WO2004008131A1

WO2004008131A1 - Sensor for anions

Info

Publication number: WO2004008131A1
Application number: PCT/GB2003/002988
Authority: WO
Inventors: Paul Derek Beer; Jason John Davis
Original assignee: Oxford University Innovation Ltd
Current assignee: Oxford University Innovation Ltd
Priority date: 2002-07-10
Filing date: 2003-07-10
Publication date: 2004-01-22
Anticipated expiration: 2005-01-10
Also published as: AU2003251327A1; GB0215987D0

Abstract

A sensor which is suitable for the detection of a negatively charged species and which comprises a monolayer attached to a metal surface, wherein the monolayer comprises, in order from the surface: (a) a metal-anchoring group; (b) a host portion comprising two or more binding groups, which groups are together capable of chelating to the negatively charged species; and (c) a detector group which is capable of undergoing a detectable change in electrochemical or optical properties upon binding of the negatively charged species by the host portion (b); the host portion (b) being connected to each of the metal anchoring group (a) and the detector group (c) by a direct bond or via a linking group. The metal surface may be on an electrode or may be the surface of a metal nanoparticle, the nanoparticle being either free in solution or assembled at an interface.

Description

SENSOR FOR ANIONS

The present invention relates to electrochemical and optical sensors for the detection, recognition and extraction of negatively charged species such as anions. The design and construction of sensors that can selectively recognise and sense negatively charged species via a macroscopic physical response is currently of great interest in supramolecular chemistry. Anion binding can be achieved using acyclic and macrocyclic amide-containing compounds in an analogous manner to the more established binding of cations by crown ethers, cryptands and spherands. The • success of this is compromised, however, by the weaker affinity of anionophores for anions compared with the affinity of cationophores for cations. The detection of anions by this method is thus more difficult than the corresponding detection of cations.

The redox-active ferrocene moiety has been exploited in the selective electrochemical sensing of anions in both organic and aqueous media. In particular acyclic, macrocyclic and calixarene amide-functionalised ferrocene derivatives have been shown to undergo substantial cathodic perturbations of the respective metallocene redox couple in the presence of a variety of anions of biological and environmental significance. However, coordination-mediated anion detection in aqueous media is challenging due to the accompanying unfavourable energetic requirements of dehydration and the low binding constants of anions, particularly to neutral amonophores.

It has now been found that surface pre-organisation within certain self- assembled monolayers can be exploited in the electrochemical and optical sensing of negatively charged species in both organic and aqueous media. Accordingly, the present invention provides a sensor which is suitable for the detection of a negatively charged species and which comprises a monolayer attached to a metal surface, wherein the monolayer comprises, in order on the surface: (a) a metal anchoring group or atom; (b) a host portion comprising two or more binding groups, which groups are together capable of chelating to the negatively charged species; and ^' (c) a detector group which is capable of undergoing a detectable change in electrochemical or optical properties upon binding of the negatively charged species by the host portion (b); the host portion (b) being connected to each of the metal anchoring group (a) and the detector group (c) by a direct bond or via a linking group. In one aspect the invention provides a sensor which is suitable for the detection of a negatively charged species and which comprises a monolayer attached to a metal surface, wherein the monolayer comprises, in order on the surface:

(a) a metal anchoring group or atom;

(b) a host portion comprising two or more binding groups, which groups are together capable of chelating to the negatively charged species; and (c) a redox active group; the host portion (b) being connected to each of the metal anchoring group (a) and the redox active group (c) by a direct bond or via a linking group. The metal surface of the sensor of the invention is typically provided on an electrode. In this embodiment the metal surface may be the actual surface of the metal electrode.

Alternatively the metal surface may be the surface of a metallic nanoparticle (including quantum dots), the nanoparticle being either free in solution or assembled at a solid interface. Typically the interface is a planar surface on which the nanoparticles are dispersed within a colloid. Thus in one embodiment the metal surface of the sensor of the invention is a colloid-modified surface. In this embodiment a colloid comprising metallic nanoparticles is dispersed on a planar surface, thereby providing the metal surface to which the monolayer of the sensor is attached.

The nanoparticles providing the surface to which the monolayer is attached may be of any suitable metal. Typically they are copper, silver or a noble metal such as gold or platinum. Preferably the nanoparticles are metallic gold nanoparticles. Metal nanoparticles have size-dependent optical properties which are not present in the bulk metal. In particular, nanoparticles of silver, gold and copper show distinct and well-defined plasmon adsorption in the visible spectrum, which _. adsorption is characterised by a large molar adsorption coefficient (Jensen et al, J. Phys. Chem. B 2000, 104, 10549). hi one embodiment of the sensor of the invention, therefore, the binding of a negative species by the host portion (b) is accompanied by a change in the optical properties of the metal surface of the nanoparticles to which the monolayer is attached. This may be in addition to any change in electrochemical and/or optical properties effected in the detector group (c) by that binding.

A sensing unit may be self-assembled on a colloid-modified surface as described below. The colloid modified surface is typically the surface of an electrode. Surface-confined colloids provide an increased electrode surface area and have the added advantage of providing a spectroscopic signature which maybe modified when a negatively charged species is bound by the host portion of the ^■ sensor.

The sensor of the invention as defined above is produced by allowing a sensing unit to self assemble on a suitable metal surface, such that a monolayer forms. The sensing unit is a chelating compound having electrochemical or optical properties, which compound comprises, in order:

(a) a group or functionality which is capable of bonding to the metal surface;

(b) a host portion comprising two or more bmding groups, which groups are together capable of chelating to a negatively charged species; and (c) a detector group which is capable of undergoing a detectable change in electrochemical or optical properties upon binding of the negatively charged species by the host portion (b); the host portion (b) being connected to each of the metal anchoring group (a) and the detector group (c) by a direct bond or via a linking group. The sensing unit may be formed using conventional chemical techniques which would be well known to a person of skill in the art.

