OPTICAL PH SENSOR

Abstract

The present disclosure relates to a pH sensor for determining the pH of an aqueous medium at least comprising a polymeric matrix comprising embedded phosphorescent nanoparticles and one or more embedded fluorescent dyes, wherein the phosphorescent nanoparticles comprise transition metal complexes having central atoms selected from the group consisting of Ru, Re, Os, Rh, Ir, Pt and the fluorescent dye comprises fluorescein derivatives according to the following formula I

##STR00001##

or charged structures thereof, wherein n is greater than or equal to 5 and less than or equal to 20, X═—O—, —OH, —OR.sup.4, —NH.sub.2, —NH— or NHR.sup.4, wherein R.sup.4 is selected from the group consisting of C1-C20 alkyl and the R.sup.1, R.sup.1′, R.sup.2, R.sup.3 are independently selected from the group consisting of H, D, substituted or unsubstituted C1-C20 alkyl and halogen. Furthermore, the present disclosure comprises a method and a system for determining pH.

Claims

1. A pH sensor for determining the pH of an aqueous medium at least comprising a polymeric matrix having embedded phosphorescent nanoparticles and one or more embedded fluorescent dyes, characterized in that the phosphorescent nanoparticles comprise transition metal complexes with central atoms selected from the group consisting of Ru, Re, Os, Rh, Ir, Pt; and the fluorescent dye is a fluorescein derivative according to the following formula I ##STR00008## or charged structures thereof, wherein n is greater than or equal to 5 and less than or equal to 20, X═—O—, —OH, —OR.sup.4, —NH.sub.2, —NH— or NHR.sup.4, wherein R.sup.4 is selected from the group consisting of C1-C20 alkyl and the R.sup.1, R.sup.1′, R.sup.2, R.sup.3 are independently selected from the group consisting of H, D, substituted or unsubstituted C1-C20 alkyl and halogen.

2. The pH sensor of claim 1, wherein n is greater than or equal to 10 and less than or equal to 18.

3. The pH sensor according to claim 1, wherein X═—OH or —O—.

4. The pH sensor according to claim 1, wherein the fluorescent dye is a 5-N-(octadecanoyl)aminofluorescein according to the following structural formula II. ##STR00009##

5. The pH sensor according to claim 1, wherein the polymeric matrix is selected from the group consisting of HYPAN, polyurethane, poly-hema, or mixtures of at least two components thereof.

6. The pH sensor according to claim 1, wherein the polymeric matrix comprises polyurethane at a weight percentage greater than or equal to 75% and less than or equal to 100%.

7. The pH sensor according to claim 1, wherein the phosphorescent nanoparticles comprise a Ru central atom.

8. The pH sensor according to claim 1, wherein the phosphorescent nanoparticles are formed by embedding Ru(dpp).sub.3Cl.sub.2 in a matrix of polyacrylonitrile.

9. A method for determining the pH of an aqueous medium, wherein the aqueous medium is contacted with a pH sensor according to claim 1 and the fluorescence properties of the composition are determined by irradiating the composition with light and recording the fluorescence response of the composition.

10. A system for determining the pH of an aqueous medium comprising a light source arranged to emit light of a specific wavelength range; a pH sensor according to claim 1; an optical sensor arranged to detect time- and wavelength-resolved light signals; and an evaluation unit arranged to determine the intensity of the time- and wavelength-resolved light signals.

Description

EXAMPLES

[0034] The production of the pH sensor according to the disclosure can be carried out in three steps, for example:

Step 1: Preparation of Luminescent Nanoparticles from Polyacrylonitrile and Ru(Dpp).sub.3Cl..sub.2

[0035] A polyacrylonitrile nanoparticle with an average particle diameter below 100 nm is prepared by dispersion polymerization. A mixture of acrylonitrile (6 ml), Ru(dpp).sub.3Cl.sub.2 (tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride, 10 mg) in DMF (2 mL), PVA (MW=80 kDa, 1 mg), AIBN (1 mg) and water (150 ml) is heated to about 60° C. and stirred for 20 hours. The resulting precipitate is separated by centrifugation and washed with 50% aqueous ethanol and water. The phosphorescent nanoparticles are then suspended in water (50 mL).

Step 2: Preparation of the Dye Solution with Fluorescent Dye Molecules

[0036] A solution 2 is prepared by dissolving 5-(N-octadecanoyl)-aminofluorescein in 90% aqueous ethanol. A concentration of 5 mM is obtained.

Step 3: Preparation of the Polymer Matrix

[0037] A solution 3 is prepared by dissolving hydrogel D4 in 90% (v/v) aqueous ethanol. A concentration of 4% (w/w) is obtained.

Step 4: Preparation of the pH Sensor

[0038] Suspension 1, solution 2 and solution 3 are mixed in a ratio of 1:4:20 (v/v/v) until a homogeneous distribution of the solutions/suspension is achieved. The homogeneous mixture is applied to a flat plate in a layer thickness of approx. 1 μm and dried under heating.

Washout of the Optical Sensor in a Blind Test

[0039] A solution of hydrogel D4 in 90% (v/v) aqueous ethanol is mixed together with an aqueous suspension of Ru nanoparticles (2% w/w). 4 samples were taken from the resulting mixture and 1 g of each was mixed with different fluorescein derivatives. The resulting samples are as follows: [0040] Sample 1 (blank): without fluorescein derivative [0041] Sample 2 (C0): 5-aminofluorescein (5 μmol). [0042] Sample 3 (C12): 5-Dodecanoylaminofluorescein (5 μmol) [0043] Sample 4 (C18): 5-octadecanoylaminofluorescein (5 μmol).

