WO2009118271A1

WO2009118271A1 - Sol-gel- derived materials for optical fluorescent ph sensing

Info

Publication number: WO2009118271A1
Application number: PCT/EP2009/053277
Authority: WO
Inventors: Colette Mcdonagh; Dorota Wencel
Original assignee: Dublin City University
Priority date: 2008-03-25
Filing date: 2009-03-19
Publication date: 2009-10-01

Abstract

A pH sensitive sol-gel based material is described. The material comprises a combination of a sol-gel matrix with a pH indicator dye.

Description

SOL-GEL DERIVED MATERIALS FOR OPTICAL FLUORESCENT pH

SENSING

Field of the Invention

The present invention relates to pH sensing and in particular to a sensing material for optical fluorescent pH sensing. The invention more particularly relates to a pH sensing material comprising a sol-gel material and sensors incorporating such materials.

Background

Accurate pH sensing is of particular importance for biological, clinical, environmental and bio-processing applications. Two exemplary techniques can be broadly classified as optical and electrochemical. Optical methods for pH measurements exhibit a number of advantages over electrochemical methods. Optical fluorescence-based pH sensing is one of the most widely used optical techniques and offers advantages such as high sensitivity and versatility with respect to detection schemes. Despite the inherent sensitivity of fluorescence, unreferenced intensity- based sensing is hampered by effects such as fluctuations in excitation source, detector drift and changes in light path through the sensor film. Furthermore, leaching and photobleaching also have an effect on the intensity reading. These effects can be largely overcome by using referenced detection, for example, lifetime-based sensing or ratiometric detection.

Such optical methods typically employ an optode which is an optical sensor device that optically measures a specific substance usually with the aid of a chemical transducer. One of the most attractive features of an optode is that it does not require a separate reference sensor and it does not suffer from electrical interferences, unlike electrochemical sensors. Optical fibres allow transmission of optical signals over large distances and, therefore, optodes based on optical fibres, are ideal for remote sensing. Optodes are also attractive for other reasons: their ease of handling, their low energy consumption, low production cost, ease of miniaturisation and possibility of non- invasive measurements, which is of a great importance for real time measurements in industry.

Most optical pH sensors consist of a proton-permeable solid matrix in which the pH indicator is encapsulated such that it is accessible to the analyte while being impervious to leaching effects. pH is measured as a function of reversible changes in the fluorescence or lifetime of the indicator, which are often influenced by the matrix- indicator interaction.

Current solutions for pH sensing do not allow for continuous optical pH monitoring over long-time periods. Furthermore, many of the known systems do not have high pH sensitivity or ease of miniaturization. Many of these systems are unstable, costly and frequently require maintenance. There is therefore a requirement for an improved optical pH sensor.

Summary

These and other problems are addressed by an optical pH sensor in accordance with the present invention that comprises a sol-gel material providing a support matrix for a pH indicator dye.

In a preferred arrangement an optical sol-gel-based pH sensor is provided which is based on ratiometric detection of the pH-dependent fluorescence of 8- hydroxypyrene-l,3,6-trisulfonic acid trisodium salt (HPTS), which has been ion- paired with cetyltrimethylammonium bromide CTAB, (HPTS-IP) and which has been physically entrapped in a hybrid sol-gel film. The microstructure of the film, which is a composite of the precursors (3-glycidoxypropyl) trimethoxysilane and ethyltriethoxysilane (GPTMS/ETEOS), has been tailored to completely encapsulate the dye thereby eliminating leaching. The polar GPTMS precursor provides a hydrophilic matrix which promotes proton permeability while the ETEOS precursor improves the adhesion and mechanical stability of the resulting sol-gel-derived layers. While it is not intended to limit the present teaching to any one set of experimental results such a sensor displays a reproducible and reversible response, a resolution of 0.02 pH units and a response time of less than 12 s. The response is stable for time periods in excess of 1 month.

Accordingly there is provided a pH sensor according to claim 1. Advantageous embodiments are provided in the dependent claims.

Brief Description Of The Drawings

These and other advantages, objects and features of the invention will be apparent through the detailed description of the embodiments and the drawings attached hereto. It is also to be understood that both the foregoing general description and the following detailed description are exemplary and not restrictive of the scope of the invention.

