrodes (the indicator electrode) is selective for a particular ion, then the half-cell potential of the indicator electrode will vary with the activity of that ion in the solution Furthermore, if the half cell potential of the other electrode (the reference electrode) remains essentially constant, then the electrical potential difference across the electrodes will be proportional to the logarithm of the activity in the solution of the ion for which the indicator electrode is selective, the precise relationship being defined by the Nernst equation.

Ion-selective electrodes include the glass electrodes conventionally used for the determination of pH where the glass is permeable to hydrogen ions but not to other ions Ion-selective electrodes of more general applicability can be made by providing the electrode with an ion-selective membrane to be placed between the electrode and the test solution Such ion-selective membranes are generally composed of a thermoplastic polymer and an ion-selective compound (otherwise referred to as an lonophore) Typically, the thermoplastic polymer is composed of a polymer together with a relatively large amount (up to 60% or more) of a plasticiser. The presence of the plasticiser represents a significant problem and restricts the applicability of the ion-selective membrane More particularly, the plasticiser had a tendency to leach out of the membrane leading to contamination of the environment in which the membrane is placed and, for example, the possibility of leaching of the plasticiser prevents electrodes with such membranes being used in a way which involves their being implanted into patients. Leaching of the plasticiser also leads to loss of the ion-selective function of the membrane since the lonophore tends to be dissolved in the plasticiser so that lonophore is lost alongside the plasticiser Leaching can also reduce the shelf life of a product including such a membrane since it can lead to the appearance of a "hue" on the surface of the membrane

The present invention seeks to overcome the problems associated with the need to use large amounts of plasticiser in the formulation of selective membranes, particularly lon- selective membranes.

According to one aspect the present invention provides a selective polymer material for use in measuring the concentration of a substance in a solution characterised in that the mateπal is a thermoplastic polymer with an acrylate backbone and a plurality of pendant lipophilic plasticising groups to provide the polymer with a Tg of - 10°C or less and in that it contains a moiety selective for the substance to be measured chemically bonded to the polymer or physically entrapped therein

According to another aspect the present invention provides a membrane for use in measuπng the concentration ot a substance in a solution, which membrane is selectively permeable to the substance to be measured, the membrane being formed of a selective polymer material as defined above.

According to yet another aspect, the present invention provides a selective electrode for use in measuπng the concentration of an ion in solution comprising a reference electrode and a polymer membrane which is selectively permeable to the substance to be measured, characterised in that the membrane is formulated from a thermoplastic polymer w ith an acrylate backbone and a plurality of pendant lipophilic plasticising groups to provide the polymer with a Tg of -10°C or less and in that it contains a lonophore selective for the substance to be measured chemically bonded to the polymer or physically entrapped therein.

The membrane according to the invention can also contain a chromoionophore and can be incorporated into an optode According to a further aspect, the present invention provides a selective optode for use in measuπng the concentration of a substance in solution comprising a polymer membrane which contains a chromoionophore and is selectively permeable to the substance to be measured, characterised in that the membrane is formulated from a thermoplastic polymer having an acrylate backbone and a plurality of pendant lipophilic plasticising groups to provide the polymer with a Tg of -10°C or less

The Tg of the polymer can be measured directly on the polymer using any suitable apparatus. Preferably polymer Tg lies in the range from -10°C to -70°C. more preferably from -30 °C to -60° C.

Preferably the lipophilic plasticising groups are C_{3 7} alkyl groups Use of C, ₇alkyl acrylates in the polymer means that the polymer is inherently soft and does not require added plasticiser, i.e. the polymer s in effect self-plasticising, so that the problem of leaching of the plasticiser does not arise. In addition, as explained in more detail below, the polymer shows considerable advantages in terms of production, versatility and incorporation of the moiety selective for the substance to be measured, preferably an lonophore.

In general the polymer used according to the invention has an acrylate backbone and is a polymer or copolymer of one or more of the following monomers propyl acrylate, butyl acrylate, pentyl acrylate, hexyl acrylate, heptyl acrylate. Subject to the requirement for Tg, the polymer may be a homopolymer or may be a co-polymer including two or more different monomer units. The different monomer units may be derived from C₃.₇alkyl acrylates as described above or the polymer may include a minor proportion of other monomer units, for example up to 10% by weight of monomer units derived from lower (C, or C₂) alkyl acrylates or methacrylates, for example methyl methacrylate.

In the case of C₆ or C₇ alkyl acrylates, it may be advantageous to use this monomer in admixture with a lower, for example C₃ or C₄, alkyl acrylate. Thus, for example, good films have been obtained using a mixture of 70% heptyl acrylate and 30% butyl acrylate. In general, straight chain alkyl acrylates are preferred. Branched chain alkyl acrylates or a- or 3-substituted monomers tend to produce a polymer of higher Tg than polymers produced from the corresponding straight chain or non-substituted monomer. If a- or /3-substituted monomers are used, for example to introduce functionality which allows attachment of a reagent to the polymer in the manner described in more detail below, then these monomers may be found to be less reactive than the corresponding unsubstituted monomer.

The properties of the polymer can be adjusted by including minor amounts of other monomers. Thus, it may be desirable to adjust the hydrophobic/lipophobic balance (HLB) of the polymer so as to be compatible with a particular ionophore or other analyte receptor which it is desired to incorporate into the polymer, this being something which it is very difficult to achieve with the prior art combination of PVC and a plasticiser. HLB can be adjusted by including monomers containing hydroxyl groups, for example hydroxyethylmethacrylate. The properties of a film made from the polymer can be altered as a result, for example, of incorporation of the ionophore and the mechanical properties of the film can be adjusted to compensate for such effects. Thus the film should not contain cracks or pinholes and difunctional or polyfunctional monomers which act as cross-linking agents can be included to add strength so that the film is soft and elastic but still tough. An example of a difunctional cross-linking agent is hexanedioldiacrylate.