An ordering of the sensing unit on the metal surface takes place such that an approximately regular single layer is formed. This layer is described as a self assembled monolayer, or SAM. Such surface pre-organisation of the sensing units significantly amplifies the electrochemical and/or optical response of the detector group (c). The sensor of the invention thus possesses a heightened sensitivity compared with previous sensors for negatively charged species having unbound sensing units.

When the sensor of the invention is in use the host portion of the monolayer binds a target negatively charged species which is present in an analyte solution to which the sensor is exposed. That binding causes a perturbation in the electrochemical or optical properties of the detector group (c). That perturbation is reflected in a change in the redox potential or UV- visible absorption spectrum and/or the luminescence spectrum, respectively, of the detector group (c). The target species can thus be detected by observing and/or measuring this change in redox potential or absorption/luminescence spectrum.

An advantage of the sensor of the invention is that, as described above, the electrochemical or optical perturbation arising from bmding of the target species is amplified. A further advantage of the sensor is that, being a solid, it can be easily separated from the analyte solution when the desired detection has taken place. This therefore provides a means for extracting a negatively charged species from an analyte solution since, after separation of the sensor from the analyte solution, the bound target species may be released from the host portion of the sensor by suitable chemical manipulation. Examples of such manipulation include adjusting the pH, washing with an appropriate competitive solvent (such as water) and/or washing with a competing chelate. If desired the sensor may be used again following release of the bound target species. The process of detection may be carried out two or more times. The sensor of the invention is thus re-usable. The sensor has the additional advantage of being robust, particularly since it is stable in a wide range of polar and non-polar solvents. It is also easily handled. When the detector group (c) of the sensor of the present invention is a redox active group, it undergoes an electrochemical perturbation when a negatively charged species is bound by the host portion (b) of the sensor. The redox active group is preferably chosen so that, when a negatively charged guest species is bound by the host portion, its redox potential is as different as possible from its redox potential when no guest species is present in the host portion. The redox active group is preferably one which is suitable for use in both orgamc and aqueous media. Typically the redox active group is a metallocene such as ferrocene or cobaltocenium. When the detector group (c) of the sensor of the present invention is a group which undergoes a detectable change in its optical properties upon binding of a target negatively charged species by the host portion (b), it is referred to herein as an optical group. The optical group is preferably chosen so that, when a negatively charged guest species is bound by the host portion, its UV-visible absorption or luminescence spectrum is as different as possible from, respectively, its UV-visible absorption or luminescence spectrum when no guest species is present. The optical group is preferably one which is suitable for use in both aqueous and organic media. Suitable examples of the optical group include metalloporphyrins, such as a zinc metalloporphyrin, more especially a zinc metallopo hyrin of formula (VII) as defined below.

The detector group (c) may be both a redox active group and an optical group. This embodiment is particularly advantageous.

The host portion of the sensor of the present invention comprises two or more binding groups which together are capable of chelating to a target negatively charged species. The host portion is thus a receptor for the negatively charged species. The stoichiometry of the host portion, or receptor, to the negatively charged species is preferably 1:1.

The binding groups of the host portion chelate to the negatively charged species when the latter is present as a guest. The constituent binding groups of the host portion may be the same or different. The electronic properties of the binding groups are influenced by any adjoining functional groups. The binding groups may thus be tailored to target a desired negatively charged species. In this way the sensor of the invention may be rendered selective for a particular desired target species. The bmding groups of the host portion are typically hydrogen bond donor groups. Suitable examples include amine, amide, thioamide, guanidinium, urea, thiourea, pyrrole and polyammonium groups. A preferred hydrogen bond donor binding group is an amide. The amide function may be oriented with either its NH moiety or its C(O) moiety nearest to the redox active group. If there are two (or more) amide groups present at the host portion, the orientation of these may be the same or different. The amide function of the host portion may be part of a peptide unit.

The structure of the host portion may be designed to create a binding cavity of the correct size to incorporate the desired target species. The size and shape of the cavity may be influenced by the structure of the detector group (c) and the metal anchoring group. The structure of the host cavity is preferably such that, when the negatively charged species is not present, intramolecular hydrogen bonding and other unfavourable intramolecular interactions are largely prevented. Furthermore, the binding groups of the host portion are preferably pre-organised for reception of the negatively charged guest species. The binding of a guest negatively charged species in the cavity provided by the host portion is typically promoted by the stabilisation afforded by the macrocyclic effect.

The negatively charged guest species may locate wholly or partially in the cavity of the host portion of the sensing unit, or it may perch wholly or partially above the cavity and hydrogen-bond on the outside of the cavity. Alternatively, or in addition, a guest species may be sandwiched between the host portions of two or more sensing units. Typically the negatively charged guest species is at least partially located in the host portion cavity of only one sensing unit, thereby providing a 1 :1 host: guest stoichiometry when the guest species is present.

The metal-anchoring portion (a) of the sensor of the present invention is any group or atom which can form a bond to the metal surface of the sensor. The metal anchoring group typically contains one or more sulphur atoms and is preferably derived from a sulphur-containing function such as a thiol, polythiol, thioether or sulphide. The metal anchoring group may be regarded as the bound residue of the group or functionality (a) of the sensing unit as defined above, from which the monolayer of the sensor is formed. Thus when group or functionality (a) in the sensing unit comprises a thiol, sulphide, disulphide, polythiol or thioether, the self assembly of the monolayer of the sensor leads to the formation of a direct sulphur- metal bond. The anchoring group (a) in that case is the covalently bound sulphur moiety S-. The metal surface preferably is, or comprises, gold or another noble metal such as silver or platinum such that the metal anchoring group provides a sulphur- metal bonding interaction.