Fig. 1 depicts the chemical structure of HPTS;

Fig. 2 graphically depicts the normalised absorption spectra of HPTS in 0.15 M phosphate buffers at different pH values;

Fig. 3 depicts the chemical structure of HPTS-IP;

Fig. 4(a) depicts an SEM image of a pH sensor film provided in accordance with the present teaching;

Fig. 4(b) depicts an AFM image of the pH sensor film;

Fig. 5(a) graphically depicts normalised excitation spectra of HPTS-IP entrapped in the sol-gel matrix (GPTMS/ETEOS) at different pH values;

Fig. 5(b) is a pH calibration plot of GPTMS/ETEOS sensor film; Fig. 6(a) graphically depicts absorption spectra of pH sensor film before measurements, after 24 h, after 3 and 4 weeks in buffer solutions at pH 7.0 and Fig.6(b) fluorescence intensity after 0 h, 1 h, 3 h, 6 h and 24 h in flow-through cell;

Fig. 7 graphically depicts the photostability of the pH sensor film;

Fig. 8 graphically depicts the reversibility of the pH sensor films; Fig. 9 graphically depicts response time of the pH sensor film;

Fig. 10 graphically depicts the reproducibility of pH sensor films; and

Fig 11 shows corrected calibration curves for GPTMS/ETEOS gel films at different temperatures, with the ionic strength (IS) of buffer solutions = 150 mM. Detailed Description Of The Drawings

Accordingly, sol-gel derived materials for optical fluorescent pH sensing that offer continuous optical pH monitoring over long-term are provided. The sol-gel materials according to the present disclosure also provide for high pH sensitivity and ease of miniaturisation (advantages over pH electrode). Moreover, the materials are stable, are available at low-cost and require low-maintenance. It is envisioned that the pH material according to the present disclosure may be used in a wide variety of commercial applications including, but not limited to bioprocess management, environmental analysis (capability of continuous, remote and underground sensing) and biomedical applications (for example in the continuous monitoring of blood pH or arising from its ease of miniaturisation and suitability for in vivo measurements).

It will be appreciated that a sol-gel is a colloidal suspension of silica particles that is gelled to form a solid. The specifics of any one sol-gel formulation will differ from those of other formulations. The present inventors have identified that the sol- gel-based formulation GPTMS/ETEOS is suitable for and particularly advantageous in the context of continuous pH sensing.

Such a gel was combined with a pH indicator dye. The pH indicator 8- hydroxypyrene-l,3,6-trisulfonic acid trisodium salt (HPTS) was selected as an exemplary dye as it is a very photostable, highly fluorescent pH indicator with a pKa of ~7.30. Using the precipitate of the ion pair (HPTS-IP) reduced leaching and improved sensor film stability results. Such an indicator dye exhibits two different pH-dependent excitation bands, corresponding to the protonated (acidic, 405 nm) form and the deprotonated (basic, 460 nm) form, and a single emission band (515 nm). The presence of the dual excitation bands facilitate the use of referenced, excitation ratiometric detection. Its pKa of ~7.4 makes it suitable for pH determination in the physiological pH range. In addition, large Stokes shift and compatibility with blue LED excitation make it a very attractive pH indicator for practical applications. HPTS-IP is more hydrophobic than HPTS and displays poor water solubility. The formulation according to the present disclosure exhibits the same pH-dependent absorption and emission maxima as the unmodified HPTS indicator so, as was detailed above, it is suitable for ratiometric measurements, which are insensitive to dye concentration, leaching and photobleaching of the fluorophore, instrument fluctuations and optical alignment. Furthermore, the formulation according to the present disclosure is a robust pH sensor achieved using physical entrapment as an immobilization method and which is resistant to dye leaching. The advantages of this method are its ease of implementation, low-cost reliability as the fabricated sensor films do not exhibit leaching. This method is easy and not time consuming to implement in contrast to covalent binding methods. The sensor film also has a short response time (tgo = 12 s) to pH changes.

The pH dye used is compatible with low-cost LEDs and can be used with inexpensive instrumentation (for example LED light sources and photodiode detectors) to provide a pH sensor detection system. The sol-gel material developed can be deposited using a wide range of printing methods which makes it suitable for low-cost mass production. Preliminary results showed that the sensor is compatible with gamma irradiation treatment, which as will be appreciated by those skilled in the art, makes it particularly useful for bioprocess monitoring applications. Heterogeneity occurred at a very fine level as shown by the AFM image in Figure 4b. The variation in height is very uniform throughout the sample area with surface roughness of 0.24 nm. This demonstrates that a pH sensor has a very smooth surface which is particularly advantageous in bioprocess monitoring as it reduces bio fouling because microorganisms find it difficult to attach. The homogeneous nature of these films is further demonstrated by the SEM image of Figure 4a which does not show any features thereby demonstrating a homogeneous material at the micrometer scale.

