WO2016071465A1

WO2016071465A1 - Luminescent ruthenium (ii) complexes and their use in ph sensors

Info

Publication number: WO2016071465A1
Application number: PCT/EP2015/075848
Authority: WO
Inventors: Guillermo Orellana Moraleda; Maximino Bedoya Gutierrez
Original assignee: Tap Biosystems (Phc) Limited
Priority date: 2014-11-05
Filing date: 2015-11-05
Publication date: 2016-05-12

Abstract

The invention provides luminescent Ru(ll) complexes that can be used in pH sensors to provide pH sensitivity over a wide pH range. The complexes have at least one polypyridyl ligand having at least one proton-accepting group with a pK_a in the range 4 to less than 6, wherein the luminescence intensity and/or luminescence lifetime of the complex varies depending on the protonation state of the proton-accepting group; and at least one polypyridyl ligand having at least one proton-donating group with a pK_a in the range 6 to 8, wherein the at least one proton-donating group is covalently linked to a ring carbon atom of the at least one polypyridyl ligand by a spacer group having the formula -(CH₂)_q- wherein q may be 1, 2, or 3, and wherein the luminescence intensity and/or luminescence lifetime of the complex varies depending on the protonation state of the proton-donating group.

Description

LUMINESCENT RUTHENIUM (II) COMPLEXES AND THEIR USE IN PH SENSORS

Field of the Invention

The present invention relates to pH sensors.

Background to the Invention

Luminescent compounds have been found to be useful for the measurement of pH, particularly where a non-invasive, non-destructive measurement is required.

Some known pH sensing systems involve the use of Ru(ll) complexes comprising polypyridyl ligands having proton-dissociable groups such as carboxyl, sulphonic, hydroxyl, ammonium or pyridinium groups on their carbon rings. When the proton dissociation is reversible, the luminescence properties of these complexes can vary depending on the proton concentration to which they are exposed. This allows the pH of an environment to be determined by irradiating the complexes with light of a wavelength at which the complexes absorb and measuring the luminescence lifetime or luminescence intensity of the complex using light emitted by the complex at the wavelength of luminescence. Luminescent Ru(ll) complexes are known to be suitable indicator dyes for chemical sensing due to their photo- and thermal stability, their long emission lifetimes and large Stokes shift between their absorption and emission bands.

For example, EP 408748 describes a luminescent probe complex comprising a polypyridine ligand having a proton-dissociable substituent on a carbon ring, and a transition metal ion selected from the Group VIII elements. Specific complexes mentioned are tris(4,4'-dicarboxy-2,2'- bipyridine)ruthenium(ll) complex, tris(4,4'- disulfonic acid-2,2'- bipyridine)ruthenium(ll) complex, tris(vasophenanthroline disulfonic acid)ruthenium(ll) complex, and tris(4,4'-dicarboxy-2,2'-bipyridine)iridium(ll) complex.

Whilst it is stated in EP 408748 that pH can be measured across a broad pH range using these probe complexes (3 to 10 in Example 1 ), in practice, the pH range in which these complexes are useful for providing an accurate pH measurement is much smaller - usually across only about two pH units. "A Wide-range Luminescent pH Sensor Based on Ruthenium(ll) Complex" (Kim et al, Bull. Korean Chem. Soc. 2009, Vol. 30, No. 3) describes the synthesis of [RuLL'₂]²⁺ where L is 4,7-dihydroxy-1 ,10-phenanthroline and L' is 4'-methyl-2,2'-bipyridine-4-carboxylic acid, and its use as a pH sensor. It is alleged by the authors that the emission intensity of this complex was strongly dependent upon pH over the broad range of pH from 3 to 9, and that this wide range of pH response was attributable to the multiple protonation-deprotonation equilibriums arising from the acidic functionalities (-CO₂H and -OH) with different pK_a values in the complex. However, since the coordinated 4,7-dihydroxy-1 ,10- phenanthroline ligand has a pK_a of approximately 2.3 in the excited state, the degree of protonation of the ligand should not vary significantly at a pH level of above 5. Moreover, protonation of the deprotonated hydroxypyridine ion ligand above pH ~ 4 in the excited state of the metal complex is not an equilibrium, but an irreversible proton transfer quenching, as has been described for other photoexcited complexes displaying low pK_a values (see, for instance, Marazuela, Moreno-Bondi and Orellana, Appl. Spectrosc. 1998, 52, 1314-1320). Such an irreversible quenching of the excited state of the luminescent Ru(ll) dye strictly follows the variations of the acid/base buffer species concentration with pH and not the hydron concentration in the medium, as a true pH sensor would require. This leads to sigmoidal plots of the luminescence intensity and lifetime of the Ru(ll) probe as a function of pH with inflection point at the buffer pK_a and an amplitude of the change that depends directly on the buffer concentration. Therefore, the use of a hydroxyl-substituted pyridine ligand cannot extend the pH range in which the complex provides useful pH sensitivity. Instead, the effect observed by the authors could be due to effects of the pH or of the buffer on the environment in which the luminescent complex is present. These effects depend not only on the pH of the solution, but also on the nature of the buffer, ionic strength and the presence of certain additives. Since these other parameters cannot be controlled in practice, such environmental effects have limited use as the basis for a pH sensor.

It is therefore an object of the invention to provide a luminescence-based pH sensing system that is effective over a broader pH range than known systems. Whilst Ru(l l) complexes can be used in solution, in many circumstances it is desirable to immobilise the Ru(ll) complexes. In EP408748, the complexes are adsorbed onto a macromolecular membrane or covalently bound to a macromolecular membrane, for example.

Known systems involving adsorption of Ru(l l) complexes onto a membrane, or entrapment of the complex within a hydrogel have been found to be prone to allowing leaching of the complex into solution. The development of further systems involving covalent bonding of the luminescent Ru(l l) complex to a polymer support would be desirable. In particular, a system where the luminescent complex is immobilised in such a way that uncontrollable environmental conditions do not influence the luminescence intensity or lifetime of the complex is desirable.

Therefore, a further object of the present invention is to provide a luminescence-based pH sensing system in which leaching of the luminescent complex is minimised. A further object is to provide a system in which luminescence intensity and lifetime are generally independent of environmental conditions other than pH.

Prior art

"Anchor-Functionalized Push-Pull-Substituted Bis(tridentate)

Ruthenium(ll) Polypyridine Chromophores: Photostability and Evaluation as Photosensitizers" (Breivogel et al, European Journal of Inorganic Chemistry, 2014, no. 16, pages 2720-2734) describes a Ru(ll) complex with the formula {[(2,2':6',2"-terpyridine)-4'-carboxylic acid][(2,2':6',2"-terpyridin)-4'-amine]} ruthenium(ll) dihexafluorophosphate (referred to in the publication as "[4](PF₆)₂"). This publication also describes a Ru(l l) complex with the formula {[(2,2':6',2"- terpyridine)-4,4',4"-tricarboxylic acid][A/,/V-dimethyl-A/,/V-dipyhdin-2-yl-(4- amino)pyridine-2,6-diamine]}ruthenium(ll) dihexafluorophosphate (referred to in the publication as "[3](PF₆)₂"). Each of these complexes has an amino substituted polypyridyl ligand, where, in contrast to the present invention, the amino group is directly attached to the polypyridyl.

"Ruthenium Polypyridyl Sensitisers in Dye Solar Cells Based on Mesoporous Ti0₂" (Reynal et al, European Journal of Inorganic Chemistry, 2011 , no.29, pages 4509-4526) describes the Ru(l l) complex {[(2,2'-bipyridine)- 4,4'-dicarboxylic acid](1 ,10-phenanthrolin-5-amine)bis(thiocyanato)} ruthenium(ll). Again, this complex has an amino substituted polypyridyl ligand, where, in contrast to the present invention, the amino group is directly attached to the polypyridyl.

Summary of the Invention

In a first aspect, the invention provides a luminescent Ru(ll) complex having:

at least one polypyridyl ligand having at least one proton-accepting group with a pK_a in the range 4 to less than 6, wherein the luminescence intensity and/or luminescence lifetime of the complex varies depending on the protonation state of the proton-accepting group; and

at least one polypyridyl ligand having at least one proton-donating group with a pK_a in the range 6 to 8, wherein the at least one proton-donating group is covalently linked to a ring carbon atom of the at least one polypyridyl ligand by a spacer group having the formula -(CH₂)_q- wherein q may be 1 , 2, or 3, and wherein the luminescence intensity and/or luminescence lifetime of the complex varies depending on the protonation state of the proton-donating group.

In a second aspect, the invention provides a polymer-bound luminescent Ru(ll) complex having:

at least one polypyridyl ligand having at least one proton-donating group with a pK_a in the range 6 to 8, wherein the at least one proton-donating group is covalently linked to a ring carbon atom of the at least one polypyridyl ligand by a spacer group having the formula -(CH₂)_q- wherein q may be 1 , 2, or 3, and wherein the luminescence intensity and/or luminescence lifetime of the complex varies depending on the protonation state of the proton-donating group,

wherein the luminescent Ru(ll) complex is bonded covalently to a polymer support through at least one of its ligands.