The metal-anchoring group may be connected to the host portion via a linking group. The linking group is typically a - C₂₀ alkylene, C₂- C₂₀ alkenylene or C₂-. C₂₀ alkynylene chain. Preferably the chain is a C₂.-C₁₀or C₂-C₈ chain, for instance a C₂-C₆ or C₃-C₅ chain. The chain is unsubstituted or substituted. Examples of suitable substituents include - C₆ alkyl, C₃ - C₈ cycloalkyl, Cj. alkoxy, halogen, -OH, -SH, nitro, amino, imino and phenyl. The chain may be interrupted by one or more heteroatoms such as O or S, by an aromatic or heteroaromatic group or by one or more unsaturated functionalities such as an imine ( -CH=N-). Examples of such an aromatic group are phenyl and naphthyl. A heteroaromatic group may be a 5- to 7-membered heteroaromatic ring containing one, two or three heteroatoms selected from O, N and S. Examples include pyridine, pyrrole, thiophene and furan. The aromatic or heteroaromatic group is unsubstituted or substituted, for instance by the substituents defined above. A C_rC₆ alkyl group may be straight or branched and is typically C₁-C₄ alkyl such as methyl, ethyl, i-propyl, n-propyl, t-butyl, s-butyl and n-butyl. A C_rC₆ alkoxy group may be straight or branched and is typically C_rC₄ alkoxy such as methoxy, ethoxy, i-propoxy, n-propoxy, t-butoxy, s-butoxy and n-butoxy. C₃ - C₈ cycloalkyl may be, for instance, cyclopropyl, cyclobutyL cyclopentyl or cyclohexyl. A halogen is fluoro, chloro, bromo or iodo. An amino group may be -NH₂ or a secondary or tertiary amine. Imino is -C=NH. A phenyl group may be unsubstituted or substituted by one or more of the substituents recited above.

The host portion is connected to the detector group (c) either by a direct bond or via a linking group. The linking group, if present, may be a linking group as defined above. In the case where the group (c) is a redox active group, the linking group is preferably chosen so that the redox potential of the redox active group is perturbed as much as possible when the guest species is present in the host portion, compared with when no guest species is present in the host portion.

Where the detector group (c) is a redox active group, it is essential for electronic communication to take place between the binding groups of the host portion and the redox active group. Where the detector group (c) is an optical group such electronic communication is preferable. The electronic communication takes place either through space or via the linking group. Preferably, for through-space communication, there is either no linking group between the host portion and the redox active group or there is a relatively short (for instance - C₄) linking group. For through-bond electronic communication the linking group typically comprises an unsaturated bond, a group containing delocalised electrons or an atom or group bearing an electron lone-pair.

An unsaturated bond is suitably provided by an alkenylene or alkynylene linking group as defined above, or by an imine group incorporated into the linking group. A group containing delocalised electrons is typically an aromatic or heteroaromatic group as defined above. An atom bearing an electron lone pair is typically a heteroatom such as O or S. For maximum through-bond electronic communication the linking group comprises a conjugated system. The metal surface of the sensor of the present invention comprises any suitable metal, for instance copper, silver or a noble metal such as gold or platinum. The metal surface may comprise a pure metal or an alloy. The alloy may be an alloy of two. or more suitable metals, for instance selected from those mentioned above, or an alloy of one metal and one or more other elements. Preferably the metal surface comprises gold; more preferably the metal surface is a gold surface. The metal surface may require cleaning and or polishing treatment prior to adsorption of the monolayer. A gold surface may be mechanically polished with a alumina or electropolished, for instance with sulphuric acid.

The self assembled monolayer of the sensor of the present invention may be regarded as comprising an assembly of identical units side by side on the metal surface, each unit comprising the constituent parts (a) to (c) of the monolayer as defined above and the constituent parts being connected together either by direct bonds or via a linking group as defined above. A single unit of the monolayer may thus be represented by the following formula (I):

Q - [X^B¹), -Y_n-A-surface]_q (I)

wherein: each X, which are the same or different, is a linking group as defined above; each B¹, which are the same or different, is a group which is, or contains, an atom or group capable of binding to a negatively charged species;

Q is a group which is capable of undergoing a detectable change in electrochemical or optical properties when said bmding to a negatively charged species takes place; each Y, which are the same or different, is a linking group as defined above;

A is an anchor group;

1 is an integer of 1 to 20; m is 0 or 1 ; n is O or 1; and q is an integer of 2 to 6.

The portion Q-K_n-CB²)^,, of formula (I) represents a chelating ligand in which the binding atoms or groups of the B¹ moieties coordinate to the mcoming negatively charged species. The latter is therefore typically accommodated as a guest within the coordination site provided by the B¹ moieties. More than one guest species may be accommodated within a single unit of the monolayer when parameter 1 is greater than 1 and there are consequently two or more co-ordination sites within the host portion represented by

The binding of the or each guest species within the host portion may be enhanced by the macrocyclic effect. The unit of the monolayer may alternatively be represented by the following formula (II):

Q-X_m-(B²)_I-Y„-A-surface (H)

wherein:

Q, X, Y, A, m, and n are as defined above;

1 is an integer of from 2 to 20; and each B² ,which are the same or different, is a group which contains an atom or group which are together capable of chelating to a negatively charged species.

In the above formulae (I) and (LT) the groups B¹ preferably are, or contain, hydrogen bond donor groups. The or each B¹ may comprise an amide or peptide unit. In a preferred embodiment each B¹, which are the same or different in a given monolayer unit, is a group of formula (i) or (ii): (i) C(O)-NH-(T)_r-

(ii) -NH-C(O)-(T)_r- wherein: r is O, 1, 2, 3 or 4; and

T is an amino acid residue or a group -C*(R')(R")- wherein C* is a chiral or achiral carbon atom and each of R' and R", which are the same or different, is

H or a group selected from C,-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl and C₆-C₁₂ aryl, the group being unsubstituted or substituted by one or more substituents and being optionally interrupted by one or more heteroatoms such as O or S, by an aromatic or heteroaromatic group or by one or more unsaturated functions such as an imine group. Examples of suitable substituents include those recited above for the linking group. Examples of embodiments of formulae (i) and (ii) include the following: -C(O)-NH-C*(R')(R")-NH-C(O)- ; -C(O)-NH-C*(R')(R")-C(O)-NH- ; -NH-C(O)-C*(R')(R")-NH-C(O)- ; and -NH-C(O)-C*(R')(R")-C(O)-NH- .

