991

REVIEW Polymer membranes in clinical sensor applications III. Hydrogels as reactive matrix membranes in fibre optic sensors M.L. Davies, SM. Murphy, C.J. Hamilton and B.J. Tighe Speciality

Materials

Research

Group, Aston University,

Aston

Triangle, Birmingham

84 7ET, UK

The potential of hydrogel copolymer membranes in clinical sensors, based on fibre optics, is addressed. The particular properties of the membranes of relevance in this application are the ease of refractive index modulation and the potential of the hydrogel to act as a permselective barrier in which a calorimetric agent may be immobilized. The results presented illustrate the complexity of calorimetric and refractive index effects together with their dependence on pH and tonicity for hydrogels of a given composition range. The incorporation of an acryloylfunctionalized reagent (bromopyrogallol red) is used to illustrate the way in which a working pH sensor based on these combined properties may be designed and fabricated. Keywords:

Membranes,

hydrogels,

fibre

optic

sensors

Received 28 January 1992; revised 17 March 1992; accepted 28 May 1992

Optical sensor technology has been one of the fastest growing fields in analytical chemistry in recent years, so much so, that a whole issue of Talantale5 was dedicated to fibre optic chemical sensors in 1988. Optical sensors generally use a fibre optic as the transducing phase of the sensor and this can be introduced in the design of the sensor either intrinsically or extrinsically6-‘. In the latter case, by definition the fibre is only used to carry light to and from the reaction cell’, lo or waveguide”-“, whereas intrinsic fibre optic sensors actively incorporate the fibre optic in one of three basic configurations’g. This paper is primarily concerned with the combination of the intrinsic inclusion of fibre optics and of hydrogel membranes in designing a working sensor. The first of the three most typical sensor configurations, uses a bifurcated fibre bundle to pass light to and from the reagent phase situated at the tip of the fibre. In order to reflect the modified incident light back towards the detector, tiny spheres are usually incorporated at the tip of the fibre close to or involved with the reagent phase. The second configuration requires only a single fibre coupled with a beam splitter in order to separate the incident and emergent radiation. The third uses the evanescent wave component or absorption of the incident light in the reagent phase, which is situated concentrically along the fibre length. In this case, Correspondence

0

1992

to Professor

Butteworth-Heinemann

0142-9612/92/140991-09

B.J. T&he.

Ltd

another parameter which can alter the intensity of the detected light is a change in refractive index of the external reagent phase. Light is transmitted along the fibre by internal reflection and at each interaction at the surface of the fibre, a component of the incident light is transmitted into the surrounding mediumz0~22. This component is known as the evanescent wave and penetrates the medium to a depth of the order of a fraction of a wavelength of the incident light. This is a useful sensing technique to use because only changes near the surface of the fibre are detected and the properties of the bulk solution around the fibre are not. This is mainly because the evanescent wave only penetrates the outer coating to a depth of a fraction of a wavelength. As long as the coating thickness is greater than this penetration depth, its specific thickness is unimportant, as is the case in the work described here. Fluorescence and colour changes can be detected” as well as changes in the refractive index of the reflecting medium23-25 and scattering of the incident light at the interfacez6. This configuration was chosen as the most appropriate for the design of a sensor based on hydrogel technology. In optical sensing, selectivity can be determined either by the reagent itself, or the selective diffusion of the sensed species to it. Membranes used in optical devices can thus serve two purposes. The first is to control diffusion into the region of the sensor that contains the Biomaterials