[0044] Each sample was placed in a 250 ml flask and dried for 24 hours. To each of the dried matrices, 100 ml of an ammonia buffer solution pH=9.5 is added and each flask was stirred at 37° C. for 72 h. The aqueous solution is then filtered and lyophilized. After cooling to room temperature, the aqueous solution is filtered off and lyophilized. The amount of material washed out is calculated according to the following formula: % (washout)=[m(residue)−m(blank)]/m(Cx).

TABLE-US-00001 Sample CO C12 C18 Wash out 7,2% 5,7% 1,2%

[0045] The amount of material washed out results both from the optical components of the system and from the matrix material itself. The washout experiments show that only an extremely small amount of the sensor is dissolved from the matrix. In addition, longer C-chains on the nitrogen seem to reduce the amount of washed out material.

Calibration of the pH Sensor

[0046] The sensor produced by process steps 1-3 was calibrated using PBS buffer solutions. The PBS buffer has an ionic strength I of 0.142 at a pH of approximately 7.4. 0.142M hydrochloric acid or 0.142 M NaOH solution was used to adjust the different pH values in the range between pH 5.0 and pH 9.0. The pH at each data point was measured after a calibration period of at least 4 minutes. Measurements were performed at room temperature (+−1° C.). A digital pH meter was used to determine the respective pH values at the calibration points.

[0047] The composition of the buffer at pH 7.4 is given via the table below.

TABLE-US-00002 Input quantity Input quantity Reagent MW in g in g in mol Na.sub.2HPO.sub.4 141.95 1.419 0.010 KH.sub.2PO.sub.4 136.08 0.244 0.001 NaCl 58.44 8.006 0.137 KCl 74.55 0.201 0.002

[0048] The results of the calibration curve are shown in FIG. 1. The curve shows the dependence of the phase angle on the pH value. The measurement points shown result from the mean value of four independent measurements. The error bars result from the standard deviation of the measurements per pH value. It can be seen that the nanoparticle, polymer and fluorophore system of the disclosure shows a very large difference in phase angle as a function of pH (FIG. 2). In other words, the system according to the disclosure is a very sensitive system, resolving even the smallest changes in pH via a large change in phase angle. This is particularly the case in the range between pH 6.0 and pH 8.0. In addition, the system shows an extremely low standard deviation for different batches, so that the system according to the disclosure is suitable for very reproducible measurements. Looking at the range around a pH of 6.5 with a delta pH of +−1.5, a slope of the phase angle as a function of pH of about 18.2 (° per pH unit with an R.sup.2 of 0.96) is found via linear regression. This spread is larger compared to commercially available systems and thus the system according to the disclosure forms an extremely sensitive optical instrument for the determination of the pH value.

Stability of the pH Sensor in Solution

[0049] The system from manufacturing steps 1-4 is used as the pH sensor. The pH sensor is placed on the exposed wall of a transparent termination piece for an optical fiber. The sensor prepared in this way is inserted into a bioreactor and by means of the sensor, the pH of the aqueous solution in the bioreactor is monitored over a period of days at room temperature. The solution was stirred during the measurement. A PBS solution with a pH of 7.0 was used as the measurement medium.

[0050] By using a buffer, the pH of the solution should actually be constant. It is found that both the measured intensity and the phase angle change over time. The intensity variation over approximately one day of measurement time is shown in FIG. 3 and the phase angle variation is shown in FIG. 4. Both the intensity and the phase angle decrease over the course of the measurement. The decrease of the measured values can be caused by the washing out of the sensor(s) (nanoparticle or fluorophore) or by bleaching, i.e. light-induced degradation, of one of the components. Further investigations show that the main part of the intensity and phase angle change can be attributed to bleaching of the nanoparticles. Surprisingly, the fluorophore according to the disclosure is much more stable against light-induced degradation compared to the nanoparticles used. In addition, further investigations have shown that the loss fraction caused by leaching is very small (see above). Overall, it can be stated that the phase angle in particular is extremely stable over the measurement time. Considering the results from the calibration, one obtains that as a function of pH, the phase angle changes by about 18° per pH unit. From the linear regression of the measurement carried out here, a change in the phase angle of approx. 0.047° per hour is obtained (regression from FIG. 4). In purely mathematical terms, this results in a change in the pH value of approx. 0.0026 per hour. This change can be described as extremely small.

[0051] These results could also be reproduced for another fluorophore, namely 5-N-(dodecanoyl)aminofluorescein (data not shown). The further fluorophore shows a very comparable spreading of the phase angle as a function of pH as well as a similar time-dependent course of the phase angle and intensity. This is surprising, since one would expect at least a different contribution due to a changed washout behavior due to the different chain lengths of the fluorophore. Thus, it becomes clear that the setup according to the disclosure with the fluorophores that can be used according to the disclosure is suitable to a high degree for the optical determination of the pH value in aqueous solutions due to the high spreading and the small amount of leaching and bleaching.

[0052] The foregoing description of the embodiments has been provided for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosure. Individual elements or features of a particular embodiment are generally not limited to that particular embodiment, but, where applicable, are inter-changeable and can be used in a select-ed embodiment, even if not specifically shown or described. The same may also be varied in many ways. Such variations are to be regarded as a departure from the disclosure, and all such modifications are intended to be included within the scope of the disclosure.