Moreover, sol-gel materials offer numerous advantages over polymers. They exhibit higher thermal, chemical and photochemical stability, along with improved mechanical strength, and are optically transparent over a broader spectral range (down to 250 nm).

The present invention relates in general to (a) a fluorescent dye, modified HPTS as shown in Figure 3, (b) a sol-gel-derived material suitable for the entrapment of the fluorescent dye and continuous optical pH sensing and (c) an optical fluorescent pH sensor that comprises the pH indicator dye entrapped in the sol-gel matrix.

The pH-sensitive fluorescent dye, 8-hydroxypyrene-l,3,6-trisulfonic acid trisodium salt (HPTS) as shown in Figure 1 , has been extensively used as an indicator for optical pH sensors. HPTS is a very photostable, highly fluorescent pH dye. With a pKa of 7.3 it is suitable for pH detection in the physiological range. In addition, water solubility, large Stokes shift, non-toxicity and compatibility with blue LED excitation make it a very attractive pH indicator for practical applications.

Depending on the pH, HPTS exhibits two different absorption maxima, one at

404 nm for the acidic form, the other at 455 nm for the basic form as shown in Figure 2. Therefore, this indicator can be used for dual excitation measurements as it exhibits pH-dependent shifts in excitation maxima. The ratio of the emission intensity at two excitation wavelengths is used as a robust measure of the pH and is particularly useful in the context of ratiometric detection techniques. It has a number of advantages over unreferenced intensity measurements. It is insensitive to dye concentration, leaching and photobleaching of the fluorophore, instrument fluctuations and optical alignment.

A key step in the development of optical pH sensors is immobilisation of a pH sensitive dye in a suitable, solid matrix. Such a matrix dictates the characteristics of the sensor. There are three methods typically used for immobilisation of indicators in a matrix: adsorption, physical entrapment and covalent binding. In the adsorption method, a pH indicator is adsorbed, e.g., via electrostatic or hydrophobic interactions, on a solid substrate. It is simple but not very reliable, since the adsorbed indicator may leach out. The covalent binding method, which involves the formation of a chemical bond between the indicator dye and the matrix, is usually complicated and time-consuming but very reliable since the indicator is not likely to leach out. The entrapment method involves the encapsulation of a pH indicator in a porous polymeric matrix. It is easy to implement and reliable but slow indicator leaching can be a problem depending on the efficiency of entrapment. The present inventors have realised that sol-gel entrapment of a pH indicator dye provides for a very efficient entrapment of the dye with the result that the final material offers the advantages of easy implementation and reliability without significant leaching. The optimum solution for the development of a robust, low-cost, easy to prepare and non-leaching optical pH sensor is to carefully tailor the sol-gel matrix to obtain efficient dye entrapment. The present inventors have realised that sol-gel materials exhibit better thermal, chemical, photochemical stability and mechanical strength than polymers.

A sol-gel-based formulation suitable for pH sensing has been developed in accordance with the present disclosure. The sol is doped with a pH sensitive dye. According to the present disclosure, the pH sensitive dye, HPTS, was ion paired with cetyltrimethylammonium bromide (CTAB), to form a HPTS derivative, HPTS-IP as shown in Figure 3. This is entrapped in a suitable sol-gel matrix to produce a pH sensor. As was discussed above, HPTS-IP is more hydrophobic than HPTS and has poor water solubility. By using such a dye, one can expect minimised leaching and better stability of the sensor films. It exhibits the same pH-dependent absorption and emission maxima as the unmodified HPTS indicator.

The developed pH sensor according to the present disclosure is suitable for pH determination from pH 5.0 to pH 8.0. It exhibits excellent reproducibility, reversibility, photostability, temporal stability, minimal leaching and short response time. Ratiometric dual excitation pH detection was employed to characterize the performance of the sensor.