The polarity of the polymer is also important in determining the performance of the membrane. For monovalent ion selective membranes with a high polarity, the migration of the counteπon into the membrane will be significant and will lead to a sub-Nernstian response whereas for a divalent ion co-transfer of a counteπon is an advantage to maintain neutrality and ideal response If the membrane is too non-polar then there may be a tendency to a super-Nernstian response Polarity can also influence selectively for mono and divalent ions Monomers such as alkyl (e.g. methyl or higher alkyl) cyanoacrylates can be used to introduce cyano functionality into the polymer thereby adjusting the polarity of the membrane

The lonophore may be incorporated into the monomer mixture used to produce the polymer and thus becomes trapped in the polymer matrix Alternatively the ionophore may be secured chemically by grafting to the polymer backbone In general, the more hydrophihc the polymer, the more the ionophore will be subject to leaching in which case it is especially advantageous to graft the ionophore to the polymer backbone

Where the ionophore is to be grafted to the polymer backbone, a co-monomer containing a suitable reactive group will need to be included in an appropriate amount when the polymer is prepared and the ionophore is grafted to the polymer backbone as a subsequent step. The nature of the reactive group will depend on the ionophore and examples include carboxyl groups (for example from acrylic or methacryhc acid), acid chlonde (for example from acryol chloride or methacryol chloπde) or epoxide groups (for example from glycidyl methacrylate) Cyano groups or ammo groups (from alkyl cyanoacrylate or alkyl aminoacrylate monomers respectively) may also be suitable as reactive groups for grafting. It is. however, necessary to ensure that the reactive groups do not have an adverse effect on the polymer film and, for example, whilst hydroxy groups may be suitable for grafting, they may also increase water uptake of the polymer to an extent that becomes unacceptable

If necessary, the ionophore itself can be provided with a suitable coupling group so that it can be grafted into the polymer directly as a monomer unit or via reaction with the reactive group introduced into the polymer backbone For example, acryloylamidobenzo (AAB) is a hgand which can be attached to the ionophore to allow it to be grafted directly into the polymer as a monomer unit and this has the advantage that the ionophore can be grafted into the polymer during the polymerisation reaction. Alternatively, a hydroxyl group can be introduced into the ionophore for reaction with an acid chloride group in the polymer or an amino group can be introduced into the ionophore for reaction with an epoxide or carboxyl group in the polymer (or vice versa). However, introducing a group into the ionophore for subsequent reaction with a reactive group in the polymer involves one or more additional steps so that the use of a ligand such as AAB is prefeπed.

Selection of the reagents provides great versatility in manipulating and fine-tuning the polymer to make this compatible with the ionophore and/or other reagents in the system and this is illustrated in more detail in the examples set out below.

In principle, the present invention is applicable to any ionophore or chromoionophore and the nature of the ionophore or chromoionophore will determine the ion or other compound for which the membrane is selective. The polymer and the ionophore or chromoionophore must be compatible but, as already indicated, there is considerable scope according to the invention for adjusting the polymer for compatibility with the ionophore or chromoionophore.

Essentially, there are three classes of ionophore commonly used at the present time for ion-selective electrodes, these being as follows:

a) neutral ionophores which are mostly antibiotics;

b) crown ethers;

c) neutral ligands.

Examples of antibiotics include valinomycin (selective for K⁺), monensin decyl ester (selective for Na⁺) and nonactin (selective for NH₄ ⁺). Crown ethers will require a side chain for grafting or at least the possibility of adding a side chain which can be activated for grafting. However, ionophores with lipophilic "tails" are easier to entrap in the polymer where grafting is not desired There are many examples of neutral hgands including-

N,N-dιcyclohexyl-N' ,N'-dιoctadecyl-3-oxapentanedιamιde (selective for Ca²⁺), tπdecylamine (selective for H"), N,N,N',N'-tetracyclohexyl-(2-butyl-2-ethyltπmethylenedιoxy)dιacetamιde (selective for Lr); carbamate or thiocarbamate hgands (selective for Pb^{2^"} or Zn²⁺)

In general, the ion selective membrane will also contain a countenon for the ion whose activity is to be measured and this can improve performance Thus, in any ion-selective membrane, transfer of the ion (say a cation) into the polymer would lead to a non- equalisation of charge (so-called Donnan exclusion failure) Lipophilic ions in the sample which can move into the polymer can overcome this by co-transfer of these ions but this in turn leads to a sub-Nemstian response. In many prior art PVC preparations, there was sufficient ionic impuπty to act as an anion already present in the polymer to neutralise incoming charge but performance was nevertheless enhanced by additional ions The polymer mateπals according to the invention are purer with respect to ionic impuπties and will thus generally need added countenon Lipophilic ions are compatible with the polymer and do not create a leaching problem The lipophilic additive may also reduce membrane resistance thus shortening response time, eliminating noisy response and improving selectivity Suitable lipophilic cations include borates such as potassium tetrakιs(p-chlorophenyl borate)

The membrane according to the invention can be used in conjunction with a chromoionophore and in this case the membrane (optode) may be selective for analytes which are not ions For example, in the optical mode in conjunction with a chromoionophore, analytes such as hepaπn, penicillin, amines and alcohols can be analysed. The membrane can also be linked into another reagent layer (for example enzyme(s)) which can indirectly transduce any number of analytes, so long as the output from the reagent layer produces a species which causes a potentiometπc (in the case of an electrode) or optical (in the case of an optode) signal. The membrane according to the invention can also be used as sensing element in any other transducer such as acoustic devices, surface plasmon resonance devices and piezoelectπc devices.

The polymer can be made by known methods for the preparation ot acrylate polymers. Polymerisation generally requires the presence of an initiator of free-radical polymerisation, for example a photoimtiator (UV) or a heat initiator UV initiators are prefeπed. Examples of heat initiators include 2.2'-azobιsιsobutryonιtnle, benzoyl peroxide and related compounds Examples of UV initiators include 2,2-dιmethoxy-2- phenylacetophenone, benzophenone, benzoyl peroxide and related compounds

The polymer can be formed m situ ad_jacent to the indicator electrode thereby reducing the number of processing steps and material costs (waste is reduced) For example, the monomer mixture, optionally in a suitable solvent, can be placed in the desired position and polymeπsed, for example where the monomer mixture includes a suitable initiator by use of UV-radiation. Adding the ionophore at the same time means that the 3 steps of polymer formation, ionophore grafting or entrapment and film deposition can take place simultaneously.