In a third aspect, the invention provides a pH sensor comprising a luminescent Ru(ll) complex according to the first aspect of the invention, or a polymer-bound luminescent Ru(l l) complex according to the second aspect of the invention, and a film support having a surface upon which the luminescent Ru(ll) complex or polymer-bound luminescent Ru(l l) complex is immobilised.

In a fourth aspect, the invention provides a reaction vessel having, adhered to an inner surface thereof, a pH sensor according to the third aspect of the invention.

In a fifth aspect, the invention provides a reaction vessel comprising, immobilised on an inner surface of the vessel, a luminescent Ru(ll) complex according to the first aspect of the invention or a polymer-bound luminescent Ru(ll) complex according to the second aspect of the invention.

In a sixth aspect, the invention provides a bioreactor comprising a reaction vessel of the invention, a light source, and a detector configured to detect the luminescence intensity or luminescence lifetime of the luminescent Ru(ll) complex.

In a seventh aspect, the invention provides the use of any of the products described above for determining the pH of a solution.

In an eighth aspect, the invention provides a process for covalently bonding a luminescent Ru(l l) complex to a polymer support, comprising providing a luminescent Ru(ll) complex according to the first aspect of the invention and reacting the luminescent Ru(ll) complex with a polymeric support to form a covalent bond.

In a ninth aspect, the invention provides a method of determining the pH of a solution comprising:

exposing a luminescent Ru(ll) complex according to the first aspect of the invention or polymer-bound luminescent Ru(ll) complex according to the second aspect of the invention to the solution;

illuminating the luminescent Ru(l l) complex or polymer-bound luminescent Ru(ll) complex so as to excite the complex;

detecting light emitted by the complex; and

determining the pH of the solution based on the luminescence intensity and/or the luminescence lifetime of the complex.

The present inventors have found that the pH range across which the pH can accurately be measured (dynamic range) can be broadened by providing, on the Ru(ll) complex, different polypyridyl ligands, each having a group having a pK_a in a different range.

Detailed Description of the Invention

Terminology

As used herein, the term "polypyridyl" refers to a compound or ligand having at least two linked pyridine rings. Polypyridyl ligands are generally bi- dentate or tri-dentate ligands that coordinate to a metal ion through the nitrogen atoms of two or three of their pyridine rings. As used herein, the term "polypyridyl" includes both polyaryls, such as bipyridyls and terpyridyls, and fused ring systems such as phenanthrolines.

As used herein, the term "proton" refers to an H⁺ cation, which is also known as a hydron.

As used herein, the term "(C_a-C_b)alkyl" wherein a and b are integers refers to a straight or branched chain alkyl radical having from a to b carbon atoms. Thus when a is 1 and b is 5, for example, the term includes methyl, ethyl, n-propyl, isopropyl, n- butyl, isobutyl, sec-butyl, t-butyl and n-pentyl.

As used herein the term "(C_a-Cb)alkylene" wherein a and b are integers refers to a saturated hydrocarbon chain having from a to b carbon atoms and two unsatisfied valences, such as -CH₂-, -CH₂CH₂-, -CH₂CH₂CH₂-, - CH₂CH(CH₃)CH₂- and -CH₂C(CH₃)₂CH₂-. For the avoidance of doubt, it is to be understood that a divalent branched chain (C_a-C_b)alkylene radical includes those wherein one of the carbons of the hydrocarbon chain is a ring carbon of a cycloalkyl ring (i.e. is a spiro centre).

As used herein the term "cycloalkyl" refers to a saturated carbocyclic radical having from 3-8 carbon atoms and includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

As used herein the term "carbocyclic" refers to a mono- or bi-cyclic radical whose ring atoms are all carbon, and includes monocyclic aryl, cycloalkyl, and cycloalkenyl radicals, provided that no single ring present has more than 8 ring members. A "carbocyclic" group includes a mono-bridged or multiply-bridged cyclic alkyl group. As used herein the term "aryl" refers to a mono-, bi- or tri-cyclic carbocyclic aromatic radical. Illustrative of such radicals are phenyl, biphenyl, napthyl, and anthryl.

As used herein the term "electropositive group" refers to a group that is less electronegative than nitrogen, such as hydrogen, optionally substituted (C_a- Cb)alkyl or optionally substituted cycloalkyl, which preferably is able to donate electrons by induction through a single chemical bond.

As used herein, the term "spacer group" refers to an aliphatic hydrocarbon chain that covalently links two atoms. The spacer group may be defined as a C_a-Cb alkylene group, or it may be defined as having the formula - (CH₂)q- wherein q is an integer.

As used herein, the symbol "^*" is used to represent the point of attachment of a group or formula to the remaining chemical structure.

Unless otherwise specified in the context in which it occurs, the term "substituted" as applied to any moiety herein means substituted with at least one substituent that does not interfere with the functionality of the Ru(ll) complex as a pH sensor, for example selected from (d-C₆)alkyl, (CrC₆)alkoxy, hydroxy, hydroxy(CrC₆)alkyl, mercapto, mercapto(C-i-C₆)alkyl, (CrC₆)alkylthio, halo (including fluoro and chloro), trifluoromethyl, trifluoromethoxy, nitro, nitrile (-CN), oxo, phenyl, -COOH, -COOR^A, -COR_A, -S0₂R^A, -CONH₂, -S0₂NH₂, -CONHR^A, -S0₂NHR^A, -CONR^AR^B, -S0₂NR^AR^B, -NH₂, -NHR^A, -NR^AR^B, -OCONH₂, -OCONHR^A, -OCONR^AR^B, -NHCOR^A, -NH^BCOOR^A, -NR^BCOOR^A, -NHS0₂OR^A, -NR^BS0₂OR^A, -NHCONH₂, -NR^ACONH₂, -NHCONHR⁶, -NR^ACONHR^B, -NHCONR^AR^B or -NR^ACONR^AR^B wherein R^A and R^B are independently a (CrC₆)alkyl group, or R^A and R^B when attached to the same nitrogen may form a cyclic amino ring such as a morpholinyl, piperidinyl or piperazinyl ring. An "optional substituent" or "substituent" may be one of the foregoing substituent groups.

The Ru(ll) complexes of the invention comprise groups that can be reversibly protonated or deprotonated in solution. These groups are represented herein in their charged state. Nevertheless, it should be understood that whether or not these groups are charged will depend on the environment in which they are present and it is, of course, not intended to exclude the possibility that these groups are present in their uncharged form. Description of Preferred Embodiments

The Ru(ll) complexes of the present invention comprise polypyridyl ligands as defined above. Of the various possible polypyridyl ligands, 2,2'- bipyridines and 2,10-phenanthrolines have been found to be most useful. 2,2'- bipyridines are especially preferred.

In the Ru(ll) complexes of the present invention, at least one polypyridyl ligand has at least one proton-accepting group, preferably two proton-accepting groups, with a pK_a in the range 4 to less than 6 wherein the luminescence intensity and/or luminescence lifetime of the complex varies depending on the protonation state of the proton-accepting group. Preferably, the at least one proton-accepting group is directly linked to a ring carbon atom of the at least one polypyridyl ligand.

Also in the Ru(ll) complexes of the present invention, at least one polypyridyl ligand has at least one proton-donating group, preferably two proton- donating groups, with a pK_a in the range 6 to 8, wherein the at least one proton- donating group is covalently linked to a ring carbon atom of the at least one polypyridyl ligand by a spacer group having the formula -(CH₂)_q- wherein q may be 1 , 2, or 3, and wherein the luminescence intensity and/or luminescence lifetime of the complex varies depending on the protonation state of the proton- donating group.

The present inventors have found that by using a spacer group having the formula -(CH₂)_q- wherein q may be 1 , 2, or 3, the pK_a values fall within the ranges stated above. Preferably, q = 1 , such that the spacer group has the formula -(CH₂)-.

This is in contrast to the compounds of the prior art, for instance, the {[(2,2':6',2"-terpyridine)-4'-carboxylic acid][(2,2':6',2"-terpyridin)-4'-amine]} ruthenium(ll) dihexafluorophosphate complex described in Breivogel et al (discussed under "prior art"). For this complex, the pK_a values of the carboxylate group and the amino group fall outside of the claimed ranges. The pK_a values of the carboxylate group and the amino group of this complex are described in "Multielectron Storage and Photo-Induced Electron Transfer in Oligonuclear Complexes Containing Ruthenium(ll) Terpyridine and Ferrocene Building Blocks" (Heinze et al, European Journal of Inorganic Chemistry, 2006, pages 2040-2050). Specifically, the carboxylate group of this complex displays a pK_a value of 2.7, and the amino group displays a pK_a value higher than 12.