Experimental formulation

The sensor films were prepared from a mixture of ETEOS- and GPTMS- derived sols combined in 1:1 molar ratio. The ETEOS-based sol was prepared by mixing ETEOS, 0.1M aqueous HCl and ethanol (EtOH) in a 1 :0.007:6.25 molar ratio. The GPTMS-based sol was prepared by mixing GPTMS, 1-methylimidazole, deionized water and EtOH in 1 :0.69:4:6.25 molar ratio. GPTMS is (3- glycidyloxypropyl)trimethoxysilane otherwise known as 3-(2,3- epoxypropoxy)propyltrimethoxysilane or silane-Y-4087 and has a molecular formula CgH₂QOsSi. ETEOS is ethyltriethoxysilane. The GPTMS/ETEOS hybrid sol was prepared by mixing the two separate sols in equal molar ratios. It will be appreciated that the molar ratio does not have to be 1 :1 as for example other ratios could also be used within the context of the present teaching The HPTS-IP doped solutions were fabricated by mixing an ethanolic solution of HPTS-IP with the prepared hybrid sol to give a final silane/dye ratio of 10^" . In a typical preparation, the precursor was combined with absolute ethanol (EtOH), followed by dropwise addition of the catalyst. The final mixture was aged for 72 h under ambient conditions.

Aging time is an extremely important factor when preparing these sols. Films prepared from sols aged for 24 h exhibited very low or no pH sensitivity and while it is not intended to limit the teaching of the present invention to any one time period it has been ascertained by the present inventors that at least 48 hours of ageing is important for adequate pH sensitivity.

Synthesis of HPTS(CTAB)2 ion pair

278 mg of CTAB (0.76 mmol) was dissolved in 25 ml of DI water at 50° C and mixed with a solution comprising 200 mg of HPTS (0.38 mmol) in 25 ml of DI water. The precipitate of the ion pair (HPTS-IP) was subsequently filtered and dried in the oven at 70 ⁰C for 12 h.

All films were formed by dip-coating using a dip-speed of 3mms^"' in a controlled environment using a computer-controlled dipping apparatus. The glass slides were treated with 30 % HNO3 for 24 h and then rinsed with copious amount of

DI water and EtOH. After deposition, pH sensor films were cured at 14O⁰C for 4 hours. The resultant thin films are very uniform as is displayed in Figure 4.

pH fluorescence measurements were acquired using a FluoroMax-2 fluorometer (Jobin Yvon, USA). All spectra were recorded from samples contained in a flow cell that was fixed at 45° with respect to the incident beam. Films on glass slides were immersed in phosphate buffer solutions that were adjusted to pH values ranging from 3.00 to 10.00. The fluorometer collected the emission intensity at 515 nm, employing excitation wavelengths of 405 nm and 460 nm. 1.5 nm passbands were used for both the excitation and emission monochromators. All measurements were performed at room temperature.

Various aspects of the pH films performance were monitored. The pH film had a pKa = 6.49 ± 0.02. Figure 5(a) depicts normalized excitation spectra of HPTS- IP entrapped in the sol-gel matrix (GPTMS/ETEOS) at various pH values and Figure 5(b) shows a pH calibration plot of GPTMS/ETEOS sensor film. As is shown by Figure 5(a) and 5(b), the pH film has a dynamic range from pH 4.0 to 9.0 with the most sensitive dynamic range occurring between pH 5 to pH 8.0 which is a particularly advantageous range for bio-processing and clinical applications. Figure 8 shows the response of the sensor over 7 measurement cycles, demonstrating the reversibility of the response. The sensor resolution, measured under the conditions of IS = 150 mM and T=25 C, was calculated to be 0.02 pH units in the pH range from 5.0 to 8.0.

Figure 6 graphically depicts absorption spectra of a pH sensor film in accordance with the present teaching before measurements, after 24 h, after 3 and 4 weeks in buffer solutions at pH 7.0 (left) and fluorescence intensity after 24 h in flow- through cell (right). Pump speed was 1 mm/s. As is shown by Figure 5, the pH films showed no leaching in pH 5.0 and 7.0 (1 month soaking) as verified with UV- Vis and fluorescence measurements. Furthermore, as is shown in Figure 7 and Figure 8, the pH sensor films showed very good photostability and excellent reversibility.