1992, Vol. 13 No. 14

992

Polymer

phase and the second to support the reagent phase. The reagent is chemically or physically immobilized either within or behind a membrane matrix’, ‘, 27-2g, or directly on to the fibre30e3’. The membrane can act as a selective barrier, only allowing the selected analyte to pass into the reagent phase beyond it, where it will thus produce some form of optical change. It is possible for the reagent to be immobilized within the selective polymer membrane which is in direct contact with the transducer (i.e. the optical fibre). Alternatively, the reagent itself may be selective and here there is no need for the membrane to exhibit permselectivity. In reality, this latter situation tends only to apply to ‘biosensors’, where highly selective reagents, such as antigens, antibodies or enzymes are used. Finally, the reagent may be immobilized directly on to the fibre optic or waveguide, thus obviating the need for a support membrane. A more detailed account of the role of particular membranes in sensor technology is contained in Part 1 of this series33. The concern of this paper lies in the principles involved in the design of a selective hydrogel membrane incorporating a chemically immobilized reagent and the incorporation of such a membrane into a reversible fibre optic sensor. Research activity in this group, has been primarily concerned with the molecular design of polymers to meet the particular requirements of the sensor in relation to its proposed application (e.g. the environment in which it is to be used). This interest follows from, and is based on, work in other biomedical applications where membrane and surface properties of polymers are important. It is not normally the case that completely new materials as such are required, since a vast range of different polymers have been synthesized and characterized in recent decades. The field of ‘speciality’ or ‘effect’ polymers consists of the design or selection of materials based upon a knowledge of the way in which polymer structure governs the properties of relevance to a particular application. To choose or design a membrane for an optical sensor, the properties or characteristics that it requires to be successfully used, must be considered. The fundamental factors in determining the selectivity and sensitivity of all sensors are the choice of the membrane or support matrix and of the reagent. Since most non-biological reagents are not highly specific, it is the selective diffusion characteristics of the membrane which determines the sensitivity and selectivity of the sensing device. The membrane can thus, in principle, govern the selective diffusion of ions to the sensor and can also be important in acting as a reagent support, without interfering to more than a minimal degree with its sensing characteristics. reagent

Hydrogels sensors

as polymer

matrices

for fibre optic

Hydrogel polymers are water-swollen polymers exhibiting a range of permeability and permselectivity characteristics which may be controlled by their monomer composition. Various aspects of the properties of these materials have been described in a series of papers from this research group34-37. Thus, hydrogels possess many advantages in relation to the requirements described, since both the Biomaterials

1992. Vol. 13 No. 14

membranes

in clinical

sensors:

M. L. Davies

et al.

rapid and selective diffusion of the analyte to the reagent are considered to be limitations in fibre optic sensing38-40 In addition, that specific hydrogels have been used for other biomedical applications, ranging -from artificial organs to contact lenses37, suggests they may provide us with sensors that could be used in vivo as well as for clinical routine applications. In both respects, the use of hydrogel membranes could prove to be highly beneficial. In the present context, useful polymers may be obtained by the copolymerization of hydroxy alkyl acrylates and methacrylates with hydrophobic monomers, such as styrene or methyl methacrylate. The resultant materials are tough, flexible, polymers, rather than soft gels. In addition, their refractive indices are controlled by monomer composition and water content and enable the design of membranes whose refractive indices interface well with that of poly(methy1 methacrylate) (PMMA) fibres. This is illustrated by the range of refractive indices obtained from copolymers of Z-hydroxyethyl methacrylate (HEMA), with styrene and with methyl methacrylate 41. In the previous paper of this series4’, the use of N-vinyl carbazole (NVC) as a photocross-linking agent was described, in the conversion of linear soluble hydrogel polymers into insoluble water-swellable membranes. In this paper we show how this procedure may be exploited, in conjunction with the unique refractive index range available in hydrogels, and ability to immobilize chemically a pH-sensitive reagent into the permselective hydrogel matrix, to form a fibre optic pH sensor.

EXPERIMENTAL Monomers procedure

and solution

polymerization

The source, purification and polymerization of Z-hydroxyethyl methacrylate (HEMA) as a linear, soluble homopolymer or as a hydrogel copolymer was described in the previous paper of this series4’. The compositions of polymers are expressed in the form 50:50 HEMA:MMA, to indicate the initial weight ratio of the monomer methyl methacrylate (MMA) in this polymerization reaction. Sensor coatings were thus produced by the photoreaction of N-vinyl carbazole (NVC) within these linear polymer systems.

Sensor test-bed

design

The sensor test-bed consisted of a fibre optic core with an appropriate membrane coating: a suitable flow-through cell and a compact, mains-driven source detector combination. Several stages of development were involved in achieving the final test-bed system. The fibre optic cable used consisted of a 0.5 mm diameter PMMA single core, with a thin fluoropolymer coating, clad in a black polythene sheath. These were removed from a section of the core, before coating with the test membranes. The flow-through cell [Figure I) was designed to allow efficient elution of previous solutions and to be easily assembled around the appropriate section of the fibre optic, without disturbing the coating or breaking the fibre. The fibre optic connections were positioned well

Polymer

membranes

in clinical

sensors:

M. L. Davies et al.

993

1 min in between. The last cycles of the chart recordings are plotted in graph form in this paper.