Alternatively, the polymer can be polymerised in the usual way, for example using photo cure or heat cure, as a sheet with the sheet then being cut to size and incorporated into the electrode.

Polymensation may be carried out in solution in a suitable solvent such as benzene, ethyl acetate or toluene. In many cases little or no solvent is required. In addition, it may not be necessary to isolate the polymer from the polymerisation mixture so that it can be applied directly as desired for example by spin coating, inkjet or screen printing

The electrode can be any type of reference electrode the essential criterion being that it should give a constant potential in the presence of a sample. Ag/AgCl electrodes are preferred. Because of the ease with which the ion-selective membranes can be constructed, a planar thick film construction can be envisaged for the sensor. The substrate can be. for example, polymer ceramic or glass and photopatterning allows a plurality of different sensor membranes to be positioned in a single substrate Only one reference is required for the whole test strip addressing each ion-selective electrode separately with respect to this reference Multiple ion arrays can theretore be envisaged.

The low cost of fabrication means that these arrays can be disposable tor routine clinical or environmental use and since they can be produced on flexible polymer substrates they may be ideal for insertion into blood vessels or other body spaces, tor example during surgery or as implants Measurement on samples which are not flat may also be an advantage and the only requirement is that a solution is also involved somewhere in the sample

The ion-selective membrane may also incorporate a hydrophihc membrane, for example of a polymer of hydroxyethylmethacrylate (polyhema) which provides an electrolyte membrane for the internal reference electrode, i e. it lies between the ion-selective electrode and the reference electrode (the bridge layer of the double junction reference) Polyhema is generally not suitable for the ion-selective membrane itself because of its rapid water uptake which could interfere with the ion-selective response However, by combining hema and another hydrophobic monomer, e.g. methyl acrylate or butyl acrylate, generally with 70% or more hema, it is possible to reduce the rapid permeation of water for the bridge layer and thus maintain the salt concentration in the bridge constant. This is a requirement for a stable solid state reference The internal polyhema layer is thus hydrated with salt solution and acts as an intermediate layer between the lon- selective membrane and the electrode, tor example the Ag/AgCl electrode Without this layer, the electrode interface is thermodynamically blocked and will sometimes drift unpredictably

The invention will now be described in more detail by reference to specific examples in which reference is made to the accompanying drawings brief description of the drawings is as follows-

Figure 1 shows performance of the electrode of Example 1. 1 Figure 2 shows performance of the electrode of Example 1.2. Figure 3 shows performance of the electrode of Example 1.3.

Figure 4 shows performance of the electrode of Example 1.4.

Figure 5 shows performance of the electrode of Example 1.5.

Figure 6 A and 6B show performance of the electrode of Example 2. 1. Figure 7 shows performance of the electrode of Example 2.2.

Figure 8 shows performance of the electrode of Example 2.3.

Figure 9 shows performance of the electrode of Example 2.4.

Figure 10 shows performance of the electrode of Example 2.5.

Figure 11 shows performance of the electrode of Example 2.6. Figure 12 shows performance of the electrode of Example 2.7.

Figures 13A to 13D show further aspects (described in Example 2.8) of the performance of the electrode of Example 2.7.

Figure 14 shows performance of the electrode of Example 2.9.

Figures 15A to 15D show further aspects (described in Example 2.10) of the performance of the electrode of Example 2.9.

Figures 16A and 16B show performance of the electrode of Example 2.11.

Figure 17 shows performance of the optode of Example 3 A.

Example 1 (Polymer Type I)

Typical Recipe n-butyl acrylate (BA) = 2.5g methylmethacrylate (MM A) = 0.3g 2,2'-azobisisobutyronitrile (AIBN) = 0.003g benzene = 3 ml ionophore (e.g. acrylolamidobenzo-15-crown-5 or 18-crown-6) = O. lg measured polymer Tg = -29.4°C.

Procedure

A: Polymerisation

The monomers, initiator and ionophore are mixed slowly with the solvent (benzene) and degassed with nitrogen for 10 minutes The mixture is heated with stirring to 1 10°C for 1 hour at which temperature it slowly thickens The temperature is then lowered to about 70°C which temperature is maintained for a further 6 hours to complete the reaction.

B. Isolation

About 5ml of benzene is added to the cooled, sticky, clear, semi-solid and the mixture stiπed vigerously 1ml of the clear sticky liquid produced is added to 100ml of petroleum ether (either 60°C or 80°C boiling fraction) with vigorous stirring until the solvent becomes clear and a stick, glue like solid is obtained The solvent is poured off and the solid washed with a further 20ml of petroleum ether The procedure is repeated with the remainder of the polymer solution until all of the polymer has been precipitated.

C. Purification

All of the precipitated polymer is redissolved in about 10ml of 1 ,4-dιoxane 100ml of distilled water is added to about 2ml of polymer solution and a white sticky polymer appears. The procedure is repeated for the remainder of the polymer solution and all of the polymer portions are mixed and dried in a freeze drier for 2 hours. The whole of the polymer purification procedure as described above is repeated and the pure polymer is then dned in a freeze drier under a vacuum of 10 ² atmospheres for 24 hours. The final appearance of the polymer is usually a colourless, clear, elastic and sticky solid although on occasions it may appear slightly translucent. Yield 50 to 70%

Possible Modifications

The following modifications may be made in the procedure described above as appropriate:

1. The solvent benzene (which is relatively toxic) may be replaced by ethyl acetate;

2. petroleum ether may be replaced by n-hexane;

3. Both the isolation and the purification steps may be omitted in which case the unpuπfied polymer mixture is diluted to the required concentration tor direct film deposition.

Fabrication of Membrane for Potassium Selective Electrode A cocktail of polymer type I (about 0.2g of the polymer) containing the appropriate lipophilic anion (for details see later) is prepared by dissolving the various components in about 1ml of dichloromethane. The cocktail is then poured onto a glass ring (QUICKFIT adapter) rested on a glass slide. The top of the ring is covered with a piece of filter paper and a heavy weight on top presses the paper against the ring. After 24 hours of evaporation in a fume cupboard, the whole is immersed in water for 2 hours following which it is possible to remove the membrane from the glass slide and the membrane is ready for testing.