Without wishing to be bound by theory, it is thought that the spacer group having the formula -(CH₂)_q- wherein q may be 1 , 2, or 3, alters the electronic properties of the complex, and so affects the electron-donating and electron- accepting properties of the electron-donating and electron-accepting groups within the complexes of the present invention, allowing the pK_a values to fall within the claimed ranges.

Where the pK_a of the proton-accepting group differs more significantly from the pK_a of the proton-donating group it is generally possible to provide a sensor that is effective over a wider range of pH. However, if the pK_a of the proton-accepting group differs from the pK_a of the proton-donating group by too great a margin, sensitivity could be lost in the middle of the range. Therefore, preferably, proton-accepting group has a pK_a in the range 4.5 to 5.5 and the proton-donating group has a pK_a in the range 6.5 to 7.5. Preferably, the pK_a of the proton-accepting group differs from the pK_a of the proton-donating group by from 1 to 3 units, more preferably by from 1.5 to 2.5 units.

As used herein, pK_a refers to the ground state pK_a of the group in the context of the specific Ru(ll) complex or pH sensing system in which it is present. This will often be quite different from the pK_a of the same group in the corresponding free polypyridyl compound (i.e. when the compound is not attached as a ligand).

The pK_a of a group can be measured by any known method. Suitable methods are described in "The Determination of Ionization Constants: A Laboratory Manual" by Adrien Albert and E. P. Serjeant (Chapman and Hall, New York). One example of a method by which the pK_a of the groups on the coordinated ligands can be measured is UV-visible absorption or emission spectroscopy.

Preferably, the proton-accepting group is a carboxylate group. Preferably, the proton-donating group is an ammonium group. More preferably the proton-accepting group is a carboxylate group and the proton-donating group is an ammonium group. The inventors have found that use of a ligand having a carboxylate group generally provides pH sensitivity in an acidic pH range and use of a ligand having an ammonium group provides sensitivity at a higher, mildly acidic, neutral and alkaline pH range. Use of these ligands together has been found to be particularly effective at providing pH sensitivity in a broad pH range.

Preferably, the proton-donating group has the formula (i):

(i)

wherein each of R¹ and R² are electropositive groups.

More preferably, the polypyridyl ligand having at least one proton- donating group includes the following pyridine ring structure (ii) (as can be seen from structure (ii) below, this pyridine ring structure consists of one of the at least one proton-donating groups, the spacer group, and one of the pyridine rings of the polypyridyl ligand structure):

wherein the nitrogen atom of the pyridine ring is coordinated to the Ru(ll) of the luminescent Ru(ll) complex;

R¹⁰ and R¹¹ are independently selected from H and a covalent bond to the remainder of the polypyridyl ligand structure, at least one of R¹⁰ and R¹¹ being a covalent bond to the remainder of the polypyridyl ligand structure;

each of R¹ and R² are electropositive groups; and

wherein q may be 1 , 2, or 3.

Preferably, R¹ and R² are independently selected from H, optionally substituted C-i-ds alkyl and optionally substituted cycloalkyl.

Preferably, R¹ and R² are independently selected from H and C-i-do alkyl optionally substituted with one or more phenyl groups. Preferably, R¹ and R² are the same or different and are each optionally substituted C-_I-C-_IO alkyl, wherein the optional substitution is preferably with one or more phenyl groups.

Preferably, R¹ and R² are the same or different and are each optionally substituted C-₁-C5 alkyl, wherein the optional substitution is preferably with one or more phenyl groups.

More preferably, R¹ and R² are the same or different and are each unsubstituted C C₅ alkyl.

Preferably, R¹ and R² are the same.

Most preferably, R¹ and R² are ethyl.

The present inventors have found that when each of R¹ and R² are the electropositive groups described above in the context of formula (i) and structure (ii), a particularly broad pH response range is achieved. This is especially the case when each of R¹ and R² are the preferable groups described above, particularly when each of R¹ and R² are ethyl.

Preferably, the polypyridyl ligand having at least one proton-accepting group includes the following pyridine ring structure (iii) (as can be seen from structure (iii) below, this pyridine ring structure consists of one of the at least one proton-accepting groups, and one of the pyridine rings of the polypyridyl ligand structure):

R¹² and R¹³ are independently selected from H and a covalent bond to the remainder of the polypyridyl ligand structure, at least one of R¹² and R¹³ being a covalent bond to the remainder of the polypyridyl ligand structure; and

R⁵ is carboxylate. The luminescent Ru(ll) complex of the invention in some embodiments has the formula (iv):

wherein R¹, R², R³ and R⁹ are independently selected from H, optionally substituted C-i-ds alkyi and optionally substituted cycloalkyi; and

wherein R⁴, R⁵, R⁶ and R⁷ are selected from H, optionally substituted C C-io alkyi, optionally substituted cycloalkyi and carboxylate, at least one of R⁴ and R⁵ being carboxylate and at least one of R⁶ and R⁷ being carboxylate.

Preferably each of R⁴, R⁵, R⁶ and R⁷ is carboxylate.

Preferably R¹, R², R³ and R⁹ are independently selected from H and d- C5 alkyi optionally substituted with one or more phenyl groups.

Preferably R¹ and R³ are the same or different and are each optionally substituted C1-C10 alkyi, wherein the optional substitution is preferably with one or more phenyl groups.

Preferably R¹ and R³ are the same or different and are each optionally substituted C1-C5 alkyi, wherein the optional substitution is preferably with one or more phenyl groups. Preferably R¹, R², R³ and R⁹ are the same or different and are each optionally substituted C-I-C-IO alkyl, wherein the optional substitution is preferably with one or more phenyl groups.

Preferably R¹, R², R³ and R⁹ are the same or different and are each optionally substituted C-i-C₅ alkyl. More preferably, R¹, R², R³ and R⁹ are the same or different and are each C1-C₅ alkyl, optionally substituted with phenyl. Even more preferably, R¹, R², R³ and R⁹ are the same or different and are each unsubstituted C C₅ alkyl.

Most preferably R¹, R², R³ and R⁹ are ethyl.

The present inventors have found that, when R¹, R², R³ and R⁹ in the context of formula (iv) are independently selected from H, optionally substituted C-1 -C-15 alkyl and optionally substituted cycloalkyl (which are electropositive groups in the context of the present invention) a particularly broad pH response range is achieved. This is especially the case when R¹, R², R³ and R⁹ are each any of the preferable groups described above, particularly when R¹, R², R³ and R⁹ are each ethyl.

The Ru(ll) complexes of the invention can be synthesised using known methods. One example of a synthesis route for a specific Ru(ll) complex, starting from commercially available starting materials (1 ) and (3), is set out below:

CI CI

The luminescent Ru(ll) complexes of the invention can be used in solution. However, in most cases it is desirable to immobilise the complex, usually on the surface of a film or on the inner surface of a reaction vessel. This can be done in a number of ways. For example, the Ru(ll) complexes can be contained within a hydrophilic hydrogel. Alternatively, the Ru(ll) complex can be immobilised by electrostatic interaction with a polymer. For example, perfluorinated ionomer networks (e.g. Nafion®), functionalised nylon (e.g. Biodyne® A, B or C from Pall Corp.), functionalised glass beads/plates (e.g. Biotage Isolute® SAX or SCX), functionalized silica gel beads/plates (e.g. Siliabond® amine or propylsulfonic acid from Silicycle), or ion-exchange polystyrene (e.g. Amberlite® or Dowex® resins) can be used. The polymer can then be adhered to the surface of a film or on an inner surface of a reaction vessel.

However, in a preferred embodiment of the invention, the Ru(ll) complex is immobilised by covalent bonding through one of its ligands to a polymeric support, and the polymeric support is then immobilised on the surface of a film support or on an inner surface of a reaction vessel.

The process for covalently bonding a luminescent Ru(ll) complex to a polymer support comprises reacting a luminescent Ru(ll) complex of the invention with the polymeric support so as to form a covalent bond.

The polymeric support can generally be any polymer to which a ligand of the Ru(ll) complex can be covalently bonded and which is capable of immobilising the Ru(ll) complex. Polymers that have been found to be particularly effective are in the form of microbeads of a hydrophobic polymer having hydrophilic polymeric chains grafted thereon. Preferably, the polymeric support is a polyethylene glycol-polystyrene graft copolymer microbead. Microbeads generally have a number-based (D[1 ,0]) average diameter in the range 5 to 50 micrometers, usually 5 to 20 micrometers, as measured by optical or electron microscopy. Polymer supports of this type have been used in the past as solid supports for peptide synthesis and are commercially available from Rapp Polymere GmbH under the name Tentagel® and are described in "Towards the Chemical Synthesis of Proteins" (E. Bayer, Angew. Chem. Int. Ed. Engl. 30 (1991) 113-129). The polyethylene glycol chains of these polymers are functionalised to allow reaction with an amine or carboxylate group so as to form a covalent bond. For example, the polyethylene glycol chains of the polyethylene glycol-polystyrene graft copolymer microbeads can be functionalised with a leaving group (Br for example) to allow nucleophilic substitution by an amine group. Covalent bonding of the Ru(ll) complexes to these polymer supports has been found to minimise or even prevent leaching of the Ru(ll) complexes into solution.