The amino acid residue is the residue of any amino acid such as a naturally occurring amino acid or an α-amino acid. The amino acid may be an aliphatic amino acid or an aromatic amino acid. The amino acid may be neutral or acidic. Specific examples of amino acids include alanine, argimne, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, allysine, citrulline, cystathionine, cysteic acid, cystine, dopa, homocysteine, homoserine, homoserine lactone, lanthionine, ornithine, 5-oxoprohne, sarcosine, thvronine and thyroxine. When the amino acid includes a chiral carbon atom it may be present in the D- or L-stereo isomeric form.

Most preferably each B¹ or each B² is -NH-C(O)- or -C(O)-NH-. In formula (II) the groups B² preferably include hydrogen bond donor groups, more preferably an amide. The groups B² must be spatially arranged so that their respective binding atoms or groups can chelate to a negatively charged guest species.

When the detector group (c) is a redox active group which is a metallocene, a single unit of the monolayer may be represented by the following formula (ffla) or (Hlb):

θ —χ_m—(B¹)ι—Y_n— A— surface

wherein:

M is a metal; and

X, B¹, Y, A, 1, m and n are as defined above.

In formula (Illb) the ferrocene moiety may be 1,2- or 1,3-disubstituted by

-X_m-(B¹),-Y_n-A-surface. Accordingly a single unit of the monolayer may be represented by the following formulae (LIIc) and (Hid.):

(Die)

A preferred embodiment of a single unit of the monolayer, when the redox active group is a metallocene, is represented by the following formula (TV):

O > — Z — Y_n — A— surface

wherein

M, Y, A and n are as defined above; and

Z is -C(O)-NH- or -NH-C(O)-.

As noted above, the metal anchoring group is preferably derived from a sulphur-containing compound, such that there is a sulphur-metal bond in the sensor of the present invention. One embodiment of a single unit of the monolayer, in which the metal anchoring group comprises a sulphur atom, is represented by the following Formula (V):

wherein

M and Z are as defined above; p is an integer of from 1 to 10; and

R¹ is H or -(CH₂)_q-S- wherein q is an integer of from 1 to 3.

When R¹ is -(CH₂)_q-S-, the S atom can additionally bind to the metal surface, such that the anchoring group comprises a plurality of sulphur atoms. In the above formulae (III) to (V), M is preferably Fe or Co⁺.

A class of sensing units used to prepare the sensor of the invention is novel. Accordingly, the invention further provides a compound of formula (Via) or (Vlb):

wherein M is a metal; B¹ is a group of formula (i) or (ii): (i) -C(O)-NH-(T)_r-

(ii) -NH-C(O)-(T)_r- whereiri: r is 0, 1, 2, 3 or 4; and T is an amino acid residue or a group -C*(R')(R")- wherein C* is a chiral or achiral carbon atom and each of R' and R", which are the same or different, is

H or a group selected from C,-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl and C₆-C₁₂ aryl, the group being unsubstituted or substituted by one or more substituents and being optionally interrupted by one or more heteroatoms, aromatic groups, heteroaromatic groups or unsaturated functions; Y is a C_rC₂₀ alkylene, C₂-C₂₀ alkenylene or C₂-C₂₀ alkynylene chain, which chain is unsubstituted or substituted and is optionally interrupted by one or more heteroatoms, by an aromatic or heteroaromatic group or by one or more unsaturated functionalities; 1 is an integer of 1 to 20; m is 0 or 1; and

R² is a group containing one or more sulphur atoms.

Preferably M is Fe. Parameter 1 is typically 1-10, more preferably 1-5, most preferably 1, 2, 3 or 4. Each B¹ is typically -C(O)-NH- or -NH-C(O)-. Y is preferably a C C₂₀ alkylene chain which is unsubstituted, substituted or interrupted as defined above. Preferably, Y is a C₂-C₁₀ or C₂-C₈ chain, for instance a C₂-C₆ or C₃- C₅ chain. Examples of suitable substituents for the linking group Y include C_j-C₆ alkyl, C₃-C₈ cycloalkyl, C_rC₆ alkoxy, halogen, -OH, -SH, nitro, amino, imino and phenyl. The chain may be interrupted by one or more heteroatoms such as O or S, by an aromatic or heteroaromatic group or by one or more unsaturated functionalities such as an imine ( -CH=N-). Examples of such an aromatic group are phenyl and naphthyl. A heteroaromatic group may be a 5- to 7-membered heteroaromatic ring containing one, two or three heteroatoms selected from O, N and S. Examples include pyridine, pyrrole, thiophene and furan. The aromatic or heteroaromatic group is unsubstituted or substituted, for instance by the substituents defined above. In one embodiment Y is a group -(CH₂)_t- wherein t is from 1 to 10, preferably 1 to 6, more preferably 2 to 6, most preferably 4.