Fabrication of Solid-State Ion-Selective Electrode Using Polymer Type I

A. Deposition of Polyhydroxyethylmethacrylate (Polyhema) Layer

About 0.02g of polyhema is dissolved in 400μl of a water/ 1 ,4-dioxane mixture (37% water). The surface of an Ag/AgCl button is cleaned with absolute ethanol and lOOμl of the polyhema solution is deposited on the button using a Gilson pipet and left to evaporate for 24 hours in a cold room (temperature 4°C). After drying, the electrode is wire bonded with silver epoxy and encapsulated with ARALDITE epoxy. The polyhema layer is hydrated with a drop of 0. 1M KC1 solution for 2 hours before the ion- selective menbrane is deposited.

B. Deposition of Polymer Type I as Potassium Selective Membrane Polymer Type I (about 0.02 - 0.04g) and the lipophilic anion (see later) are dissolved in 100μl of dichloromethane. Once a homogeneous solution is obtained, the cocktail is deposited on the hydrated polyhema layer using a Gilson pipette. Slow evaporation of the solvent is allowed to occur in a cold room (temperature 4°C). After 24 hours drying, the solid-state potassium sensitive electrode is conditioned in 0.01M KC1 for 2 hours before potentiometric evaluation is carried out.

Results

1. Polymer type I with immobilised potassium ionophore 18C6

Membrane recipe: Non-plasticised polymer = 0. 15g (BA = 90 wt% , MMA = 6.4 wt%)

Immobilised 18-crown-6 = 1.9 wt%

Potassium tetrakis(p-chlorophenyl borate) = 1.7 wt% (60 mol%) measured polymer Tg < -29.4 °C. Performance of the electrode is shown in Figure 1.

Sensitivity = 58mV/decade

Linear range = 0.01 - lOOmM

Detection limit = about 0.005 mM

Selectivity (Separate Solution method): Cation Selectivity Coefficient

Li -3.5

Ca -3.8

Mg -4.5

Na -1.6 NH₄- -1.2

2. Polymer type I with immobilised potassium ionophore 15C5

Membrane recipe:

Non-plasticised polymer = 0.25g (BA = 88.6 wt% , MMA = 7 wt%) Immobilised 15-crown-5 = 3.8 wt%

Potassium tetrakis(p-chlorophenyl borate) = 0.6 wt% (10 mol%) measured polymer Tg = -20.9°C.

Performance of the electrode is shown in Figure 2.

Sensitivity = 57.6mV/decade Linear range = 0.01 - lOOmM

Detection limit = about 0.005 mM

Selectivity (Separation Solution method):

Cation Selectivity Coefficient

Li -3.7 Ca -4. 1

Mg -4. 1

Na -1.7 NH₄ ⁺ -1.4

3. Potassium ion-selective solid-state electrode based on polymer type I with immobilised potassium ionophore 15C5 evaluated under long term dry storage conditions

Membrane composition (drop coat technique):

Polyhema = 5.7 mg (conditioned in KC1 0. 1M for 3 hours)

Polymer = 24.4mg (BA = 91.2 wt% , MMA = 6.2 wt%)

Immobilised 15-crown-5 = 3.5 wt% Lipophilic anion = 0. 12 wt% (3 mol %) (potassium tetrakis ( ?-chlorophenyl)borate) measured polymer Tg = -20.9°C.

Performance of the electrode is shown in Figure 3 ( ■ = day 1 , α = day 3, ^♦ = day 8,

O = day 25).

Changes in selectivity coefficients and sensitivity are as follows: Day 1 Day 3 Day 8 Day 25

Li -3.3 -2.7 -2.2 -1.9

Ca -4.5 -4.3 -3.4 -3. 1

Mg -4.3 -4.2 -3.3 -3.2

Na -2. 1 -1.8 - 1.4 - 1.2

NH₄ ⁺ -1.6 - 1.5 -1.2 - 1

Slope (mV/dec) 57.2 60.5 49.8 52

SD 1. 1 0.8 1.2 1.6

4. Potassium ion-selective solid-state electrode based on polymer type I with immobilised potassium ionophore 18C6 evaluated under long term dry storage conditions

Membrane composition (drop coat technique): Polyhema = 6.3 mg (conditioned in KC1 0. 1M for 3 hours) Polymer = 42.7mg (BA = 91 wt% , MMA = 7 wt%) Immobilised 18-crown-6 = 1.8 wt%

Lipophilic anion = 0.23 wt% (10 mol%) (potassium tetrakis ( -chlorophenyl borate) measured polymer Tg = -30. 1 °C. Performance of the electrode is shown in Figure 4 (■ = day 1 , □ = day 2, ♦ = day 5, 0 = day 11).

Changes in selectivity coefficients and sensitivity

Day 1 Day 2 Day 5 Day 1 Sensitivity (mV/dec) 55.6 55.6 53.6 47 SD 0.4 0.6 1.5 1 r2 0.9998 0.9996 0.998 0.999

Selectivity coefficient Li⁺ -3.2 -3.2 -3.2 Ca²⁺ -4.1 -4.2 -3.7

Mg^2* -4.5 -4.3 -4

Na⁺ -1.6 -1.8 - 1 .5

NH₄- -1.2 - 1.3 - 1 . 1

5. Potassium ion-selective solid-state electrode based on polymer type I with entrapped valinomycin evaluated under long term dry storage conditions

Membrane recipie (drop coat technique):

Polyhema = 5.7 mg (conditioned in KC1 0. 1 M for 3 hours)

Polymer = 31mg (BA = 91 wt% , MMA = 6.6 wt%) Entrapped valinomycin = 1.9 wt%

Potassium tetrakis(p-chlorophenyl borate) = 0.5 wt% (53 mol %) measured polymer Tg < -29.4°C.