Figure 9 depicts the response time of the pH sensor film. The film showed a short response time (t₉₀ = 12 seconds from pH 5.0 to pH 7.0 and from pH 7.0 to pH 5.0). We define the sensor response time as so-called tgo time, the time taken for the intensity to achieve 90% of the final value when the pH is changed from pH 5.0 to pH 7.0. These pH values were chosen as they are on either side of the pKa' value of 6.49. The response time is dependent on film thickness and the flow rate/injection time of the buffer solutions. In order to eliminate the fill time of the flow cell, buffer solutions at pH 5.0 and 7.0 were injected directly into the flow cell through a short section of tubing.

As was mentioned above a typical response under these conditions for films of thickness ~lμm is about 12s. It will be understood that the specifics of the measurement parameters relate to the thickness of the film and the response time could be further improved by using thinner sol-gel films. The choice of film thickness usually represents a compromise between signal-to-noise ratio and response time. The signal-to-noise ratio in our experiments is sufficiently high to facilitate thinner films and hence achieve shorter response times.

The pH film according to the present disclosure also shows good temporal stability as is depicted below in Table 1.

Table 1 pKa' values before, during and after storage.

No significant pKa' changes were recorded over a 4 week period (films kept in buffer at pH 7.0 and in ambient).

The pH film according to the present disclosure also displayed excellent sensor resolution r = 0.02 pH units. Furthermore, as shown in Figure 10, the pH films show excellent reproducibility. The calculated relative standard deviation of pKa' for 5 films was 0.32%, which demonstrates the high level of reproducibility in pH response.

Effect of temperature on pH response was also investigated. As shown in Figure 11, a negligible temperature effect was measured when temperature was varied between 25 ⁰C and 42 ⁰C. A pH error of 0.06 pH units (averaged over 5 independent measurements) was measured for a temperature change between 25 ⁰C to 37° C and the response shifted by 0.11 pH units when temperature was changed from 25 ⁰C to 42 ⁰C. Therefore, it will be appreciated that the exposure of material provided in accordance with the teaching of the present invention to small temperature changes should have a negligible effect on their pH response.

A factor that is commonly underestimated in the case of optical pH sensors is the cross-sensitivity towards ionic strength (IS) because it affects accuracy of pH sensors. In this work, the influence of IS was investigated by monitoring the sensor response at different ionic strengths: 50 mM, 100 mM, 150 mM, 200 mM and 300 mM, and potassium chloride was employed as a background electrolyte. Figure 12 clearly shows that there is a dependence of the pH response on the IS. The largest variation, when IS is varied from 50 mM to 300 mM, yields a pH error of 0.5 pH units. A change from 150 mM to 200 mM (relevant in bioprocess monitoring) yielded a maximum pH error of 0.15 pH units. Depending on the sensitivity of the application, this implies that a recalibration of the pH sensor may be required at different ionic strengths.

It will be appreciated that what has been described is a high performance optical ratio metric, sol-gel-based pH sensor which, when a combination of key performance parameters is considered, compares very well to similar sensor systems which have been reported in the literature. The sensor in an exemplary arrangement is based on the ratiometric detection of the pH-dependent fluorescence of HPTS which has been ion-paired with CTAB to reduce its solubility in water and hence minimise dye leaching. Furthermore, the indicator has been completely physically encapsulated in a novel hybrid GPTMS/ETEOS sol-gel matrix which, due to the degree of organic-inorganic polymerisation and cross-linking, results in a dense microstructure which eliminates dye leaching while allowing ingress of H⁺ ions. The sensor is reproducible and reversible, has a resolution of 0.02 pH units in the pH range from 5.0 to 8.0, a response time of 12s for a film thickness of lμm and displays temporal stability of at least 1 month. Self-referenced ratiometric detection ensures that the sensor is immune to drifts such as photobleaching effects. The dynamic range of the sensor is from pH 5.0 to 8.0 and the dye is completely encapsulated in the sol-gel matrix. The dynamic range and performance of the sensor are compatible with a range of applications such as bio-processing and clinical measurements. While many reported optical pH sensor studies present optimum sensor performance for selected parameters, a sol-gel based pH sensing material in accordance with the present teaching provides high performance in all of the key sensor parameters, namely, reproducibility, resolution, stability, response time and leaching characteristics.

While the specifics of the pH material described have been with reference to specific sol-gel formulations and indicator dyes it will be appreciated that modifications can be made to that described herein without departing from the scope of the present teaching.