plug connector

Synthesis of reagent-functionalized and polymer tubing

PVC

cell

body

Solution Rubber marker

inlet

Peristaltic connector

+

pump

tubing Coated section of fibre

Figure 1

The flow-through

cell.

away from the main cell body so that solution did not leak into the bulk heads and alter the optical interface. Finally, the complete cell, together with the incorporated fibre optic, must be easily constructed and assembled into the test-bed. The source/detector system (Figure 2) was designed to run off the mains with little noise in the output signal and to operate in the visible region. The light source was an ultra-bright green LED (light emitting diode] with peak output at 565 nm, providing a single wave band source. The output signal was smoothed across the input of the chart recorder, which provided a visual on-line response. Thus, rise-time, response time, fluctuations and noise were all recorded.

Procedure

for test-bed

runs

Buffer solutions (pH 2.565) were made from 0.1 M citric acid and 0.2 M dipotassium hydrogen phosphate solutions. The probes were conditioned by soaking in dipotassium hydrogen phosphate overnight. Approximately 25 ml buffer was eluted through the cell at each change in pH. To determine the reversibility of the sensor, the sample solutions were cycled from pH 6.5 to 2.5 and back to 8.5. Where an appreciable response to changing pH was apparent, this was repeated for up to four consecutive cycles. The recorder was allowed to run for 3 min at each change in pH and was switched off for no more than

monomer

Reagents chosen for incorporation into a fibre optic sensor can be either calorimetric or fluorimetric. Bromopyrogallol red (BPR; Figure 3) is an appropriate pHsensitive molecule, which may be derivatized to form an acryloyl monomer (AcBPR; Figure 4). BPR (0.5 g) was dissolved in a mixture of 100 ml acetone and 1 ml triethylamine contained in a conical flask and magnetically stirred. To this mixture, 1.0 ml of 20% acryloyl chloride in acetone was added dropwise and the solution left to react for 1 h. The remaining excess triethylamine was removed by addition of 1.5 ml HCl. After filtering off the resultant triethylamine hydrochloride, acetone was removed in the rotary evaporator and the product redissolved in water, poured into a separating funnel and salted out of the aqueous phase, into isobutyl alcohol. The organic solvent was then removed by rotary evaporation under reduced pressure. In a typical experiment to provide a membrane-coated fibre optic sensor, a linear copolymer of the reagent, HEMA and MMA was first produced using solution polymerization at 60°C for 6 h, in approximately 250 ml ethanol with 1% a-azobisisobutyronitrile (AZBN) as initiator. After the linear polymer had been extracted, 3 g linear polymer and 0.03 g NVC were dissolved in 15 g methanol. The end of the fibre optic was dipped into the coating solution which was then allowed to run over an exposed section of the fibre (approx. 1 cm length) and dried. This was repeated until several dry layers of linear polymer incorporating NVC were obtained. To provide an insoluble cross-linked polymer matrix, this coating was photoreacted under UV for approximately 1 h. After subsequent hydration of the membrane layer, the fibre optic was incorporated into the test-bed described. The composition of the fibre coating is typically described in the form (40:60 HEMA:MMA):lO% AcBPR, indicating the percentage by weight of the monomer feed in the solution polymerization reaction. The weight of the acryloyl monomer, AcBPR, is given as a percentage of the total weight of the other component monomers. OH

OH

+5v

-+A 1K

_)_I

5.6MG 1OKn

LED

OV ‘z Light passes via fibre optic

Figure 3

Bromopyrogallol

red.

OH+CICOCHZ=CH

F

47kn 355 zJ!+z+

T

6 I

7

Figure 4 Figure 2

$H + HCI F=O 0

z-

-15v

CHZ=

f~;;c$:;

The source/detector

system.

monomer

The reaction mechanism of bromopyrogallol red.