Preferably, the luminescent Ru(ll) complex that is reacted with the polymeric support comprises an ammonium group as its proton-donating group, the polymeric support that is reacted with the luminescent Ru(ll) complex comprises polyethylene glycol chains functionalised with a leaving group and the reaction involves nucleophilic substitution of the leaving group of the polyethylene glycol chain for an ammonium group of the Ru(ll) complex (although it will be appreciated that the ammonium group needs to be present to some extent as an amine for the reaction to occur). The leaving group is preferably selected from iodine, bromine, chlorine, tosyl, brosyl, mesyl, triflyl, methylsulfyl and hydroxyl.

Preferably, the reaction forms an ammonium group. If the reaction forms a quaternary ammonium group, then preferably one of the substituents of the quaternary ammonium group is subsequently eliminated by treatment with a base to form a tertiary amine. This allows the linking group to contribute to the pH sensitivity of the bound complex.

It is also possible for the reaction to form an amide group, although this is less preferred, because the amide linker group cannot be formed where the starting amine has bulky substituents, or is tertiary. Furthermore, the amide linker itself is not basic, so cannot act as a proton-donating or proton-accepting group. In such circumstances a separate proton-donating or proton-accepting group needs to be present on the ligand to provide pH sensitivity.

In some cases, where the proton-accepting group with a pK_a in the range 4 to less than 6 is a carboxylate group, the immobilisation process can result in attachment of the carboxylate group to the polymer support by formation of an ester linkage. This type of linkage is less preferred, because it is relatively easily hydrolysed under acidic or basic conditions. Therefore, where another linkage, such as an amine or amide linkage, is also present, it is preferred to hydrolyse the ester linkages before use of the polymer-bound luminescent Ru(ll) complex in a sensor.

Within the polymer-bound luminescent Ru(ll) complex, the at least one polypyridyl ligand having at least one proton-donating group preferably has two proton-donating groups.

Within the polymer-bound luminescent Ru(ll) complex, the at least one polypyridyl ligand having at least one proton-accepting group preferably has two proton-accepting groups. Preferably, the at least one proton-accepting group is directly linked to a ring carbon atom of the at least one polypyridyl ligand.

Preferably, within the polymer-bound luminescent Ru(ll) complex, q = 1 , such that the spacer group has the formula -(CH₂)-.

Preferably, within the polymer-bound luminescent Ru(ll) complex, the least one polypyridyl ligand having at least one proton-donating group is a 2,2'- bipyridyl ligand.

Preferably, within the polymer-bound luminescent Ru(ll) complex, the least one polypyridyl ligand having at least one proton-accepting group is a 2,2'- bipyridyl ligand.

Preferably, within the polymer-bound luminescent Ru(ll) complex, the proton-donating group has the formula (i):

wherein each of R¹ and R² are electropositive groups. More preferably, within the polymer-bound luminescent Ru(ll) complex, the polypyridyl ligand having at least one proton-donating group includes the following pyridine ring structure (ii) (as can be seen from structure (ii) below, this pyridine ring structure consists of one of the at least one proton-donating groups, the spacer group, and one of the pyridine rings of the polypyridyl ligand structure):

(ii)

each of R¹ and R² are electropositive groups; and

wherein q may be 1 , 2, or 3.

Preferably, R¹ and R² are independently selected from H, optionally substituted C-i-ds alkyl, optionally substituted cycloalkyl and * Y PS, wherein PS represents a polymer support and Y represents a spacer that covalently links the Ru(ll) complex to the polymer support.

Preferably R¹ and R² are independently selected from H and C-i-do alkyl optionally substituted with one or more phenyl groups.

Preferably R¹ and R² are the same or different and are each optionally substituted d-do alkyl, wherein the optional substitution is preferably with one or more phenyl groups.

Preferably R¹ and R² are the same or different and are each optionally substituted C1-C5 alkyl, wherein the optional substitution is preferably with one or more phenyl groups. More preferably, R¹ and R² are the same or different and are each unsubstituted C C₅ alkyl.

Preferably R¹ and R² are the same.

Most preferably, R¹ and R² are ethyl.

Preferably, within the polymer-bound luminescent Ru(ll) complex, the polypyridyl ligand having at least one proton-accepting group includes the following pyridine ring structure (iii) (as can be seen from structure (iii) below, this pyridine ring structure consists of one of the at least one proton-accepting groups, and one of the pyridine rings of the polypyridyl ligand structure):

R⁵ is carboxylate.

The polymer-bound luminescent Ru(ll) complex preferably has the formula (v):

wherein R¹, R², R³ and R⁸ are independently selected from H, optionally substituted C₁-C15 alkyi, optionally substituted cycloalkyi and * Y PS, wherein PS represents a polymer support and Y represents a spacer that covalently links the Ru(ll) complex to the polymer support; and

wherein R⁴, R⁵, R⁶ and R⁷ are selected from H, optionally substituted d- C-io alkyi, optionally substituted cycloalkyi and carboxylate, at least one of R⁴ and R⁵ being carboxylate and at least one of R⁶ and R⁷ being carboxylate.

Preferably, each of R⁴, R⁵, R⁶ and R⁷ is carboxylate.

Preferably, R¹, R², R³ and R⁸ are independently selected from H and Ci- C₅ alkyi optionally substituted with one or more phenyl groups.

Preferably, R¹ and R³ are the same or different and are each optionally substituted C₁-C₁₀ alkyi and R⁸ is H, wherein the optional substitution is preferably with one or more phenyl groups.

Preferably, R¹ and R³ are the same or different and are each optionally substituted C₁-C5 alkyi, wherein the optional substitution is preferably with one or more phenyl groups. Preferably, R¹, R² and R³ are the same or different and are each optionally substituted C-I-C-IO alkyl and R⁸ is H, wherein the optional substitution is preferably with one or more phenyl groups.

More preferably, R¹, R² and R³ are the same or different and are each optionally substituted C-1-C5 alkyl and R⁸ is H, wherein the optional substitution is preferably with one or more phenyl groups.

Most preferably, R¹, R² and R³ are ethyl and R⁸ is H.

In another preferable embodiment, R¹, R² and R³ and R⁸ are ethyl.

Y can be any spacer that covalently links the Ru(ll) complex to a polymeric support and is conveniently a C1 -C5 alkylene group. Preferably, Y is linked to a polyethylene glycol chain of the polyethylene glycol-polystyrene graft copolymer microbead.

In one preferred embodiment, ^* Y PS can be represented by

wherein n is from 1 to 5, m has an average value in the range 20 to 200 and X represents a polystyrene chain of the polyethylene glycol-polystyrene graft copolymer microbead. More preferably, m has an average value in the range 50 to 100 and most preferably the average value of m is in the range 60 to 80. The average value of m can be determined by finding the weight average molecular weight of the polyethylene glycol chains before they are grafted onto the polystyrene bead by light scattering or by size exclusion chromatography. The weight-based average value of m can then be calculated directly.

According to one aspect of the present invention, pH sensors are provided, which comprise a luminescent Ru(ll) complex as described above, or a polymer-bound luminescent Ru(ll) complex as described above, and a film support having a surface upon which the luminescent Ru(ll) complex or polymer- bound luminescent Ru(ll) complex is immobilised.

When the sensor comprises a luminescent Ru(ll) complex that is not covalently bound to a polymer support, the complex can be immobilised by other means. In a preferred embodiment, the luminescent Ru(ll) complex is immobilised by being contained within a hydrophilic hydrogel that is adhered to the surface of the film support. The hydrophilic hydrogel is preferably selected from hydrophilic polyurethane hydrogel, polyacrylamide hydrogel, silicone hydrogel, polyvinylalcohol hydrogels, hydroxy- or amine-substituted methacrylates cross-linked with ethyleneglycol dimethacrylate and co-polymers of the monomers included in the above polymer materials. The most preferred hydrophilic hydrogel is a hydrophilic polyurethane hydrogel.

When the sensor comprises a polymer-bound luminescent Ru(ll) complex, the polymer-bound complex can nevertheless be contained within a hydrophilic hydrogel. However, it has been found that the hydrogel need not be used and the polymer-bound luminescent Ru(ll) complex can be adhered directly to the surface of the film support with an adhesive. Acrylic adhesives have been found to be particularly suitable for this purpose.