Performance of the electrode is shown in Figure 5 (■ = day 19, □ = day 8, ♦ = day

16, 0 = day 22). Changes in selectivity coefficients and sensitivity

Day 8 Day 16 Day 19 Day 22

Sensitivity (mV/dec) 60. 1 57.6 61.7 56.5

SD 0.7 0.9 1 2 r2 0.998 0.999 0.996 0.991

Selectivity coefficient

Li⁺ -4.1 -3.5 -3.4

Ca ⁺ -4.6 -4.2 -4 Mg²⁺ --44..55 --44..22 -4.

Na⁺ --33..88 --33..66 -3.4

NH₄- --11..88 --11..66 -1.8

Special Features/Advantages

The above ion-selective electrodes based on polymer type I show the following special features/advantages:

• use of non-plasticised polymer to form the ion-selective membrane

• ionophores such as crown ethers can be grafted onto the polymer backbone • isolation of the polymer from the polymerisation mixture may not be required so that the film can be deposited directly for example by spin coating or ink jet/screen printing.

Example 2 (Polymer Type II)

Typical Recipe

Inner layer: hydroxyethyl methacrylate = 3mg

2,2-dimethoxy phenylacetophenone = 1 wt% Ion-selective film: n-butyl acrylate = 90 wt% hexanedioldiacrylate = 0. 1 - 0.3 wt%

2,2-dimethoxy phenylacetophenone = 1 .6% .

Measured polymer Tg = -44. 1 °C.

Amount of ionophore varies depending on the type of ionophore.

Amount of lipophilic additive varies depending on the ionophore.

Procedure

A. Fabrication of Polyhema Layer

The surface of a Ag/AgCl button is wiped clean of dust with absolute ethanol. A solution of hydroxyethyl methacrylate which contains 1 wt% of the photoinitiator 2,2- dimethoxy phenylacetophenone (DMPP) is prepared and 3mg of this solution is deposited on the button using a Gilson pipet. Without delay the button is placed in a UV exposure unit (RS) and the unit is flashed with a stream of nitrogen before the lamp is switched on for a duration of about 5 minutes with continued flashing with nitrogen during which time polymerisation occurs. At the end of 5 minutes, polymerisation is completed to form a hard, glassy, transparent polymer film. The UV unit is switched off and the polyhydroxymethacrylate (polyhema) film rinsed with a little absolute ethanol. The film is dried at room temperature for several minutes before a drop of salt solution (depending on the sensor type) is added on the film and 2 hours are allowed for hydration.

B. Recipies for ion-selective films

Depending on the type of ion sensing film, the monomer, ionophore and lipophilic additive used to prepare the ion-selective film can vary both in type and concentration as follows: Monomers: n-butyl acrylate methyl methacrylate n-heptyl acrylate cyanoethylacrylate

Ionophores: potassium: acrylamidobenzol 5-crown-5

(requires THF/benzene as solvent) valimomycin sodium: bιs[(12-crown-4)methy.]dodecylmethylmalonate (Sodium ionophore VI) hydrogen: tπdodecylamine (Hydrogen ionophore I) calcium: N.N-dicyclohexyl-N'N'-dioctadecyl-S-oxapentamide (Calcium ionophore IV) lithium: 6,6-dibenzyl-14-crown-4

(Lithium lonophore VI) Additives: potassium tetrakιs( -chlorophenyl)borate sodium tetrakιs(tπfluoromethylphenyl)borate.

In general, lipophilic ionophores appear to be more compatible with the n-butyl acrylate employed (good solubility in the monomer). The amount of cross-linkers and photoinitiator needed also depends on the monomer used. Heptyl acrylate which yields a very soft polymer requires more cross-linkers to strengthen the film. Exposure time is affected by the monomers and cross-linkers used as well as the solvent and photoinitiator. Details of the recipies for various photocured ion-selective films are given below.

C. Fabrication of Ion-Selective Film

A cocktail which contains the monomer or monomers, cross-linker hexanedioldiacrylate, photoinitiator DMPP, ionophore and lipophilic additive is prepared and all of the components should form a homogeneous solution. About lOμl of this solution is drop coated on top of the hydrated polyhema layer with a Gilson pipet. The button is then exposed to UV radiation in an UV exposure unit for about 6 minutes (for most cross- linked acrylate films) under continuous purging with nitrogen gas. The film formed is usually clear, soft and tacky but elastic. The surface of the film is rinsed with several drops of pertoleum ether to remove unreacted substances.

The solid state electrode is ready for testing after encapsulation with ARALDITE epoxy or fixed on to a static cell with an O-ring seal.

Possible Modifications

Alternative initiators can be used including benzoyl peroxide and benzophenone.

1. Potassium ion-selective solid-state electrode based on polymer type II with immobilised 15C5 evaluated under constant exposure of 0.1M KC1

Polyhema layer: hydroxyethyl methacrylate = 6mg 2,2-dimethoxy phenylactophenone = 1 wt%

Duration of UV irradiation = 10 minutes

Conditioned in KC1 for 2 hours

Composition of photocured membrane (total weight 26mg): n-butyl acrylate = 83.9 wt% methyl methacrylate = 12 wt%

AAB 15C5 = 2.5 wt% (in 90μl THF) potassium tertakis( -chlorophenyl borate) = 0.3 wt% (8.6 mole %) 2,2-dimethoxy phenylacetophenone = 0.9 wt% UV exposure time = 40 minutes measured polymer Tg < -20.9 °C.

Performance of the electrode is shown in Figures 6 A and B. Sensitivity (mV/decade):

1 hr Day 2 Day 3 Day 10 Day 17 Day 25 Mean SD Day 31-dry

54.4 53.4 55.5 54 53.3 52.6 53.8 1 .01 48.4

2. Potassium ion-selective solid-state electrode based on polymer type II with entrapped valinomycin - reproducibility of four electrodes

Polyhema layer: hydroxyethyl methacrylate = 3mg

2,2-dimethoxy phenylactophenone = 1 wt%

Duration of UV irradiation = 7 minutes Conditioned in KC1 for 2 hours

Composition of ion-selective membrane (total weight 15mg): n-butyl acrylate = 96 wt% hexanediol diacrylate = 0.09 wt% valinomycin = 2.1 wt% potassium tertakis( -chlorophenyl borate) = 0.59 wt% (60 mole %)

2,2-dimethoxy phenylacetophenone = 1 wt%

UV exposure time = 7 minutes measured polymer Tg = -42.6°C.