R² is generally a group containing one or more thiol, sulphide, disulphide or thioether functions. Preferred examples include the dithiol -C(SH)-(CH₂)₂SH and the disulphide group:

The compounds of formulae (Via) and (Vlb) in which -(B¹),- is -NH-C(O)- are prepared by any suitable process for the synthesis of compounds of this type. An example of such a process comprises condensing l,l'-diamino ferrocene, 1,2- diamino ferrocene or 1,3-diamino ferrocene with 2 moles of a carboxylic acid of formula R²-(Y)_m-COOH, or an activated derivative thereof, wherein R², Y and m are as defined above for formulae (Via) and (VTb). An example of the preparation of such compounds is described in Example 1 which follows. Likewise the compounds of formula (VI) in which X is -C(O)- H- are prepared by any suitable process for the synthesis of compounds of this type. An example of such a process comprises condensing l,l'-dicarboxy ferrocene, 1,2-dicarboxy ferrocene or 1,3- dicarboxy ferrocene, or an activated derivative thereof, with 2 moles of an amine of formula R²-(Y)_m-NH₂. Activated derivatives in both cases include acid halides and acid anhydrides.

The compounds of formulae (Via) and (Vlb) wherein the groups B¹ comprise a peptide unit are generally prepared by chemical synthesis. A peptide unit is built up by condensation of the constituent amino acids in the order in which they occur in the peptide structure. The peptide may be obtained with a free carboxy or amide group at its C-terminus. Solid phase or solution phase methods may be employed. When solid-phase synthesis is used, the constituent amino acid sequence is built up from the C-terminal amino acid which is bound to an insoluble resin. The desired peptide is then cleaved from the resin. In solution-phase synthesis the peptide may be built up from the C-terminal amino acid. The carboxy group of this acid remains blocked throughout by a suitable protecting group, which is removed at the end of the synthesis.

In either synthetic technique the amino acids added to the reaction system typically have a protected amino group and an activated carboxy group. An amino group may be protected by the fluoren-9-ylmethoxycarbonyl (Fmoc) or t- butoxycarbonyl (Boc) group. A carboxy group may be activated as a pentafluorophenyl or l-oxo-2-hydroxy-dihydrobenzotriazine ester. The condensation steps may be effected in the presence of dicyclohexylcarbodiimide (DCCI) or 1- hydroxybenzotriazole (HOBt). Side chains present in the amino acids may be protected using suitable protected derivatives. The sensor of the present invention is produced by synthesising a sensing unit corresponding to the desired monolayer and then allowing the units to self assemble on a metal surface. The sensing unit may, for instance, be a compound of formula (Via) or (VTb) as defined above.

When the sensor of the invention is in use, a negatively charged species is bound by the host portion of the monolayer. Recognition of that bound species then occurs, as described above, by the detection of a perturbation in the optical or electrochemical properties of the detector group (c).

The perturbation in electrochemical properties is measured by any conventional means, for instance by cyclic voltammetry. When the redox active group is a ferrocene, the voltametric response shows a cathodic perturbation when the guest negatively charged species is chelated within the host portion of the monolayer. When the host portion comprises hydrogen bond donor groups, the redox potential of the ferrocene is perturbed by the electronic communication of the electronic changes associated with hydrogen bond donation to the guest negatively charged species.

The host portion of the monolayer preferably comprises amide groups, as noted above. More preferably the amide groups are the peptide linkages of peptide units. Robust monolayers which have particularly selective anion recognition properties are produced as a result. When the amide functionalities are directly bonded to a ferrocene, the hydrogen-bond mediated recognition of specific anions or oxoanions is accompanied by a significant perturbation of the ferrocene redox potential. The anchoring of this unit to a metal surface in the sensor of the invention, and providing that sensor on an electrode, permits this electrochemical response to be significantly amplified by the surface pre-organisation of the host. This phenomenon of "surface sensing amplification" , whereby the metal- bound monolayer gives a significantly higher level of anion sensing than the corresponding free compound, or sensing unit, is demonstrated by a comparison of the data in Table 3 of Example 5 with those in Table 2 of Example 4. These data highlight the significant advantages associated with preorganisation of the host pseudo macrocycle on a surface. Specifically, in Table 3 the redox responses are more cathodically shifted by (approximately) 90 and 60 mV for phosphate and chloride, respectively. Cathodically shifted waves were all characterised by greater peak separation (and often lower faradaic current) than observed in the precursor ferrocene couple.

A preferred class of sensing units, used to prepare a sensor of the invention in which the detector group (c) is a metallopoφhyrin, is defined by the following formula VTJ):

wherein:

W is a group of formula -X^B^-Y^R²;

M is a metal; and

X, m, B, 1, Y, n and R² are as defined above.

Typically M in formula (VII) is Zn. Typically W in formula (VJJ) is of the structure:

wherein B¹ and A are as defined above.

When W has the following structure the compounds of formula (VJJ) are novel:

wherein n is an intege of from 1 to 4.

The compounds of formula VJI are prepared by any suitable process for the synthesis of compounds of this type. For example the preferred compound of formula VH may be produced by the condensation of,α,α,α,α-atropisomer of 5,10,15,20-meso-tetrakis (o-aminophenyl) porphyrin and four equivalents of thioctic acid in the presence of DCCI and HOBt, followed by reaction with Zn(OAc)₂. A sensor of the invention may be formed by allowing the compound of formula (VJJ) to self assemble on a metal surface, for instance on the surface of metal nanoparticles which are either free in solution or contained in a colloid which is dispersed on a surface.

When the sensor of the present invention is in use, the host portion (b) binds a target negatively charged species. The binding of the target species causes perturbation of the electrochemical or optical properties of the detector group (c), allowing recognition of the target species. Importantly, surface pre-organisation of the sensing units of the monolayer enhances anion coordination and therefore the resulting perturbation of the electrochemical or optical properties of the sensor.

Optical perturbation is measured by any conventional means, for example by UV- visible absorption specfroscopy. UV-visible titrations are conveniently carried out. Binding of the target negatively charged species leads to a change in the absorption spectrum. For example, when the sensing unit is a compound of formula VII and the guest species is bromide, the absorption spectrum shows an increasing absorbance at approximately 570nm when the host portion binds to the guest species.