For example, the person of skill in the art of pH dyes will appreciate that there are a large number of dyes available for selection and use in determination of pH. However, of this large number of available dyes the present inventors have selected a sub-set based on the application of the pH sensor for pH determination in the physiological pH range. The important criteria are: an appropriate pKa, absorption in the visible region in order to use cheap light sources, large Stokes shifts, no toxicity, high quantum yield and photostability. The most frequently used pH indicators are: 8- hydroxypyrene-l,3,6-trisulfonic acid sodium salt (HPTS), fluorescein derivatives, seminaphthorhodafluor (SNARF) and seminaphtho fluoresceins (SNAFL) dyes and hydroxycoumarines. While these could therefore be used instead of the HPTS of the preferred arrangements, Fluoresceins, SNARF and SNAFL dyes are not as photostable as HPTS. In addition, SNARF and SNAFL indicators are extremely expensive and most coumarins are excitable in the range from 300 to 400 nm. HPTS, and more particularly its ion paired formulation, is therefore a desirable preferred dye for use in the context of the present teaching.

However depending on the application, the limitations of these other materials when compared to the performance of HPTS may not have a detrimental effect on the performance of the sensor and these materials could also therefore be used instead of or in conjunction with the HPTS. Furthermore one or more other dyes could be used in order to extend the working range of the HPTS-IP-based sensor. This can be accomplished by using indicators with two pKa values or by using a group of similar dyes with different pKa values In addition, tuning of the pKa can be achieved by co- immobilisation of surfactants in the sol-gel matrix. Therefore, one can cover different range of pH by judicious selection of appropriate indicators dyes for immobilisation in the sol-gel matrix. It will be understood that by providing the pH sensitive material in a sol-gel form it may be applied onto a substrate in one or more techniques such as for example spin-coating, dip-coating, spray-coating, gravure and screen printing, knife coating, ink-jet printing, pin-printing, micro-array deposition and stamp printing. While the pH sensor material has been described in an exemplary arrangement as being provided as a film it will be appreciated that the teaching of the present invention should not be construed as so limited. For example discrete spots, strips or other patterns of the sensor material could be provided depending on the specific application requirements. Suitable substrate materials include those formed from glass, plastics e.g. zeonor, zeonex, polycarbonate. By providing the substrate with an adhesive surface such as for example in the form of an adhesive label it is possible to adhere the pH sensor to a vertical or horizontal surface within the test environment. The pH sensor could also be provided as a coating on an optical fiber. The geometric form of the substrate could be modified to be for example one or more of the following: planar, curved, rigid and flexible substrates.

A pH sensor such as that described heretofore can be used in a variety of different applications including for example:

Environmental: fresh water and marine, gas-phase (for detection for example of noxious gases), pH of rain water;

Bioprocess: in head-space or in liquid phase, as free-standing demountable, probe-based sensor or printed on inside of transparent bioprocessor e.g. small volume disposable bioprocessors; Biomedical: blood, saliva, other body fluids, cell diagnostics as above, point of care;

Food quality/freshness: food packs, food processing.

If provided as part of a sensor system, such a pH sensor will provide on excitation a source of florescence which may be detected by one or more optical or electronic sensors provided relative thereto. A particularly advantageous sensing device comprises one or more LEDs and one or more photodiodes, which may be arranged relative to the sol-gel material to capture any generated fluorescence resultant from excitation of the pH indicator dye. While a sensing system may be configured to solely provide an output in response to pH fluctuations, such a system may be modified to provide a multi- analyte sensor having additional sensing platforms configured to provide outputs based on one or more of an O₂ sensor, a CO₂ sensor, a temperature sensor, a humidity sensor. The individual outputs of the dedicated sensors could be cross referenced to one another or could be provided as independent assessments of specific criteria that are being sensed.

It will be appreciated that an exemplary arrangement of a pH sensitive sol-gel based material has been described. The material comprises a combination of a sol-gel matrix with a pH indicator dye. By stabilizing the indicator dye within a sol-gel material the present inventors provide a stable platform that may be used for extended periods.

While exemplary arrangements and results have been described herein, it will be understood that these are provided to assist in an understanding of the present teaching and that modification can be made without departing from the scope of the claimed invention. Furthermore, the words comprises/comprising when used in this specification are to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

Claims

1. An optical fluorescent pH sensor comprising: a pH indicator dye entrapped in a sol-gel matrix.

2. The pH sensor of claim 1, wherein the pH indicator dye is 8- hydroxypyrene-l,3,6-trisulfonic acid trisodium salt (HPTS).