to synthesize

the acryloyl

Biomaterials 1992, Vol. 13 No. 14

Polymer

994

RESULTS AND DISCUSSION Hydrogel-coated index, monomer water content

fibres: the effect of refractive composition, pH change and

The refractive index of a hydrogel polymer is dictated in the dehydrated state by its monomer composition and then in the hydrated state by its equilibrium water content [EWC) which in turn is dependent on the pH, temperature and tonicity of the hydrating media34s43. These, therefore, are likely to be factors that alter the output characteristics of a fibre optic sensor incorporating a hydrogel polymer as the core coating. Because of these interdependencies, it is important first to establish the effects on the sensor response of hydrophobic and hydrophilic monomer content, the influence of the crosslinking agent (NVC), and pH change. The effects of refractive index changes in the test solution are also studied. Evanescent wave theory predicts that, as the refractive index of the coating becomes closer to that of the fibre core [and thus the critical angle decreases),the amount of light which is totally internally reflected will increase and so therefore will the output of the detector. This implies that a hydrogel of low water content with a high hydrophobic monomer (i.e. MMA) content would result in an increase in optical sensitivity of the device and a reduction in the effects of water content change on the relative change in refractive index of the hydrogel. The refractive indices and water contents of some HEMA:MMA copolymer membranes which were covalently cross-linked with lo/o ethylene glycol dimethacrylate are given in Table 14’. As the water content is reduced, as a result of an increased proportion of hydrophobic monomer, a ‘cut off’ in ion transport will result. This has been shown to occur for simple alkali and of around 50:50 alkali earth cations41 5 at a composition HEMA:MMA. Varying combinations of HEMA and MMA, from 50% HEMA upwards, were chosen for initial studies. The gels swell isotropically and linear swell factors (i.e. from dry to hydrated membrane) can be calculated directly from the volume fraction of water in the hydrated gel. Thus for p(HEMA), with a water content of 37W, the linear swell factor is 1.2 and decreases progressively with MMA incorporation.

effect of hydrophobic monomer constituents on sensor response

membranes

Davies et a/.

Ratio HEMA:MMA

EWC (%)

Refractive index

0:ioo 25175 40:60 50:50 60:40 70:30 9O:lO loo:o

5 10 13 16 22 31 37

1.490 1.485 1.482 1.480 1.477 1.465 1.447 1.435

in membrane water content with pH is observed. Conversely, as the percentage of hydrophilic monomer increases, so does the amplitude of the output signal or the ‘sensitivity’ of the device. On the basis of refractive index, if monomer composition alone is considered, we would expect the output to increase with hydrophobic monomer content because as the refractive index approaches that of the PMMA core (i.e. as the percentage of MMA in the hydrogel increases) the reflecting efficiency of the fibre optic over this coated section, and thus the intensity of the output, should improve. The observed effect may be a result of tonicity change in the buffer solution. Thus, whereas the refractive indices of the dehydrated membranes, or membranes hydrated in water, would decrease with increasing MMA content, this is not necessarily true in the buffer. The sensitivity of the device is observed to improve with a decrease in MMA which implies that the refractive index change in the hydrogels has become more sensitive to the tonicity changes in the buffer solutions as the percentage of hydrophilic monomer (i.e. HEMA) increases. N-vinyl carbazole itself is a photosensitive hydrophobic monomer and is useful in facilitating the formation of ‘photolocked’ hydrogel coatings on sensor devices. However, its presence could potentially affect membrane characteristics and hence the sensor response. Coating solutions of linear p(HEMA) containing varying concen65-

+

# I)-

A series of linear polymer solutions, containing 20% (wt/wt) of the hydrogel linear polymer and 1% NVC (photocrosslinking agent) in methanol, were produced. The linear polymers used were: [a) 70:30 HEMA:MMA; (b] 60:40 HEMA:MMA; and (c) 50:50 HEMA:MMA. These solutions were applied to the fibre optics and the sensor output measured, as described in the experimental section. Results are shown in Figure 5. It is immediately apparent from these results that the monomer composition of the membrane does have some effect on the response of the probe. The ‘noise’ in the response subsides with the increase in hydrophobic monomer content and is hardly visible in the case of the 50:50 HEMA:MMA membrane. Additionally, the output of the sensor decreases. At this composition, little change

3-

1992, Vol. 13 No. 14

sensors: M.L.

Table 1 Refractive indices and equilibrium water contents (EWC) of various HEMA:MMA cross-linked hydrogel membranes

The

Biomaterials

in clinical

#

$

2P 1 n 0

2

m

n 1

I

II

I

I

6

l I

n I

8

I

I

10

pH buffer

Figure 5 The effects on sensor output of varying the HEMA:MMA ratio of the fibre coating. The reversibility of the sensor is described by the duality of each set of points. +, 70:30 HEMA:MMA; Cl, 60:40 HEMA:MMA; n , 50:50 HEMA:MMA. 5% N-vinyl carbazole.