Two or more sensors of the present invention may be used together for the detection of two or more different negatively charged species in a given analyte solution. The present invention accordingly further provides a sensor assembly comprising an array of two or more sensors of the invention as defined above, wherein each sensor is tailored to recognise a different negatively charged species. A means for the differential detection and extraction of negatively charged species is thus provided. The sensors of the assembly may be provided on a support.

The sensor of the invention may be used to detect, recognise or extract any negatively charged species. The negatively charged species may be an anion, for instance a halide, nitrate, phosphate, dihydrogen phosphate, sulphate, carboxylate, perrhennate or radioactive pertechnetate. The anion may be an oxoanion. The negatively charged species may alternatively be a species such as a negatively charged biological species. Examples of the latter include RNA, DNA, ADP and ATP.

The sensor of the present invention may be used to detect or extract a negatively charged species, for instance as described above, which is present in an analyte. The present invention accordingly provides a process for assaying an analyte for the presence of a negatively charged species. The process comprises contacting the analyte with a sensor of the invention as defined above or an assembly of the invention as defined above and observing, in the case where the detector group (c) is ^{^} a redox active group, any subsequent perturbation in the electrode potential of the redox active group(s) or, in the case where the group (c) is an optical group, any subsequent perturbation in optical absorption and/or luminescence spectrum of the optical group(s), the sensor or assembly being tailored to detect one or more negatively charged species that are known, or suspected, to be present in the analyte. The sensor has detection levels in the micromolar to millimolar (10^"6M - 10^"3M) range, which is physiologically useful.

The analyte may be an organic or aqueous solution. Thus the solvent may be an organic solvent, for instance acetonitrile or dichloromethane, or it may be water. The analyte may be, for instance, a biological solution or a solution of environmental significance. Examples of the latter include biological and environmental waste solutions containing one or more negatively charged species which it is desired to detect or extract. Negatively charged species. in this context may be, for instance, radioactive negatively charged species such as radioactive anions.

In a medical context the analyte may comprise a body fluid such as blood and the sensor may be used in a method of diagnosis, for instance by detecting the presence of biologically significant negatively charged species in the fluid.

The SAM of the sensor of the present invention is stable in a wide range of polar and non-polar solvents. The particular solvent chosen for operation of the sensor will depend on the particular species which it is desired to detect. As described above, the sensor may be reused by releasing the guest species from the host portion by washing or pH adjustment. As an- alternative, the sensor may be chemically broken down and the components (for instance the metal) used to make new sensors or used for other purposes.

The present invention will be further described in the following Examples with reference to the accompanying Figures, in which:

Figure 1 is a schematic representation of a single unit of a gold surface-bound monolayer of the sensor of the present invention, with a negatively charged guest species bound by the two amide groups which make up the host portion;

Figure 2 shows a plurality of the units depicted in Figure 1, forming a self- assembled monolayer on the gold surface; and

Figure 3 shows the voltametric response of a sensor of the invention in the absence (A) and presence (B) of H₂PO₄ ^" (8 mM). The x axis represents current (x 10^'7 A) and the y axis represents mV vs. Ag/AgNO₃.

Figure 4 shows schematically the bmding of a sensing unit of formula (5) of Example 6 to a gold surface of a nanoparticle, forming the porphyrin-modified gold nanoparticles (6).

EXAMPLE 1: Preparation of Sensing Units

1 , 1 '-bis substituted ferrocene amide derivatives (2) and (3) were prepared as depicted in the following scheme. Thus, 1,1-diamino ferrocene (1) was condensed with 2 moles of the activated thioctic acid derivative in DCC and dichloromethane at room temperature for 3 hours, to give compound (2) in 75% yield. Treatment of compound (2) with NaBH₄ (4 equivalents) in ethanol/methanol at room temperature for 24 hours gave the polythiol, compound (3), in 80% yield.

(2) (3)

EXAMPLE 2: Formation of SAM on a gold electrode

Sensors of the invention were prepared as follows. Home-made and commercial polycrystalline gold electrodes were cleaned exhaustively by mechanical polishing on alumina and electropolishing in IM ultra-pure H₂SO₄. These were rinsed in pure (Elga) water and AnalR alcohol. They were then immersed in argon- degassed mM solutions (dry dichloromethane) of, respectively, compounds (2) and (3) of Example 1, overnight at 5°C and in the dark. Mono layers of compounds (2) and (3) self assembled on the gold macroelectrodes as a result. Calculated coverages of 5 - 8 x 10¹⁴ molecules/cm² (3 - 4.5 x 10 moles/cm²) were broadly consistent with a good molecular adlayer. Capacitive analyses (50 - 70 μF/cm²) indicated that molecular packing was not as tight as typically observed in simply alkylthiols and or that the monolayers were not defect-free. Monolayers of the thiol (3) were typically of better quality than those of the disulphide (2). EXAMPLE 3: Determination of stability constants

Various tetrabutylammomum anion salts (the chloride, carboxylate and dihydrogen phosphate) were added to a mixture of acetonitrile-d3 and deuterochloroform (CD₃CN/CDC1₃₎ 2: 1 v/v) Η NMR solutions of the free compound (2) described in Example 1. This resulted in significant perturbation of the amide protons. The resulting titration curves suggested a 1:1 receptor: anion stoichiometry. The computer program EQNMR (MJ Hynes, J. Chem. Soc. Dalton Trans., 1993, 311) was used to determine stability constant values in the mixture. The results are set out in Table 1 below... The results show that the selectivity preference was AcO^" >H₂PO₄ ^" >C1^", a trend that reflects the decreasing basicity of the anion guest.

TABLE 1

Εrrors <10%.

EXAMPLE 4: Diffusive voltammetry of compound (2).