The sensor preferably comprises an opacifier. The use of an opacifier can also assist with absorption of light by the luminescent Ru(ll) complex. Suitable opacifiers include Ti0₂ and Si0₂ particles, white poly(tetrafluoroethylene) particles, and white hydrophilic polycarbonate. Ti0₂, Si0₂ and poly(tetrafluoroethylene) particles can be mixed with the polymer- bound luminescent Ru(ll) complex before application to the film support, or dispersed in the hydrophilic hydrogel when present. In a preferred embodiment, however, the sensor comprises an opacifier that forms a coating over the luminescent Ru(ll) complex, over the polymer-bound luminescent Ru(ll) complex, or over the layer of hydrophilic hydrogel. An opacifier in this form is particularly preferred when the sensor comprises a polymer-bound luminescent Ru(ll) complex that is adhered directly to the surface of the film support with an adhesive. In these circumstances, the coating can help to prevent the polymer- bound luminescent Ru(ll) complex from becoming detached from the surface of the film support. White hydrophilic polycarbonate is preferably used as the opacifier when it is coated on in this way.

The sensor can then be used by adhering it an inner surface of a reaction vessel. Thus, in a further aspect, the invention provides a reaction vessel having, adhered to an inner surface thereof, such a pH sensor.

A sensor can also be formed directly on the surface of a reaction vessel. Thus in a further aspect, the invention provides a reaction vessel comprising, immobilised on an inner surface of the vessel, a luminescent Ru(ll) complex according to the invention or a polymer-bound luminescent Ru(l l) complex according to the invention.

When the luminescent Ru(l l) complex is not covalently bound to a polymer support, the complex can be immobilised on the surface of the reaction vessel by other means. In a preferred embodiment, the luminescent Ru(l l) complex is immobilised by being contained within a hydrophilic hydrogel that is adhered to the inner surface of the vessel. One particularly suitable hydrogel for this purpose is a hydrophilic polyurethane hydrogel.

When a polymer-bound luminescent Ru(ll) complex is immobilised on the surface of the reaction vessel, the polymer-bound complex can nevertheless be contained within a hydrophilic hydrogel. However, it has been found that the hydrogel need not be used and the polymer-bound luminescent Ru(ll) complex can be adhered directly to the surface of the vessel with an adhesive. Acrylic adhesives have been found to be particularly suitable for this purpose.

The use of an opacifier can also assist with absorption of light by the luminescent Ru(l l) complex when the luminescent Ru(l l) complex or polymer- bound luminescent Ru(l l) complex is immobilised on an inner surface of a reaction vessel. Suitable opacifiers, include TiO₂ and SiO₂ particles, white poly(tetrafluoroethylene) particles and white hydrophilic polycarbonate. TiO₂, SiO₂ and poly(tetrafluoroethylene) particles can be mixed with the polymer- bound luminescent Ru(ll) complex before application to the surface of the reaction vessel, or dispersed in the hydrophilic hydrogel when present. In a preferred embodiment, however, the sensor comprises an opacifier that forms a coating over the luminescent Ru(ll) complex, over the polymer-bound luminescent Ru(ll) complex, or over the layer of hydrophilic hydrogel. An opacifier in this form is particularly preferred when a polymer-bound luminescent Ru(ll) complex is adhered directly to the surface of the reaction vessel with an adhesive. In these circumstances, the coating can help to prevent the polymer- bound luminescent Ru(ll) complex from becoming detached from the surface of the reaction vessel. White hydrophilic polycarbonate is preferably used as the opacifier when it is coated on in this way.

The reaction vessels described above are particularly useful as part of a bioreactor system that further comprises a light source, and a detector configured to detect the luminescence intensity or luminescence lifetime of the luminescent Ru(ll) complex or polymer-bound luminescent Ru(ll) complex. In an embodiment, the bioreactor system further comprises an optical fibre configured to transmit light from the light source to the reaction vessel. The optical fibre can also be configured to transmit light emitted by the luminescent Ru(ll) complex to the detector. Alternatively, the bioreactor system can comprise a further optical fibre configured to transmit light emitted by the luminescent Ru(ll) complex to the detector. The wide response range over which pH can be measured means that the bioreactor systems of the present invention allow measurement of pH in both microbial and mammalian cell cultures.

The pH of a solution can be measured by a method comprising exposing a luminescent Ru(ll) complex of the invention or a polymer-bound luminescent Ru(ll) complex according to the invention to the solution; irradiating the luminescent Ru(ll) complex or polymer-bound luminescent Ru(ll) complex so as to excite the complex; detecting light emitted by the complex; and determining the pH of the solution based on the luminescence intensity and/or the luminescence lifetime of the complex.

The lifetime of the luminescent excited state of the indicator dye, also called luminescence lifetime or emission lifetime (r), can be measured by several known techniques (see, for instance, J. N. Demas, "Excited State Lifetime Measurements", Academic Press, New York, 1983; A. Juris and M. Maestri, in "The Exploration of Supramolecular Systems and Nanostructures by Photochemical Techniques", P. Ceroni (Ed.), Springer, Science+Bussiness Media, Dordrecht, The Netherlands, 2012; pp. 167-184). These techniques are broadly classified into "time-resolved' and "phase-sensitive" techniques.

Time-resolved emission measurements are based on monitoring the decay of the luminescence from the photoexcited indicator dye after its excitation with a short pulse of light (typically from a laser or pulsed xenon lamp source) of a wavelength comprised within any of the absorption bands of the luminescent indicator dye in the near-infrared, visible or ultraviolet regions of the electromagnetic spectrum. The time-dependent electrical signal from the photon detector, 1(f), elicited by the arrival of the emitted photons from the indicator dye and proportional to the indicator emission intensity, is converted into a digital format with a fast transient digitiser or any other device capable of recording the fast-decaying signal in real time, for the data analysis. Once in digital format, the indicator emission decay is usually fitted to a multi-exponential equation of the type:

where the number of exponentials (n) in the above equation is typically between 1 and 4, A is an offset value that accounts for the signal background, B is the time-independent "pre-exponential factor" and t is the time elapsed since the excitation light pulse was fired. Mathematical fit of the digitized experimental decay curve to the above equation, using well known algorithms such as the weighted linear least squares, Marquardt-Levenberg, grid-search, or the like, starts always with n = 1 , and subsequent exponential terms are added until an acceptable quality of the fit is achieved as judged by one or more of the following parameters: the χ² statistical parameter, visual inspection of the good ness-of -fit (coincidence between the experimental and fitted data), the Durbin-Watson parameter, the autocorrelation function, etc. The best fit allows determination of the A and B, parameters of the above equation.

Alternatively, other methods based on the analysis of the emission decay curve may be used, one of the most popular being the so-called "rapid lifetime determination" (see, for instance, K. K. Sharman, et al. Anal. Chem. 1999, 71, 947-952). Commercial instruments to measure emission lifetimes by time- resolved techniques include, for instance, the Edinburgh Instruments (Livingston, UK) LP920 laser kinetic spectrometer or the Luzchem (Gloucester, Canada) LFP-11 1 laser flash photolysis system.

Also in the time domain, but based on a different principle of the emission detection, are methods to determine the emission lifetime of an indicator dye using single photon timing (SPT, also called "time-correlated single photon timing" or TC-SPC) detection of the emission decay upon pulsed excitation. These methods consist on measuring the arrival time of a single photon of the light emitted by the indicator dye after each pulse of excitation light was fired. After proper calibration of the instrument, the emission decay can be reconstructed from the measured histogram of the probability of finding a single photon emitted at each time after firing the excitation pulse of light. Once reconstructed, the decay curve fitting techniques are routinely used. Several manufacturers market instruments for measuring τ using the SPT technique, such as Horiba Scientific (Longjumeau, France) Fluoromax-4 and Fluorolog Fluorometers, Edinburgh Instruments (Livingston, UK) FS5, FLS980 and LifeSpecll spectrometers.

Unlike "time-resolved" procedures, "phase-sensitive" methods are based on excitation of the indicator dye (either immobilised or in solution) with a sinusoidally-modulated excitation light of a wavelength that can be absorbed by the indicator dye. In this case, the luminescent emission from the photoexcited indicator dye will also be modulated with the same frequency than that of the excitation light. However, due to the finite lifetime of the emissive excited state, the two sine waves will be shifted ("phase shift angle" or just "phase shift") with respect to each other by an angle ( ) given by the equation:

where / is the modulation frequency of the excitation source selected by the user (around 1/r) to be in the optimum region (maximum slope) of the tan trigonometric function and r is the emission lifetime defined above. Alternatively, the variation of the modulation of the emission wave (instead of the phase shift) with the luminescence lifetime can be employed as the analytical signal. There are commercial instruments that can measure the emission phase shift, such as the ISS (Champaign, IL, USA) ChronosFD Fluorometer, Horiba Scientific (Longjumeau, Francia) MF² Fluorometer, Presens GmbH (Regensburgh, Germany) Microx and Fibox transmitters, etc.