Polymer

membranes

in clinical

sensors:

995

M. L. Davies et al.

trations of NW, were chosen for study for several reasons. On the basis of the work already discussed, the sensitivity of the device would be relatively high for HEMA and from the membranes discussed earlier, it would also be expected that the reversibility and stability of the response would be poor. Thus, any positive contributions made to sensor response, as a result of the presence of NVC would become more evident. Linear polymer solutions of 20% p(HEMA), dissolved in approximately 5 ml methanol, and containing 5, 12 and 20% NVC (calculated as a wt% on the linear polymer content) were therefore prepared. Probes were constructed from these solutions and results obtained as described previously. The results are illustrated in Figure 6. The addition of increasing amounts of the photocrosslinking agent seems to stabilize the response, since the membrane appeared to come rapidly to equilibrium in the case of 12 and 20% NVC but where only 5% NVC was present the response of the probe was erratic and prone to noise and drift (not shown). This reinforces the points made earlier: in the case of both hydrophobic monomers, MMA and NVC, the effect of their presence is generally to stabilize the response of the probe, to improve reversibility and to reduce the fluctuations of the output signal. Although increasing proportions of MMA reduce the amplitude of the output, where NVC is concerned, the sensitivity increases as the percentage NVC increases from 12 to 20% for HEMA membranes, The range of pH to which the probes respond does not seem to change in either case between the limits of 2.5 and 8.5 but the level of sensitivity (or the difference in output at these two pH values) alters with hydrophobic monomer content. The output of the coating containing 12% NVC is higher than that containing 20% NVC. Again, this implies that the refractive index is probably altering as a result of tonicity change in the buffer medium. Having established the sensor response due to refractive index changes in a simple hydrogel membrane, the study of reagent-containing systems can now be used to show any superimposed calorimetric effects.

Bromop~ogalloi red-modi~ed hydrogel membranes: optimization of pH sensitize Since there appears to be a strong effect of tonicity on the response of hydrogel-coated fibre optic sensors, it is important to establish whether a response along the fibre, to colour change around the fibre core can be achieved and whether this effect dominates sensor response, or merely contributes to an overall response which is created by changing tonicity. These relative responses must then be studied with uncoated fibres in buffered solutions, and with hydrogel-coated fibres in the presence and absence of copolymerized reagent monomer. Further, the use of membranes of very low water content may reduce the effects of refractive index change with tonicity and thus result in a sensor with a true calorimetric response. In an attempt to identify the contributions of both colour change and refractive index change (due to the tonicity of the test solutions) the effects of these variables were first studied around a bare fibre optic core. The appropriate buffered solutions were eluted through the cell as described in the experimental section. The response, solely due to changing refractive index of the buffer was then determined using plain buffer solutions. No change in response was observed with the change in refractive index of the plain buffer solutions. A purely calorimetric response was obtained by using buffer solutions containing the reagent bromopyrogallol red. The most noticeable feature of the calorimetric response, shown in Figure 7 is that it is only sensitive from pH 5 upwards, peaking at pH 7. Thus, when the reagentmodified linear polymers are used as fibre optic coatings, this may result in an increase in sensitivity at or around pH 7, as a result of a significant calorimetric contribution to the overall response. To distinguish between the effects on the fibre optic response which are due to calorimetric and to refractive index changes in the membrane, two coatings of 70:30 HEMA:MMA and (70:30 HEMA:MMA):S% AcBPR were studied. Initial experiments were carried out on an adapted spectrophotometer, to provide a multiwavelength source/dete~tor system. Buffer solutions were eluted 56

c t

54

I 2

I

1

I

I

6

4 pH

I

I

8

1

0

I

10

buffer

Figure8 The effects on sensor output of varying the N-vinyl carbazole (NVC) content in HEMA coatings. The reversibility of the sensor is described by the duality of each set of points. +, 12% NVC; Cl, 20% NVC.