Performance of the electrode is shown in Figure 7. Selectivity coefficients:

Mean (n ^: =4) SD SD/Mean (%

Li -3.9 0. 1 2.5

Na -3.8 0.05 1.3

Ca -4.9 0.05 1

Mg -4.6 0.05 1. 1

NH₄ - 1.9 0.07 3.6 Calibration slope = 59.0 (SD =0.61) mV/decade Artificial serum assay: Serum ionic composition:

NaCI 140mM, KCl 2.8mM, KH₇PO₄ 1.3mM, CaC 2.5mM, MgCl₂ 2.3mM. Actual 4.1 mM K⁺

Found 4. 1 (SD =0.22)mM (n =4)

3. Potassium ion-selective solid-state electrode based on polymer type II with immobolised 15C5 Polyhema layer: hydroxyethyl methacrylate = 6mg

2,2-dimethoxy phenylactophenone = 1 wt%

Duration of UV irradiation = 10 minutes.

Conditioned in KCl for 2 hours. Composition of ion-selective film (total weight 20mg): n-butyl acrylate = 93.2 wt% hexanediol diacrylate = 0. 12 wt% acrylolamidobenzo 15C5 = 4.4 wt% in 200μl THF:benzene (1 : 1) potassium tertakis(^-chlorophenyl borate) = 0.95 wt% (13.5 mole %) 2,2-dimethoxy phenylacetophenone = 1.3 wt% duration of irradiation = 10 minutes measured polymer Tg = -42.6°C.

Performance of the electrode is shown in Figure 8.

Sensitivity (mV/decade) = 61.9 (SD 1.2), r2 = 0.999, linear range = 0.01 - lOOmM Selectivity coefficients: Li⁺ -2.8. Ca²⁺ -4.3, Mg^{2 +} -4.2, Na⁺ -1.7.

4. Sodium ion-selective solid-state electrode based on polymer type II with entrapped sodium ionophore IV - reproducibility of 3 electrodes

Polyhema layer: hydroxyethyl methacrylate = 3mg

2,2-dimethoxy phenylactophenone = 1 wt% Duration of UV irradiation = 7 minutes Conditioned in NaCI 0. 1M for 2 hours Composition of ion-selective film (total weight l l mg): n-butyl acrylate = 94.7 wt% hexanediol diacrylate = 0. 1 1 wt% 2,2-dimethoxy phenylacetophenone = 0.72 wt% Na ionophore IV = 4.3 wt% sodium tertakis(trifluorophenyl borate) = 0.22 wt% (9 mole %) Duration of irradiation = 8 minutes measured polymer Tg = -42.6°C. Performance of the electrode is shown in Figure 9. Selectivity coefficient:

Mean SD SD/Mean (%)

Li -2.8 0.1 3.5

K -1.5 0.06 7

Ca -3.2 0. 1 3. 1

Mg -3.4 0.06 1.8

NH₄ -2.3 0.2 8.6

Calibration slope = 58. (SD =2.65) mV/decade. Artificial serum assay:

Serum ionic composition:

NaCI 140mM, KCl 2.8mM. KH₂PO₄ 1.3mM, CaC 2.5mM. MgCl, 2.3mM.

Actual 140 mM Na⁺

Found 150 (SD = 10)mM (n =5)

5. Performance of sodium ion-selective solid state electrode based on polymer type II with entrapped sodium ionophore IV - response with time under dry storage conditions

Electrode as described in 4 above measured polymer Tg = -42.6°C.

Performance of the electrode is shown in Figure 10.

Changes in sensitivity: 72

Day 3 Day 10 Day Day 1 16

Slope 61.5 56.3 55.5 53.4

Range: 0.01 - 100 mM Na^* Selectivitv coefficients

Day 3 Day 10 Day 16 Day 1 16 Mean SD

Li -2.8 -2.7 -2.6 -2.7 -2.7 0. 1

Ca -3.2 -3.3 -3.2 -3.3 -3.2 0.05

Mg -3.4 -3.5 -3.8 -3.4 -3.5 0.2

K - 1.6 -1.6 - 1.7 - 1.6 - 1 .6 0.05

NH₄ -2.4 -2.4 -2.4 -2.4 -2.4 0

6. Performance of sodium ion-selective solid state electrode based on polymer type II with entrapped sodium ionophore IV - response with time after exposure to NaCI solution 0.1M

Electrode as described in 4 above except n-butyl acrylate = 94.9 wt% hexanediol diacrylate = 0. 19 wt%

2,2-dimethoxy phenylactophenone = 0.7 wt&

Na ionophore IV = 3.8 wt% sodium tetrakis (trifluorophenyl borate) = 0.38 wt% (14 mol %) measured polymer Tg = -42.6°C.

Performance of the electrode is shown in Figure 1 1 .

Sensitivity changes:

Day 1 Day 3 Day 7 Day 24 Day 36 Mean SD

Slope 59.4 57.9 57.8 62 59.8 59.4 1.7

Range: 0.01 - • 100 mM Na⁺

Selectivity coefficients

Day 1 Day 3 Day 7 Day 24 Day 36 Mean SD

Li -3 -3.2 -3.2 -3. 1 -3. 1 0. 1

Mg -3.8 -3.3 -3.8 -3.8 -3.8 -3.7 0.2

Ca -3.7 -3.4 -3.7 -3.8 -3.6 -3.6 0. 1

NH₄ -2.7 -2.7 -2.8 -2.8 -2.7 -2.7 0.05 K -1.8 - 1.9 -1.9 - 1.8 - 1.9 - 1.9 0.05

7. Hydrogen ion-selective solid-state electrode based on polymer type II with entrapped hydrogen ionophore I Polyhema layer: hydroxyethyl methacrylate = 3mg

2.2-dimethoxy phenylactophenone = 1 wt%

Duration of UV irradiation = 7 minutes

Conditioned in trisHCl 0. 1 M (pH7) for 2 hours Composition of ion-selective film (total weight lOmg): n-butyl acrylate = 89.2 wt% hexanediol diacrylate = 0.21 wt% hydrogen ionophore I = 7. 1 wt%

2,2-dimethoxy phenylacetophenone = 1.4 wt% potssium tertakis( -chlorophenyl borate) = 2. 1 wt% (32 mole %) duration of UV irradiation = 7 minutes measured polymer Tg = -42.6° C.