The organic-phase diffusive voltammetry of the compound (2) of Example 1 in CH₃CN/CH₂C1₂ was studied initially at polished edge-plain graphite electrodes. The diffusive electrochemical experiments were carried out in a 3-electrode cell designed in-house. This comprised a working compartment, Pt gauze counter electrode and a luggin capillary connected to the reference compartment. Calomel and mercury sulphate reference electrodes were used in aqueous solution. A regularly calibrated, silver/silver nitrate reference was used in orgamc solvent (5 - 7 mV vs SCE). All solutions were carefully de-gassed with pure argon prior to scanning. Working electrodes were 2 to 3 mm diameter circular disks, 1 mM diameter gold wires or a 3 mm diameter gold rod. The latter were annealed in a pure butane flame prior to electro polishing.

Scans were carried out at 295K in 100 mM tetrabutylammomum tetrafluoroborate (2:1 v/v CH₂C1₂:CH₃CN) supporting electrolyte. Faradaic currents and half- wave potentials for compound (2) were as expected. The addition of molar excesses of tetrabutylammonium (TBA) anion salts (chloride, bromide and dihydrogen phosphate) to solutions of compound (2) led to significant cathodic perturbations of the ferrocene wave. The results are shown in Table 2 below; wherein Ep_a-Ep_c is the peak separation reference to Ag/AgNO₃; the diffusive half wave potential E_1/2 is defined as (Ep_a-Ep_c)/2 is -20 mV; and ΔE denotes the coordination induced cathodic shift in half wave potential in the presence of 8-9mM anion.

TABLE 2: Diffusive Solution Responses of (2)

^a Error ± 5 mV.

The results show that the addition of molar excesses of TBA anion salts to solutions of compound (2) led to significant cathodic perturbation of the ferrocene wave. It was thus shown that, by directly linking the amide group to the ferrocene ring, hydrogen bonded anion recognition events were accompanied by significant cathodic perturbations of the ferrocene wave, particularly with the dihydrogen phosphate anion.

EXAMPLE 5: Voltammetry of SAMs on gold electrode Sensors of the invention produced as described in Example 2 were submitted to the following voltametric experiments using solutions of anions. 1. A sensor comprising compound (2) was scanned at 295K in 100 mM tetrabutylammom^'um tetrafluoroborate (2:1 v/v CH₂C1₂:CH₃CN) supporting electrolyte. The addition of molar excesses of TBA anion salts (chloride, bromide and dihydrogen phosphate) to the sensor led to significant perturbations in the ferrocene wave, which were greater than those observed for compound (2) diffusively ( compare Table 2 in Example 4 above). The results are shown in Table 3 below, wherein the SAM E_1/2 is -20 mV vs Ag/AgNO₃ and ΔE denotes the co-ordination induced cathodic shift in half wave potential in the presence of 8-9 mM anion.

TABLE 3: Monolayer Responses of (2)

^b Error ± 10 mV.

The structure of the sensor with the bound anion in situ is shown in the accompanying Figures 1 and 2.

2. The following voltametric experiments using the sensors of Example 2 were carried out in the presence of perrhennate in both aqueous and organic medica. (a) Sensors comprising SAMs of compound (2) or compound (3) had identical half wave potentials (-20mV vs Ag/AgNO₃) in an organic solvent (7:3 v/v CH₂C1₂:CH₃CN)- Aqueous scans were carried out at 296K in the presence of 100 mM NaClO₄ supporting electrolyte. Under such conditions the half wave potentials of the sensors (SAMs on gold electrode ) were -200 mV vs Hg HgSO₄.

The sensors comprising SAMs of compound (2) or (3) in water, when scanned as described above, gave reproducible cathodic shifts (15-20 mV) in the presence of perrhennate, which is a comparatively poorly hydrated anion. (b) In competition experiments carried out with SAMs of (2) and (3) with molar equivalents of perrhennate and dihydrogen phosphate in CH₃CN:CHC1₃, H₂PO₄' was selectively detected. In water, however, perrhennate was selectively detected.

EXAMPLE 6: Preparation of novel sensing units

The amide-disulphide functionalized porphyrin (4) was prepared in 37% yield from the condensation reaction of α,α,α,α-atropisomers of 5,10,15,20-meso-tetrakis (o-aminophenyl) porphyrin and four equivalents of thioctic acid in the presence of DCCI and HOBt. Reaction of (4) with Zn(OAc)₂ produced the metalloporphyrin derivative (5) in 75% yield.

EXAMPLE 7: Preparation of sensor nanoparticles

Gold nanoparticles were synthesised via the Brust method, using dodecanethiol as the protecting thiol. The metalloporphyrin (5), prepared in Example 6, was added to a stirred chloroform solution of dodecanethiol-protected gold nanoparticles. The solution was stirred for 20 hours, the solvent removed and the product recrystallised from chloroform and diethyl ether. Non-covalently bound (5) was removed by washing the resulting solid exhaustively with methanol and acetone until the liquor ran colourless. Porphyrin-modified gold nanoparticles (6) (see Figure 4) were obtained and characterised by ^lΗ and ¹³C NMR, UV-visible spectroscopy, elemental analysis, mass spectroscopy and thin layer chromatography. .

The number of porphyrin receptor moieties per nanoparticle was estimated, using transmission electron microscopy (TEM)-derived nanoparticle diameters and elemental analysis, to be 200-400 (depending on particle size).

EXAMPLE 8: Determination of binding constants

Binding constants were determined by an analysis of data provided by UV visible spectroscopy carried out during titration of the free metalloporphyrin (5) and the nanoparticle-bound metalloporphyrin (6) with a number of anions in dichloromethane and DMSO solutions. The porphyrin chromophore characteristics were monitored during the addition of anions (as their tetrabutylammonium (TBA) salts), directly into spectrochemical cells at 293K. The stability constants for 1 : 1 complexes were then calculated using the Specfit© program.