46 9 2

4

6 pH (buffet-

8

10

+ BPRI

Figure7 Sensor output to both pH and thus colour change of a buffered solution containing bromopyrogallol red (BP!+ bare fibre). Biomaterials

1992, Vol. 13

No. 14

996

Polymer

through the cell in order of decreasing pH, since it was noted that the more rapid response was in this direction of changing pH. The response of the probe to each solution was taken over a range of wavelengths 350750 nm. It was apparent that the effect due to changing the pH of the buffer was most noticeable at 565 nm, therefore plots of the responses at this wavelength were constructed (Figure 8). The most prominent feature of the response for the reagent-modified membrane is its near-linearity over this wide range of changing pH. This suggests that the presence of the reagent exerts a stabilizing influence on the response. The shape of the response does not, however, relate to the calorimetric response obtained earlier from buffered solutions of the reagent BPR. It is worthy of note that congo red and other indicators, chemically bound on to porous membranes by Jones and Porter similarly exhibited an extended range of sensitivity3’. To reduce the refractive index effects and thus increase the contribution made by the calorimetric reagent to the overall response, systems of lower water content were then studied. Linear, low-water-content polymers were made, with refractive indices close to that of MMA. Although the cut-off point for transport was found to be 50:50 HEMA:MMA, earlier studies where the reagent was only encapsulated had revealed that it served to produce a more permeable network. It was therefore thought possible to balance this effect by increasing the ratio of MMA to HEMA. The effects of varying the percentage of NVC in each linear polymer coating was also studied. The linear polymers tested which illustrate this change were: (a) (40:60 HEMA:MMA):5% AcBPR; and [b) (25:75 HEMA:MMA):Sk AcBPR. Since these were membranes of very low water content, they were coated thinly on to the fibre to maximize ionic transport through the hydrated matrices. The fibres were coated with solutions of 0.5 g linear polymer in approximately 3 ml methanol, containing 5-20% NVC. The coating solutions were prepared as described, with a range of variations in NVC content for each of the linear polymer coating solution compositions. Each sensor response was tested

membranes

in clinical

sensors:

M.L.

Davies et al.

over a range of pH values, provided by citric acid/ dipotassium hydrogen phosphate buffer solutions. The results were taken as described earlier and are shown in Figures 9 and 10. From these figures it can be seen that the hydrogel coatings possess a near-reversible nature with respect to their changing water contents with pH. However, the sensitivity is low but it .does show that the membranes possess adequate permeability, even at these high concentrations of MMA. The response of the probes made from coating solutions of (40:60 HEMA:MMA):5% AcBPR [Figure 9) and (25:75 HEMA:MMA]:5% AcBPR [Figure 101show that a large increase in the sensitivity of the membrane with changing pH has resulted from an increase in the MMA content from 60 to 75%. This may be due to an increase in optical sensitivity as the refractive index of the membrane approaches that of the fibre core, offsetting the decrease in sensitivity which would correspond to a lowering of the membrane water

I

01

I

I

4

2

I

I

6

I

I

8

I

10

pH buffer

Figure 9 The effects of varying the N-vinyl carbazole (NVC) content of 40:60 HEMA:MMA (5% reagent monomer) coatings on sensitivity and reversibility. The reversibility of the sensor is described by the duality of each set of points. n , 20% NVC; 0, 13% NVC; l ,5% NVC.

* *

*

I

2

30 1 2

I

I

I

4

I

6

I

I

I

I

6

I

I

8

I

I

10

I

8

pH buffer

Figure 8 Sensor output with coatings of 70:30 HEMA:MMA (1% /V-vinyl carbazole) with (8) and without (+) 5% copolymerized reagent monomer at 565 nm. Biomatecials 1992, Vol. 13 No. 14

I

4

pH buffer Figure 10 The effect on sensor output to a variation in the Nvinyl carbazole (NVC) content of 2575 HEMA:MMA (5% reagent monomer) coatings on sensitivity and reversibility. The reversibility of the sensor is described by the duality of each set of points. Cl, 6% NVC; +, 11% NVC; a, 18% NVC.

Polymer

membranes

in clinical

sensors:

M. L. Davies et al.

content with addition of a hydrophobic monomer. From these graphs, it can be seen that the reversible nature of the hydrogel coating is improving with increasing NVC content and at a concentration of 18% NVC we have achieved the construction of a probe which possesses the characteristics of a reversible on-line response to pH. The response obtained is evidently achieved as a result of a complexity of dynamic factors which affect the refractive index of the coating resulting broadly from the monomer composition and the effect of the test environment upon its water content and therefore structure. When probes were made of linear polymers of HEMA:MMA of varying ratios, these indicated that transport should stop at a concentration of 50:50 HEMA:MMA whereas the ‘calorimetric’ probes exhibited transport at water contents less than 12%. The reagent is therefore playing some part in the sensor response if only altering the transport properties of the membrane. Water content and hence refractive index change is still the major factor contributing to response even at these very low membrane water contents. Since the membranes were observed to be opaque, they are macroporous and/ or multiphase systems. Either a macroporous structure or a block copolymer containing sections of high water content may be responsible. This explains why transport is observed at such a low concentration of HEMA only when the reagent monomer is copolymerized into the system. Additionally, photographs of the membranes taken under a fluorescence microscope showed that the membranes were multiphase systems with NVC evenly distributed throughout the matrix.