Performance of the electrode is shown in Figure 12.

Slope = 57 mV/decade. Linear pH range = 5 - 1 1.

For purposes of comparison, a comparable electrode incoφorating an ion-selective plasticised PVC film containing the same ionophore gave a slope = 57.8 mV/decade and a linear pH range = 4.5 - 1 1.0.

8. Cation interferences on hydrogen ion selective (pH) solid-state electrode

The electrode was as described in 7 above measured polymer Tg = -42.6° C. The results are shown in Figures 13A to 13D. Figure 13A = 0. 1M Ca, slope = 53, pH = 5 - 1 1 Figure 13B = 0. 1M Li, slope = 51. 1 , pH = 5.5 - 10 Figure 13C = 0. 1M Na, slope = 51 .2, pH = 5 - 10 Figure 13D = 0. 1M K, slope = 51. 1 , pH = 5 - 9.0. Selectiv ity coefficient Log K

Present invention Required for Plasticised PVC

(mix 0. 1M) blood assay (mix. soln. 1M)

Ca^{2 +} < -10 < -7.7 < - 1 1.3

Li^* < -ιo

Na⁺ < -10 < -8.5 - 10.4

K- < -9.9 < -7.0 -9.8

9. Calcium ion-selective solid-state electrode based on polymer type II with entrapped calcium ionophore IV

Polyhema layer: hydroxyethyl methacrylate = 3mg

2,2-dimethoxy phenylactophenone = 1 wt%

Duration of UV irradiation = 7 minutes

Conditioned in CaCl₂ 0. 1M for 2 hours

Composition of ion-selective film (total weight lOmg): n-butyl acrylate = 74.4 wt% n-heptyl acrylate = 18.3 wt% hexanediol diacrylate = 2.0 wt%

2,2-dimethoxy phenylacetophenone = 0.92 wt%

Calcium ionophore IV = 3.5 wt% potassium tertakis(trifluorophenyl borate) = 0.9 wt% (41 mole %)

Duration of UV irradiation = 15 minutes measured polymer Tg = -44. 1 °C.

Performance of the electrode is shown in Figure 14 (slope = 33.6 mV/decade)

Selectivity coefficient

Present invention Sep Requi ired for Plasticised PVC mix soln (0. 1M) soln blood Ca assay

Li ⁺ < -4.0 -4.3 -5.8

Mg²⁺ -4.2 -4.3 < - 1.9 -4.4

Na⁺ < -3.5 -3.3 < -3.6 -5.9

K⁺ -2.9 -3 < -0.6 -7.5 10. Interferences of cations on Ca responses by mixed solutions method

Results are shown in Figures 15 A to 15D: Figure 15A = 0. 1M Mg Figure 15B = 0. 1M K Figure 15C = 0. 1M Na Figure 15D = 0. 1M Li.

11. Lithium ion-selective solid-state electrode based on polymer type II with entrapped lithium ionophore VI Polyhema layer: hydroxyethyl methacrylate = 3mg

2,2-dimethoxy phenylactophenone = 1 wt%

Duration of UV irradiation = 7 minutes

Conditioned in lithium chloride 0.1M for 2 hours Composition of ion-selective film (total weight lOmg): n-butyl acrylate = 91.7 wt% hexanediol diacrylate = 0.22 wt%

2,2-dimethoxy phenylacetophenone = 1.4 wt% lithium ionophore VI = 4.5 wt% potassium tertakis( -chlorophenyl borate) = 2. 1 wt% (54 mole %)

UV exposure = 15 minutes measured polymer Tg = -42.6° C.

Performance of the electrode is shown in Figures 16A and 16B:

Figure 16A = lithiun ion response (slope = 60 mV/decade) Figure 16B = response to changes in pH (mean = 161.4 sd = 1.4)

Selectivity coefficients

Present invention Required for Plasticised PVC

(Sep Soln) blood Li assay (mix. soln.)

0.1M 0.5M 0.05M

Ca²- -3.2 -2.7 < -6.6 -4.7 (0.5M)

Mg²⁺ -3.3 -2.9 < -3.5 -4.3 (0.5M)

Na⁺ - 1 .6 - 1 .8 < -4.3 -2.4 (0.05M) K⁺ -1 4 - 1 7 < -2 8 -2 3 (0 05M)

Special Features/Advantages

The above ion-selective electrodes based on polymer type II show the following special features/advantages use of non-plasticised polymer to form the ion-selective membrane the ion-selective film can be prepared in situ in a 3 in 1 process with simultaneous polymer formation, ionophore grafting or entrappment and film deposition good polymer adhesion • fabrication of solid-state electrodes using photocure techniques the polymers can be prepared directly from the monomers and the use of ohgomers or other ready made solid polymers is not necessary short duration of film preparation (usually < 10 minutes) little or no solvent is needed during film production • the polymer matrix can be manipulated with different monomers for grafting and for multiple ion sensing

Example 3 (Potassium Ion-Selective Optode Based on Methacrylic-Acrylic Polymers)

Fabrication Procedure

The appropπate polymer (see below) is mixed with a chromoionophore, e.g 9- (dιethylamιno)-5-octadecanolylιmιno-5H-benzo[a]phenoxazιne (lipophilic Nile Blue), an lonophore (either immobolised or not) and a lipophilic anion, e g potassium tetrakis [3,5-bιs(tπfluoromethyl)phenyl] borate and dissolved in a solvent such as dichloromethane, tetrahydrofuran or chloroform (4 - 7 mg of the mixture per ml of solvent) The polymer solution mixture is then spin coated on to a glass slide and the thickness of the film can vary from 0 5 to 2 microns After a drying time of 1 hour, the film is ready to be used as an ion sensing film

The film can be used in conjunction with a spectrophotometer with the response monitored in the absorbance mode Before use, the film is exposed to HC1 (about 1M) to convert the chromoionophore to us protonated form The presence of the ion for which the ionophore is selective will change the absorbance from wavelength 660 nm to 540 nm, i.e. from the protonated form of the chromionophore to the non-protonated form.