Examples

Example 1a: Synthesis of c/s-dichlorobis (2,2'-bipyridyl-4,4'-dicarboxylic acid) ruthenium(ll)

This complex was synthesized using Graetzel's procedure (see P. Liska, et al, J. Am. Chem. Soc. 1988, 110, 3686-3687 and Md. K. Nazeeruddin, et al, Inorg. Chem. 1999, 38, 6298-6305). RuCI₃-3H₂0 (Alfa Aesar, Germany) (526 mg, 2.0 mmol) was dissolved in 50 mL of DMF under argon. After the solution was stirred for 15 min, 50 mL more of DMF was added. To this solution, 2,2'- bipyridine-4,4'-dicarboxylic acid (Alfa Aesar, Germany) ligand (946 mg, 3.9 mmol) was added. The flask content was refluxed at 175 °C with vigorous stirring for 3 h in the dark and under argon atmosphere. The reaction progress was monitored by UV-vis spectroscopy until the relative intensities of the absorption maxima (in ethanol) at 565, 414 and 316 nm were 1 :1.05:3.33, respectively. Then, the reaction mixture was allowed to cool to room temperature and filtered through a sintered glass funnel. The DMF solvent was evaporated completely in a rotary evaporator under vacuum. The resulting solid product was washed with a 1 :4 mixture of acetone and diethyl ether. The solid was stirred again in 100 mL of 2 M HCI for 4 h in the darkness and the mixture was filtered through a sintered glass funnel. The solid was dried under vacuum (0.1 mbar) for 24 h to yield 1.03 g (80%) of a dark purple solid.

Example 1 b: Synthesis of 4,4'-bis(bromomethyl)-2,2'-bipyridine

4,4'-Bis(hydroxymethyl)-2,2'-bipyridine (TCI, Japan) (900 mg, 4.2 mmol) was dissolved in a mixture of 48% HBr (Sigma-Aldrich, Germany) (20 mL) and 98% concentrated sulfuric acid (6.7 mL). The resulting solution was refluxed for 6 h, allowed to cool to room temperature and then 40 mL of water was added. The pH was adjusted to 7.0 with NaOH solution and the resulting precipitate was filtered, washed with purified water and air-dried. The product was dissolved in chloroform (40 mL) and filtered. The solution was dried over magnesium sulfate and evaporated to dryness to yield 960 mg (68%) of a light yellow powder. ¹H NMR (300 MHz, CDCI₃; δ/ppm): 4.50 (4H, s); 7.38 (2H, d); 8.45 (2H, s); 8.68 (2H, d).

Example 1 c: Synthesis of 4,4'-bis((diethylamino)methyl)-2,2'-bipyridine

4,4'-Bis(bromomethyl)-2,2'-bipyridine (350 mg, 1.02 mmol) and anhydrous potassium carbonate (281 mg, 2.04 mmol) were added to benzene (10 mL) that had been flushed previously with argon. Then, a mixture of diethylamine (2 mL, Sigma-Aldrich) and benzene (3 mL) was added to the bipyridine solution. The resulting reaction mixture was heated at 45 °C overnight. After cooling the reaction mixture to room temperature, the potassium carbonate was removed by filtration and discarded, and the filtrate was rotary evaporated under vacuum to dryness to yield a raw solid product. The latter was purified by column chromatography on silica, starting with 10:1 hexane/ethyl acetate as the eluant. Finally, a 1 % solution of 2.0 M ammonium hydroxide in CH₃OH was used to elute the product from the column. The product fraction was collected and rotary evaporated to dryness. Then, it was re-dissolved in ethyl acetate and dried under CaS0₄ overnight, filtered and the filtrate evaporated under vacuum to yield 280 mg (84%) of a viscous light yellow- coloured product. ¹H NMR (300 MHz, CDCI₃; δ/ppm): 8.6 (2H, d); 8.2 (2H, d); 7.3 (2H, dd); 3.6 (4H, s), 2.5 (8H, c), 1.0 (12H, t). Example 1 d: Synthesis of [(4,4'-bis((diethylamino)methyl)-2,2'-bipyridine)bis(2,2'- bipyridyl-4,4'-dicarboxylic acid)lruthenium(ll) (abbreviated as Ru(DCB)?DEAMB) To a solution of 292 mg (0.44 mmol) c/s-dichlorobis(2,2'-bipyridyl-4,4'- dicarboxylic acid)ruthenium(ll) in 5 mL of an aqueous NaOH solution (72 mg, 1.80 mmol) was added a solution of 148 mg (0.47 mmol) of diethylaminomethylbipyridine (1c) in 5 mL of methanol. The mixture was heated at reflux in the dark under argon atmosphere for 21 h. The solvents were removed by rotary evaporation under vacuum and the crude dark red residue was dissolved in a minimum amount of water, loaded on a SP-Sephadex C-25 (GE Healthcare, USA) column and eluted with pure water. The pure fractions of the target product were collected in the last coloured band and rotary evaporated to dryness. ¹H NMR (300 MHz, D₂0; δ/ppm): 9.1 (4H, s); 8.7 (2H, s); 7.9 (8H, m); 7.7 (2H, m); 7.5 (2H, m); 3.4 (4H, s); 2.6 (8H, c); 1.1 (12H, t).

Example 1 e: Immobilisation of r(4,4'-bis((diethylamino)methyl)-2,2'- bipyridine)bis(2,2'-bipyridyl-4,4'-dicarboxylic acid)lruthenium(ll) on TentaGel® beads.

A 40-80 mg amount of TentaGel® M Br beads (RAPP Polymere, Germany) was weighed in a 2 mL Eppendorf (original) tube and 2.5 μί per mg of beads of an aqueous 5 mg/mL solution of Ru(ll) dye was added. The TentaGel® beads suspension into the (closed) Eppendorf tubes was heated at 80 °C in an oven for 18 h. Then, 400 μί of water was added to the mixture, centrifuged at 13400 rpm for 3 min and the supernatant was removed. The centrifugation step was repeated with several amounts (400 μί each) of water until the supernatant became colourless. The dyed beads were suspended in 400 μΙ_ of a 50 mM pH-1 1.5 phosphate buffer solution containing also 50 mM of KCI. The dyed TGMB suspension was magnetically stirred at 250 rpm while heated at 80 °C for 18 h. Then, the mixture was centrifuged at 13400 rpm for 3 min and the supernatant was removed. The centrifugation step was repeated with several amounts (400 μΙ_ each) of water amounts until the supernatant appeared colourless. After removing the supernatant, the alkali-treated dyed beads were stored wet and in the dark at room temperature. Example 1f: Formation of a pH sensor

Appropriate amounts of Polidisp® 2731 aqueous emulsion (Resiquimica, S.A., Porto, Portugal) and purified water (1 :9 v/v) were thoroughly mixed. A 5 μΙ_ drop of the mixture was deposited and spread to a ca. 4-mm circle over biaxially oriented thermally stabilised 175 μιη Mylar® (Goodfellow, USA) sheet and allowed to dry for 30 min at room temperature. Then, 10-20 μΙ_ of the dyed wet beads were mixed with 5 vol of purified water and homogenized. A 3 μΙ_ drop of the diluted dyed beads suspension was deposited, spread to a ca. 3-mm circle over the Polidisp glue and allowed to dry for 30 min at room temperature. The pH sensing spot was finally covered with a 4-mm disk of hydrophilic white polycarbonate (Pall, USA, ref. 66629) as opacifying layer. Finally, 5 mm discs of the pH-sensitive spots were cut with a die.

Example 2: Synthesis of [(4,4'-bis((n-butylamino)methyl)-2,2'-bipyridine)bis(2,2'- pipyridyl-4,4'-dicarboxylic acid)lruthenium(ll) (abbreviated as Ru(DCB)?BAMB) [(4,4'-Bis((n-butylamino)methyl)-2,2'-bipyridine)bis(2,2'-bipyridyl-4,4'-dicarboxylic acid)]ruthenium(ll) was synthesised using the method described in Example 1 , except that n-butylamine was used in place of diethylamine. The complex was immobilised on Tentagel® beads using the method described in Example 1 e and a pH sensor was formed with the bound complex using the method described in Example 1f.

Example 3: Synthesis of i(4,4'-bis((amino)methyl)-2,2'-bipyridine)bis(2,2'- bipyridyl-4,4'-dicarboxylic acid)lruthenium(ll) (abbreviated as Ru(DCB)?AMB) [(4,4'-Bis((amino)methyl)-2,2'-bipyridine)bis(2,2'-bipyndyl-4,4'-dicarboxyli acid)]ruthenium(ll) was synthesised using the method described in Example 1 , except that ammonia was used in place of diethylamine. The complex was immobilised on Tentagel® beads using the method described in Example 1 e and a pH sensor was formed with the bound complex using the method described in Example 1f.

Example 4: Synthesis of [(4,4'-bis((dibenzylamino)methyl)-2,2'- bipyridine)bis(2,2'-bipyridyl-4,4'-dicarboxylic acid)lruthenium(ll) (abbreviated as Ru(DCB)?DBAMB)

[(4,4'-Bis((dibenzylamino)methyl)-2,2'-bipyridine)bis(2,2'-bipyridyl-4,4'- dicarboxylic acid)]ruthenium(ll) was synthesised using the method described in Examples 1 a-1 d, except that dibenzylamine was used in place of diethylamine.