TABLE 4

Comparative binding constants of porphyrin receptors (5) and (6) iα 1:1 dichloromethane:acetonitrile with tetrabutylammonium salts of the respective anions. The binding constants, given in log₁₀, were calculated using Specfit©. Errors + 0.1.

The data obtained in CH₂C1₂ solution (Table 4) is indicative of extremely strong complex formation between (5) and (6) and both Cl^" and H₂PO₄\ Table 4 also displays the striking result that the metallopoφhyrin-functionalised nanoparticle (6) exhibits significantly larger magnitudes of stability constant, in comparison to (5), for Bf, I^" and NO₃ ^" anions studied. This observation is further reinforced by the results of anion complexation studies with Cl^" and H₂PO₄ ^"carried out in the competitive solvent DMSO (Table 5). Particularly striking are the respective chloride binding affinitieSjWhich highlights remarkable enhancements of anion binding strength for the surface-confined metalloporphyrin.

TABLE 5

Comparative binding constants of porphyrin receptors (5) and (6) in DMSO with tetrabutylammonium salts of the respective anions. The binding constants, given in log₁₀, were calculated using Specfit©. Errors + 0.1.

Zinc metallopoφhyrins self-assembled onto gold nanoparticles exhibited an enhanced anion binding affinity compared with the receptor free in solution.

Claims

1. A sensor which is suitable for the detection of a negatively charged species and which comprises a monolayer attached to a metal surface, wherein the monolayer comprises, in order from the surface:

(a) a metal-anchoring group;

(b) a host portion comprising two or more binding groups, which groups are together capable of chelating to the negatively charged species; and (c) . a detector group Which is capable of undergoing a detectable change in electrochemical or optical properties upon binding of the negatively charged species by the host portion (b); the host portion (b) being connected to each of the metal anchoring group (a) and the detector group (c) by a direct bond or via a linking group.

2. A sensor according to claim 1 wherein the metal surface is provided on an electrode.

3. A sensor according to claim 1 wherein the metal surface is the surface of a metallic nanoparticle (including quantum dots), the nanoparticle being either free in solution or assembled at an interface.

4. A sensor according to claim 3 wherein the surface of the nanoparticle is capable of undergoing a change in optical properties upon binding of the negatively charged species by the host portion (b).

5. A sensor according to any one of the preceding claims wherein the group (c) is a metallocene or a metallopoφhyrin. .

6. A sensor accordmg to claim 5 wherein the metallocene is ferrocene or cobaltocenium.

7. A sensor according to claim 5, wherein the metallopoφhyrin is a zinc metallopoφhyrin.

8. A sensor according any one of the preceding claims, in which the host portion comprises at least one hydrogen bond donor group.

9. A sensor according to any one of the preceding claims, in which the host portion comprises an amide, thioamide, polyamide, polythioamide, urea, thiourea, pyrrole or guanidinium group, or a peptide unit.

10. A sensor according to any one of the preceding claims, in which the metal surface is, or comprises, copper, gold, platinum or silver.

11. A sensor according to any one of the preceding claims, in .which the metal- anchoring group is a sulphur-containing moiety.

12. A sensor assembly comprising an array of two or more sensors as defined in any one of the preceding claims, wherein each sensor is tailored to recognise a different negatively charged species.

13. An assembly according to claim .12 wherein the sensors are provided on a support.

14. A process for the production of a sensor as defined in any one of claims 1 to 11, which process comprises synfhesising the units of the monolayer and allowing the monolayer to self assemble onto a metal surface.

15. A process for assaying an analyte for the presence of a negatively charged species, which process comprises contacting the analyte with a sensor as defined in any one of claims 1 to 11 or an assembly as defined in claim 12 or

13 and observing the change in electrochemical or optical properties of the group (c), the sensor or assembly being tailored to detect, respectively, one or more negatively charged species that are known or suspected to be present in the analyte.

16. A process according to, claim 15 wherein the analyte is a biological solution or a solution of environmental significance.

17. Use of a sensor as defined in any one of claims 1 to 11 or an assembly as defined in claim 12 or 13 for the detection of negatively charged species in . an analyte.

18. A process according to claim 15 or 16 or a use according to claim 17, wherein the negatively charged species is selected from halides, nitrate, phosphate, sulphate, carboxylate, perrhennate, radioactive anions, ATP, ADP, DNA and RNA.

19. A compound of the following formula (Via) or (Vlb):

wherein

M is a metal;

B¹ is a group of formula (i) or (ii): (i) -C(O)-NH-(T)_r- (ii) -NH-C(O)-(T)_r- wherein: r is O, 1, 2, 3 or 4; and

T is an amino acid residue or a group -C*(R')(R")- wherein C* is a chiral or achiral carbon atom and each of R' and R", which are the same or different, is H or a group selected from C C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl and C₆-Ci₂ aryl, the group being unsubstituted or substituted by one or more substituents and being optionally interrupted by one or more heteroatoms, aromatic groups, heteroaromatic groups or unsaturated functions; . Y is a -C_JO akylene, C₂-C₂₀ alkenylene or C₂-C₂₀ alkynylene chain is unsubstituted or substituted, and may be interrupted by one or more heteroatoms, by an aromatic or heteroaromatic group or by one or more unsaturated functionalities;

1 is an integer of 1 to 20; m is O or 1; and R² is a group containing one or more sulphur atoms.

20. A compound according to claim 19 wherein M is Fe or Co⁺, Y is Cj - C₆ alkylene, m is 1 and R² is a group containing one or more thiol, polythiol, sulphide, disulphide, or thioether functions.

21. A compound of formula (VJJ) :

wherein each W is

in which n is an integer of 1 to 4.

10 22. A compound according to claim 21 wherein n is 4.

23. A sensor according to claim 1 wherein the monolayer comprises sensing units derived from a compound of formula (VII) as defined in claim 21 or 22.