BPR-modified membranes: potassium salts

response

997

5

6

7

8

pH

10

9

11

salt solution

Figure 11 The response of the sensor to the change in pH for four different potassium salts: 0, KSCN; n , KOH; 6, K,HPO,; 0, K2S04.

8

8

8 8

to various

The porosity of the hydrogel matrices studied indicates that these low-water-content systems might lend themselves to the detection of tonicity changes, via refractive index. Since the coating which showed the most promise in terms of its reversible and rapid response was (25:75 HEMA:MMA):5% AcBPR:lB?h NVC, this was the coating chosen for further studies. If the sensor is responding to a change in the water content of the coating and therefore to refractive index change, the water-structuring ability of the ions will play a part in determining the sensor output. In addition, the pH of the test solution will also have an effect. In fact, this response may well override any effects brought about by the water-structuring ability of the constituent ions of the test solution. It is important therefore to note the pH changes with changing salt concentration, To test the response of the sensor to different potassium salts, standard solutions of I&SO,, K,HPO,, KOH and KSCN, were made and each was successively diluted to provide a concentration range, shown in Figure 12. The sensor was constructed with the coating solution containing the linear polymer (25:75 HEMA: MMA):5% AcBPR:lB% NVC. The pH of each solution was measured before testing the response and the output was plotted against both pH and -log[K+] (i.e. p[K+]). The results of the plots are shown in Figures 11 and 12. The plots of pH against output have been normalized such that the value of the output is zero at pH 7. The plots of -log[K+) against output were curve-fitted to a second-

b.0

2.0

4.0

6.0

8.0

10.0

-log [K+]

Figure 12 The response of the sensor to the change in p[K+] for four different potassium salts: 0, KSCN; +, K,HPO,; 0, K,SO,; a, KOH.

order polynomial and normalized such that the output is given as zero at p[K+] = 0. Figure 11, illustrates that since the different responses do not follow the same curve this cannot be purely an effect of pH change. The results are interesting in that they tend to illustrate the dependence on the acidity of the anion. Thus it appears that the pH change in the solutions determines the general shape of the response curve but that the tonicity change in the solutions also has a superimposed effect. Figure 12 shows the response curves for the four potassium salts, plotted as a function of potassium concentration. Each salt exhibits a unique response which is both a function of changing pH and tonicity and thus refractive index of the hydrogel membrane.

CONCLUSIONS Perhaps the most significant single factor that emerges from these studies is the importance of refractive index Biomaterials

1992, Vol. 13 No. 14

998

as a potential means of monitoring the change in environment of a hydrogel membrane coated on to a fibre optic sensor. The work described further illustrates that it is feasible to bind chemically a functionalized reagent into a hydrogel membrane and still retain both the calorimetric sensing ability of the reagent and the rapid transport characteristics of the membrane. Chemical encapsulation of the indicator, and thus the elimination of leaching, is an impo~ant step forward in improving the life-time and the feasibility of calorimetric devices as clinical sensors. The configuration of the optical fibre, chosen to use the evanescent wave component of the source radiation, allows thin coatings of hydrogels to be applied to the fibre optic. Another benefit of using this configuration was that it improved the reproducibility of the device. This is mainly because the evanescent wave only penetrates the outer coating to a depth of a fraction of a wavelength. As long as the coating thickness is greater than this penetration depth, its specific thickness is unimportant. PMMA fibre optics, initially chosen because of their compatible refractive index, specifically with MMAcontaining hydrogels, offered a versatile base for the construction of a relatively simple and inexpensive testbed, which together with the low-water-content hydrogel coating and PMMA fibre combination, provides a system which is both sensitive and stable, with a reversible and rapid response. The nature of the sensor response is not directly calorimetric since the changing refractive index of the coating, with tonicity, results in a more prominent effect than that of the colour change. The presence of the calorimetric reagent, however, has proved to be of impo~ance in the type of response associated with each sensed species, most likely due to the action of the sulphonic acid group present on the reagent as a cation exchanger. If the reagent were not present in the membrane, we would not expect to obtain any response at such low water contents (