Example 5:

The pH responses of the pH sensors synthesised in Examples 1 , 2, and 3 were determined by measuring the variation in luminescence lifetime with pH as phase-sensitive measurements in 50 mM phosphate buffer of an average 200 mosM (KCI). The pH was increased from a minimum value of 3.5, and the variation in luminescence lifetime was measured. The pH value at which the luminescence lifetime stopped increasing with pH was determined for each pH sensor. The results are summarised below (with the upper limit of the "pH response range" corresponding to the point at which the luminescence lifetime stopped increasing with pH) and are shown in Figure 1.

The results demonstrate that hese pH sensors display sensitivity to pH across the pH response ranges given in the table above.

Example 6:

The pH response for the Ru(ll) complex synthesised in Example 4 was determined by measuring the variation in luminescence lifetime with pH as time- resolved emission measurements in air-equilibrated 50 mM phosphate buffer solution. As in Example 5, the pH was increased from a minimum value of 3.5, and the variation in luminescence lifetime was measured. The pH value at which the luminescence lifetime stopped increasing with pH was determined. The pH response range was measured as 3.5-8.5, and the results are shown in Figure 2.

Example 7

The pK_a values for the Ru(ll) complexes synthesised in Examples 1 d, 2, 3 and 4 were measured in air-equilibrated 50 mM phosphate buffer solution by measuring the variation in luminescence lifetime as time-resolved emission measurements. The results are summarised below, and shown in Figure 2, where the pK_a values are the inflection points of the sigmoidal curves.

Figure 3 displays the results of Figure 2 in comparison to the pK_a values of tris(2,2'-bipyridine-4,4'-dicarboxylic acid)ruthenium(ll), as a comparative example. The pK_a values of the comparative tris(2,2'-bipyridine-4,4'-dicarboxylic acid)ruthenium(ll) complex were determined using the same method as that used for Examples 1 d, 2, 3 and 4, and are in agreement with those reported in "Dependence of the photophysical and photochemical properties of the photosensitizer tris(4,4'-dicarboxy-2,2'-bipyridine)ruthenium(ll) on pH" (J. Photochem. Photobiol. A: Chem. 1995, 86, pages 89-95). The comparative tris(2,2'-bipyridine-4,4'-dicarboxylic acid)ruthenium(ll) complex displayed two acid-base equilibriums: at pK_a 0.7 and 3.7.

Claims

1. A luminescent Ru(ll) complex having:

2. A luminescent Ru(ll) complex according to claim 1 , wherein the at least one polypyridyl ligand having at least one proton-accepting group, has two proton-accepting groups.

3. A luminescent Ru(ll) complex according to claim 1 or claim 2, wherein the at least one polypyridyl ligand having at least one proton-donating group, has two proton-donating groups.

4. A luminescent Ru(ll) complex according to any of claims 1 to 3, wherein the at least one proton-accepting group is directly linked to a ring carbon atom of the at least one polypyridyl ligand.

5. A luminescent Ru(ll) complex according to any of claims 1 to 4, wherein the proton-accepting group is a carboxylate group.

6. A luminescent Ru(ll) complex according to any of claims 1 to 5, wherein the proton-donating group is an ammonium group.

7. A luminescent Ru(ll) complex according to any of claims 1 to 6, wherein q = 1 , such that the spacer group has the formula -(CH₂)-.

8. A luminescent Ru(ll) complex according to any of claims 1 to 7, wherein the least one polypyridyl ligand having at least one proton-donating group is a 2,2'-bipyridyl ligand.

9. A luminescent Ru(ll) complex according to any of claims 1 to 8, wherein the least one polypyridyl ligand having at least one proton-accepting group is a 2,2'-bipyridyl ligand.

10. A luminescent Ru(ll) complex according to any of claims 1 to 9, wherein the proton-donating group has the formula (i):

wherein each of R¹ and R² are electropositive groups.

11. A luminescent Ru(ll) complex according to any of claims 1 to 10, wherein the polypyridyl ligand having at least one proton-donating group includes the following pyridine ring structure (ii):

each of R¹ and R² are electropositive groups; and wherein q may be 1 , 2, or 3.

12. A luminescent Ru(ll) complex according to claim 10 or claim 1 1 , wherein R¹ and R² are independently selected from H, optionally substituted C-i-ds alkyl and optionally substituted cycloalkyl.

13. A luminescent Ru(ll) complex according to any of claims 10 to 12, wherein R¹ and R² are independently selected from H and C-I -C-IO alkyl optionally substituted with one or more phenyl groups.

14. A luminescent Ru(ll) complex according to any of claims 10 to 13, wherein R¹ and R² are the same or different and are each optionally substituted C-I -C-IO alkyl.

15. A luminescent Ru(ll) complex according to claim 14, wherein R¹ and R² are the same or different and are each optionally substituted C1-C₅ alkyl.

16. A luminescent Ru(ll) complex according to any of claims 10 to 15, wherein R¹ and R² are the same.

17. A luminescent Ru(ll) complex according to claim 16, wherein R¹ and R² are ethyl.

18. A luminescent Ru(ll) complex according to any of claims 1 to 17, wherein the polypyridyl ligand having at least one proton-accepting group includes the following pyridine ring structure (iii):

wherein the nitrogen atom of the pyridine ring is coordinated to the Ru(ll) of the luminescent Ru(ll) complex; R¹² and R¹³ are independently selected from H and a covalent bond to the remainder of the polypyridyl ligand structure, at least one of R¹² and R¹³ being a covalent bond to the remainder of the polypyridyl ligand structure; and

R⁵ is carboxylate.

19. A luminescent Ru(ll) complex according to any preceding claim having the formula (iv):

wherein R¹, R², R³ and R⁹ are independently selected from H, optionally substituted C-i-ds alkyl and optionally substituted cycloalkyi; and

wherein R⁴, R⁵, R⁶ and R⁷ are selected from H, optionally substituted d- C-io alkyl, optionally substituted cycloalkyi, and carboxylate, at least one of R⁴ and R⁵ being carboxylate and at least one of R⁶ and R⁷ being carboxylate.

20. A luminescent Ru(ll) complex according to claim 19, wherein each of R⁴, R⁵, R⁶ and R⁷ is carboxylate.

21. A luminescent Ru(ll) complex according to claim 19 or claim 20, wherein R¹, R², R³ and R⁹ are independently selected from H and d-do alkyl optionally substituted with one or more phenyl groups.

22. A luminescent Ru(ll) complex according to any of claims 19 to 21 , wherein R¹ and R³ are the same or different and are each optionally substituted d-C-io alkyl.

23. A luminescent Ru(ll) complex according to claim 22, wherein R¹ and R³ are the same or different and are each optionally substituted C1-C5 alkyl.

24. A luminescent Ru(ll) complex according to any of claims 19 to 23, wherein R¹, R², R³ and R⁹ are the same or different and are each optionally substituted C1-C10 alkyl.

25. A luminescent Ru(ll) complex according to claim 24, wherein R¹, R², R³ and R⁹ are the same or different and are each optionally substituted C1-C5 alkyl.

26. A luminescent Ru(ll) complex according to claim 25, wherein R¹, R², R³ and R⁹ are ethyl.

27. A polymer-bound luminescent Ru(ll) complex having:

28. A polymer-bound luminescent Ru(ll) complex according to claim 27, wherein the at least one polypyridyl ligand having at least one proton-accepting group, has two proton-accepting groups.

29. A polymer-bound luminescent Ru(ll) complex according to claim 27 or claim 28, wherein the at least one polypyridyl ligand having at least one proton- donating group, has two proton-donating groups.

30. A polymer-bound luminescent Ru(ll) complex according to any of claims 27 to 30, wherein the at least one proton-accepting group is directly linked to a ring carbon atom of the at least one polypyridyl ligand.

31. A polymer-bound luminescent Ru(ll) complex according to any of claims 27 to 30, wherein the proton-accepting group is a carboxylate group.

32. A polymer-bound luminescent Ru(ll) complex according to any of claims 27 to 31 , wherein the proton-donating group is an ammonium group.

33. A polymer-bound luminescent Ru(ll) complex according to any of claims 27 to 32, wherein q = 1 , such that the spacer group has the formula -(CH₂)-.

34. A polymer-bound luminescent Ru(ll) complex according to any of claims 27 to 33, wherein the least one polypyridyl ligand having at least one proton- donating group is a 2,2'-bipyridyl ligand.

35. A polymer-bound luminescent Ru(ll) complex according to any of claims 27 to 34, wherein the least one polypyridyl ligand having at least one proton- accepting group is a 2,2'-bipyridyl ligand.

36. A polymer-bound luminescent Ru(ll) complex according to any of claims 27 to 35, wherein the proton-donating group has the formula (i):

R²

R

N^+'

(i)

wherein each of R¹ and R² are electropositive groups.