Advantages of the Polymer as Ion-Selective Optode Membrane

• no plasticiser is required and the ionophore can be immobilised so that leaching of the membrane components can be avoided which is particularly important if a thin membrane is needed to reduce response time good adhesion on glass surfaces this mode of ion-sensing does not require reference electrode so that sensor design can be further simplified in contrast to other polymer membranes such as PVC which can only function as optode membranes at a pH below about 5. the film gives good response in the pH range close to 7, i.e. coinciding with the physiological range, thereby making the sensor suitable for possible medical applications.

Recipies for Potassium Ion-Selective Optode Membranes

Membrane A: *Polymer I (11 wt% MMA, 89 wt% BA) = 87. 1 wt%

Lipophilic Nile Blue = 2 wt%

Valimomycin ionophore = 6.2 wt%

Lipophilic anion = 4 7 wt% measured polymer Tg = -20 9°C.

Membrane B. ^xPolymer I (11 wt% MMA, 89 wt% BA) = 82 4 wt%

Lipophilic Nile Blue = 1 9 wt%

BME44 lonophore = 1 1 4 wt%

Lipophilic anion = 4 3 wt% measured polymer Tg = -20.9°C

Membrane C "Polymer I (7 wt% MMA. 91 wt% BA, 1 9 wt% immobilised 18

Crown 6) = 95.4 wt%

Lipophilic Nile Blue = 1.8 wt%

Lipophilic anion = 2.8 wt% measured polymer Tg = -30. 1 °C. Membrane D: *Polymer I (12.5 wt% MMA, 81 wt% BA. 6.5 wt% immobilised

15 Crown 5) = 91.3 wt% Lipophilic Nile Blue = 2.5 wt% Lipophilic anion = 6.2 wt% measured polymer Tg = -20.9°C.

"Polymer I prepared by solution polymerisation as described above

Results The performance of optode membrane A above is shown in Figure 17. The film thickness is approximately 1.5 microns and the figure shows change in absorbance (xO.OOl) vs time in minutes (■ = O. IM KCl, □ = O. IM NaCI. ♦ = O. IM CaCl,, 0 = O. IM LiCl).

Claims

CLAIMS.

1. A selective polymer material for use in measuring the concentration of a substance in a solution characterised in that the material is a thermoplastic polymer with an acrylate backbone and a plurality of pendant lipophilic plasticising groups to provide the polymer with a Tg of - 10°C or less ana in that it contains a moiety selective for the substance to be measured chemically bonded to the polymer or physically entrapped therein

2. A selective polymer material as claimed in claim 1 wherein the lipophilic plasticising groups are C₃ -alkyl groups

3. A selective polymer material as claimed in claim 1 or 2 wherein the polymer has a Tg in the range from -10°C to -70°C.

4. A selective polymer material as claimed in claim 3 wherein the polymer has a Tg in the range from -30 °C to -60°C.

5. A selective polymer material as claimed in any of claims 1 to 4 wherein the polymer contains up to 10 weight % of monomer units derived from C, or C₂ alkyl acrylates or methacrylates

6. A selective polymer material as claimed in any of claims 1 to 5 wherein the polymer includes a co-monomer having a reactive group suitable for grafting and the moiety selective for the suostance to be measured is grafted onto said reactive group.

7. A selective polymer material as claimed in any of claims 1 to 5 wherein the polymer includes the moiety selective for the substance to be measured incoφorated therein directly as a monomer unit

8. A selective polymer material as claimed in claim 7 wherein the moiety selective for the substance to be measured is incoφorated into the polymer as a monomer unit by means of the hgand acryloylamidobenzo.

9 A selective polymer material as claimed in any ot claims 1 to 8 wherein the substance to be measured is an ion and the moiety selective therefor is an ionophore

10 A membrane for use in measuring the concentration ot a substance in a solution and which is selectively permeable to said substance, the membrane being formed of a selective polymer material as claimed in any of claims 1 to 9

11 A selective electrode tor use in measuring the concentration of an ion in solution comprising a reference electrode and a polymer membrane which is selectively permeable to the substance to be measured, characterised in that the membrane is formulated from a thermoplastic polymer with an acrylate backbone and a plurality of pendant lipophilic plasticising groups to provide the polymer with a Tg of - 10°C or less and in that it contains a ionophore selective for the substance to be measured chemically bonded to the polymer or physically entrapped therein.

12. An electrode as claimed in claim 11 wherein the lipophilic plasticising groups are C₃.₇ alkyl groups.

13. An electrode as claimed in claim 1 1 or 12 wherein the polymer has a Tg the range from -10°C to -70°C

14. An electrode as claimed in claim 13 wherein the polymer has a Tg in the range from -30 °C to -60 °C

15 An electrode as claimed in any of claims 1 1 to 14 wherein the ionophore is grafted onto reactive groups in the polymer or is incoφorated into the polymer directly as a monomer unit

16 An electrode as claimed in any of claims 1 1 to 15 wherein the membrane also contains a lipophilic countenon

17 An electrode as claimed in any of claims 1 1 to 16 which is an Ag/AgCl electrode. j l

18 An electrode as claimed in any of claims 1 1 to 17 including a hydrophihc membrane as an electrolyte membrane for the internal reference electrode

19 An electrode as claimed in claim 18 wherein the hydrophihc membrane is polyhydroxyethyl methacrylate

20 A selective optode tor use in measuring the concentration of a substance in solution compπsmg a polymer membrane which contains a chromoionophore and is selectively permeable to the substance to be measured, characterised in that the membrane is formulated from a thermoplastic polymer having an acrylate backbone and a plurality of pendant lipophilic plasticising groups to provide the polymer with a Tg of -10°C or less

21 A selective optode as claimed in claim 20 wherein the lipophilic plasticising groups are C_{3 7} alkyl groups

22 A selective optode as claimed in claim 20 or 21 wherein the polymer has a Tg in the range from -10°C to -70 °C

23 A selective optode as claimed in claim 22 wherein the polymer has a Tg in the range from -30 °C to -60 °C