3. The pH sensor of claim 2, wherein the pH indicator dye, HPTS, is ion paired with cetyltrimethylammonium bromide (CTAB) to form HPTS-IP.

4. The pH sensor of claim 1, wherein the sol-gel matrix comprises (3- glycidoxypropyl)trimethoxysilane (GPTMS) and ethyltriethoxysilane (ETEOS) .

5. The pH sensor of claim 4 wherein the GPTMS and ETEOS are provided in a 1 :1 ratio.

6. The pH sensor of claim 1 wherein the sol-gel matrix comprises (3- glycidoxypropyl)trimethoxysilane (GPTMS) and monoalkylsiloxanes of the form (C_nH_2n+O-Si-OR₃ (n = 1 to 6, R = methyl or ethyl).

7. The pH sensor of any preceding claim wherein the pH indicator dye entrapped in the sol-gel matrix is provided as a film.

8. The pH sensor of claim 7 comprising a substrate, the film being provided at one or more discrete locations on the substrate.

9. The pH sensor of claim 7 or 8 wherein the film has a thickness of about lμm and exhibits a tgo response time of about 12 seconds in transitions from from pH 5.0 to pH 7.0 and from pH 7.0 to pH 5.0

10. The pH sensor of any preceding claim comprising a flexible substrate, the sol-gel being provided on a surface of the substrate.

11. The ph sensor of claim 10 wherein the sol-gel is provided at distinct regions on the surface of the substrate.

12. The pH sensor of any preceding claim comprising an adhesive backed substrate, the sol-gel being provided on a surface of the substrate, the adhesive backing of the substrate allowing in use for the adhesion of the sensor to horizontal or vertical surfaces within a test environment.

13. The pH sensor of any one of claims 1 to 6 further comprising a substrate, the pH indicator dye entrapped in the sol-gel matrix being printed or deposited onto the substrate using one or more of the following techniques:

Spin-coating, dip-coating, spray-coating, gravure, screen printing, knife coating,

Ink-jet printing, pin-printing, micro-array deposition, and/or stamp printing.

14. The pH sensor of claim 13 wherein the substrate is selected from one or more of the following materials: glass, plastics, adhesive labels, and or fibers.

15. The pH sensor of claims 13 or 14 wherein the substrate is configured to be planar, or curved, or rigid or flexible in form.

16. The pH sensor of any preceding claim having a resolution of 0.02 pH units in the pH range from 5.0 to 8.0.

17. The pH sensor of any preceding claim wherein the sol-gel silane/pH indicator dye ratio is provided in the range of 10^~2 to 10^~4, desirably about 10^~3.

18. A ratio metric pH sensor comprising the pH sensor of any preceding claim.

19. The ratio metric pH sensor of claim 18 wherein the pH indicator dye exhibits first and second different pH-dependent excitation bands

20. A pH sensor system comprising: a) a pH sensor as claimed in any of claims 1 to 19, the pH indicator dye being responsive to an excitation source to fluoresce; b) a detector provided relative to the pH sensor and configured to detect any fluorescence resultant from excitation of the pH indicatory dye.

21. The system of claim 20 wherein the detector comprises a photodiode.

22. The system of claim 20 or 21 wherein the excitation source comprises an LED.

23. The system of any one of claims 20 to 22 further comprising one or more of an O₂ sensor, a CO₂ sensor, a temperature sensor, a humidity sensor .

24. A method of fabricating a pH sensor comprising: Combining (3-glycidoxypropyl)trimethoxysilane (GPTMS) with ethyltriethoxysilane (ETEOS) to form a sol-gel;

Ion pairing hydroxypyrene-3,6,8-trisulphonic acid (HPTS) with cetyltrimethylammonium bromide (CTAB), to form HPTS-IP; mixing an ethanolic solution of the HPTS-IP pH sensitive dye with the formed sol-gel to form a pH sensitive material.

25. The method of claim 11 wherein the formed pH sensitive material has a silane/dye ratio of between 10^"2 and 10^"4, desirably about 10³.

26. The method of claim 24 or 25 comprising dip coating the pH sensitive material onto a substrate to form a thin film.