37. A polymer-bound luminescent Ru(ll) complex according to any of claims 27 to 36, wherein the polypyridyl ligand having at least one proton-donating group includes the following pyridine ring structure (ii):

each of R¹ and R² are electropositive groups; and

wherein q may be 1 , 2, or 3.

38. A polymer-bound luminescent Ru(ll) complex according to claim 36 or claim 37, wherein R¹ and R² are independently selected from H, optionally substituted C-i-ds alkyl, optionally substituted cycloalkyl and * Y PS, wherein PS represents a polymer support and Y represents a spacer that covalently links the Ru(ll) complex to the polymer support.

39. A polymer-bound luminescent Ru(ll) complex according to any of claims 36 to 38, wherein R¹ and R² are independently selected from H and d-do alkyl optionally substituted with one or more phenyl groups.

40. A polymer-bound luminescent Ru(ll) complex according to any of claims 36 to 39, wherein R¹ and R² are the same or different and are each optionally substituted C-i-C ₀ alkyl.

41. A polymer-bound luminescent Ru(ll) complex according to claim 40, wherein R¹ and R² are the same or different and are each optionally substituted C1 -C5 alkyl.

42. A polymer-bound luminescent Ru(ll) complex according to any of claims 36 to 41 , wherein R¹ and R² are the same.

43. A polymer-bound luminescent Ru(ll) complex according to claim 42, wherein R¹ and R² are ethyl.

44. A polymer-bound luminescent Ru(ll) complex according to any of claims 27 to 43, wherein the polypyridyl ligand having at least one proton-accepting group includes the following pyridine ring structure (iii):

R⁵ is carboxylate.

45. A polymer-bound luminescent Ru(ll) complex according to any of claims 27 to 44, wherein the polymeric support is a polyethylene glycol-polystyrene graft copolymer microbead.

46. A polymer-bound luminescent Ru(ll) complex according to any of claims 27 to 45, wherein the luminescent Ru(ll) complex has the formula (v):

wherein R¹, R², R³ and R⁸ are independently selected from H, optionally substituted C1-C15 alkyl, optionally substituted cycloalkyi and * Y PS, wherein PS represents a polymer support and Y represents a spacer that covalently links the Ru(ll) complex to the polymer support; and

wherein R⁴, R⁵, R⁶ and R⁷ are selected from H, optionally substituted d- C-io alkyl, optionally substituted cycloalkyi and carboxylate, at least one of R⁴ and R⁵ being carboxylate and at least one of R⁶ and R⁷ being carboxylate.

47. A polymer-bound luminescent Ru(ll) complex according to claim 46, wherein each of R⁴, R⁵, R⁶ and R⁷ is carboxylate.

48. A polymer-bound luminescent Ru(ll) complex according to claim 46 or claim 47, wherein R¹, R², R³ and R⁸ are independently selected from H and d-

C5 alkyl optionally substituted with one or more phenyl groups.

49. A polymer-bound luminescent Ru(ll) complex according to any of claims 46 to 48, wherein R¹ and R³ are the same or different and are each optionally substituted C1-C10 alkyl and R⁸ is H.

50. A polymer-bound luminescent Ru(ll) complex according to claim 49, wherein R¹ and R³ are the same or different and are each optionally substituted C1-C5 alkyl.

51. A polymer-bound luminescent Ru(ll) complex according to any of claims 46 to 50, wherein R¹, R² and R³ are the same or different and are each optionally substituted C-i-C ₀ alkyl, preferably C C₅ alkyl, and R⁸ is H.

52. A polymer-bound luminescent Ru(ll) complex according to claim 51 , wherein R¹, R² and R³ are ethyl and R⁸ is H.

53. A polymer-bound luminescent Ru(ll) complex according to any of claims 46-48, wherein R¹, R², R³ and R⁸ are ethyl.

54. A polymer-bound luminescent Ru(ll) complex according to any of claims 46 to 53, wherein Y is linked to a polyethylene glycol chain of a polyethylene glycol-polystyrene graft copolymer microbead.

55. A polymer-bound luminescent Ru(ll) complex according to any of claims 46 to 54, wherein Y represents a C1-C5 alkylene group.

56. A polymer-bound luminescent Ru(ll) complex according to any of claims 46 to 55, wherein ^* Y PS can be represented by

wherein n is from 1 to 5, m has an average value in the range 20 to 200, preferably in the range 50 to 100, more preferably in the range 60 to 80 and X represents a polystyrene chain of the polyethylene glycol-polystyrene graft copolymer microbead.

57. A pH sensor comprising a luminescent Ru(l l) complex according to any of claims 1 to 26, or a polymer-bound luminescent Ru(l l) complex according to any one of claims 27 to 56, and a film support having a surface upon which the luminescent Ru(l l) complex or polymer-bound luminescent Ru(ll) complex is immobilised.

58. A pH sensor according to claim 57, wherein the luminescent Ru(ll) complex or polymer-bound luminescent Ru(l l) complex is contained within a layer of hydrophilic hydrogel that is adhered to the surface of the film support.

59. A pH sensor according to claim 58, wherein the hydrogel is a hydrophilic polyurethane hydrogel.

60. A pH sensor according to claim 57, wherein the sensor comprises a polymer-bound luminescent Ru(ll) complex according to any of claims 27 to 56 that is adhered directly to the surface of the film support with an adhesive, preferably with an acrylic adhesive.

61. A pH sensor according to any of claims 57 to 60, wherein the sensor further comprises an opacifier that forms a coating over the luminescent Ru(l l) complex, over the polymer-bound luminescent Ru(ll) complex, or over the layer of hydrophilic hydrogel.

62. A pH sensor according to claim 61 , wherein the opacifier is a white hydrophilic polycarbonate.

63. A reaction vessel having, adhered to an inner surface thereof, a pH sensor according to any of claims 57 to 62.

64. A reaction vessel comprising, immobilised on an inner surface of the vessel, a luminescent Ru(l l) complex according to any of claims 1 to 26 or a polymer-bound luminescent Ru(ll) complex according to any of claims 27 to 56.

65. A reaction vessel according to claim 64, wherein the luminescent Ru(ll) complex or polymer-bound luminescent Ru(l l) complex is contained within a layer of hydrophilic hydrogel that is adhered to the surface of the reaction vessel.

66. A reaction vessel according to claim 65, wherein the hydrogel is a hydrophilic polyurethane hydrogel.

67. A reaction vessel according to claim 64, wherein the vessel comprises a polymer-bound luminescent Ru(ll) complex according to any of claims 27 to 56 that is adhered to the surface of the reaction vessel with an adhesive, preferably with an acrylic adhesive.

68. A reaction vessel according to any of claims 64 to 67, wherein the vessel further comprises an opacifier that forms a coating over the luminescent Ru(l l) complex, over the polymer-bound luminescent Ru(ll) complex, or over the layer of hydrophilic hydrogel.

69. A reaction vessel according to claim 68, wherein the opacifier is a white hydrophilic polycarbonate.

70. A bioreactor system comprising a reaction vessel according to any of claims 63 to 69, a light source, and a detector configured to detect the luminescence intensity or luminescence lifetime of the luminescent Ru(ll) complex or polymer-bound luminescent Ru(ll) complex.

71. Use of the luminescent Ru(ll) complex according to any of claims 1 to 26, a polymer-bound luminescent Ru(ll) complex according to any of claims 27 to 56, a pH sensor according to any of claims 57 to 62, a reaction vessel according to any of claims 63 to 69, or a bioreactor according to claim 70 for determining the pH of a solution.

72. A process for covalently bonding a polymer-bound luminescent Ru(ll) complex to a polymer support, comprising providing a luminescent Ru(l l) complex according to any of claims 1 to 26 and reacting the luminescent Ru(ll) complex with a polymeric support to form a covalent bond.

73. A process according to claim 72, wherein the proton-donating group of the luminescent Ru(ll) complex is an ammonium group;

wherein the polymeric support that is reacted with the luminescent Ru(ll) complex comprises polyethylene glycol chains functionalised with a leaving group; and

wherein the reaction involves nucleophilic substitution of the leaving group of the polyethylene glycol chain for an ammonium group of the Ru(ll) complex.

74. A process according to claim 73, wherein the leaving group is selected from iodine, bromine, chlorine, tosyl, brosyl, mesyl, triflyl, methylsulfyl and hydroxy I.

75. A process according to any of claims 72 to 74, wherein the polymeric support is a polyethylene glycol-polystyrene graft copolymer microbead.

76. A process according to any of claims 72 to 75, wherein the product of the reaction is a polymer-bound luminescent Ru(ll) complex according to any of claims 27 to 56.

77. A method of determining the pH of a solution comprising:

exposing a luminescent Ru(ll) complex according to any of claims 1 to 26 or polymer-bound luminescent Ru(ll) complex according to any of claims 27 to 56 to the solution;

illuminating the luminescent Ru(ll) complex or polymer-bound luminescent Ru(ll) complex so as to excite the complex;

detecting light emitted by the complex; and