GB2467142A

GB2467142A - Electrode comprising two or more amphiphatic molecules and a redox active group

Info

Publication number: GB2467142A
Application number: GB0901085A
Authority: GB
Inventors: Sarah Ann Goodchild; Nicola Elizabeth Cant; Piero Migliorato; Man Yi Ho; Peng Li; Pedro Miguel De Lemos Correia Estrela
Original assignee: UK Secretary of State for Defence
Current assignee: UK Secretary of State for Defence
Priority date: 2009-01-23
Filing date: 2009-01-23
Publication date: 2010-07-28
Also published as: GB0901085D0

Abstract

Electrochemical electrode comprising a self assembled monolayer having two or more amphiphatic molecules, one of which have a redox active group and the other having a recognition element. The redox active group can be ferrocene, ferricyanide, hexaammineruthenium, a metallic protein, a nucleic acid or a DNA aptamer. The first and second amphiphatic molecules can also both be n-alkanethiolates.

Description

Electrode and Electrochemical Sensor The present invention relates to an electrode comprising a solid conducting substrate having thereon a self-assembled monolayer which comprises a first amphipathic molecule which comprises a recognition element specific to a target analyte and a second amphipathic molecule which comprises a redox-active group, methods for preparation of the electrode, an electrochemical cell comprising the electrode, and uses of the electrode and electrochemical cell in the detection of a target analyte.

As used herein, the term "self-assembled monolayer (SAM)" refers to a thin organic monolayer of spontaneously adsorbed amphipathic molecules on the surface of a solid substrate (e.g. a metal or a metal oxide). An amphipathic molecule has hydrophilic and lipophilic properties, and commonly a hydrophilic head' group and a lipophilic tail' group. The self-assembly process is initiated by a strong chemical interaction between the hydrophilic head' group and the substrate. The lipophilic tail' groups are subsequently aligned parallel to each other on the substrate surface through the interplay of van der Waals, steric, repulsive and electrostatic forces to create the SAM. Self assembly is achieved by dipping a solid substrate into a solution containing the amphipathic molecules. The outer surface of a SAM, i.e. the surface opposite that adsorbed to the solid substrate, is available for interaction with the surrounding environment. The shape and physical properties of a self-assembled monolayer can be controlled and modified on a molecular level.

SAMs have been formed from a variety of molecular species including fatty acids, trichlorosilanes and trialkoxysilanes on glass, metal and silicon substrates and carboxylic acids and alkyl phosphates on metal oxide substrates. Surfaces of metal and metal oxide substrates in particular readily adsorb organic materials because of a reduction of the free energy at the interface between the metal/metal oxide and the ambient environment upon adsorption. Gold is often used as the substrate, and alkylthiols as the amphipathic molecules. SAMs formed by alkanethiol molecular species on gold offer particular advantages due to the strong specific interaction of gold with sulfur. Moreover, gold is a relatively inert metal and thus can resist oxidation and atmospheric contamination. The predictable adsorption of sulfur to gold allows the formation of tightly packed monolayers even in the presence of many functional groups on the alkanethiol molecular species. Long-chain alkanethiols in particular form a densely packed, crystalline or liquid-crystalline monolayer due to strong molecular interactions (van der Waals forces) between the long carbon chains.

A SAM was first produced and characterised by R. G. Nuzzo et at, J. Am. Chem. Soc., 1983, 105, 4481 -4483. The SAM was composed of disulfides on gold. These monolayers have been shown to provide model surfaces for investigating and/or mimicking biological interactions, such as cell signalling, cell adhesion, and protein interactions. Changing as little as one atom of a group at the outer surface of a SAM can dramatically alter macroscopic properties such as wettability and biocompatibility, and thus dramatically effect biological interactions. Review articles on SAMs and their applications include 0. Finklca, Elcctroanalytical chemistry: a series of advances 1996, 19, 109-335 andJ. C. Love, Chem. Rev. 2005, 105, 1103-1169.

SAMs have been studied by a multitude of surface analysis techniques including atomic force microscopy (AFM), Fourier transform infra red (FTTR), surface Plasmon resonance (SPR) and scanning tunnelling microscopy (STM) providing a wealth of information enabling the design of SAMs tailored to a particular application.

Preparation of alkanethiol based SAMs is a simple process. For example, a gold-coated substrate immersed in a dilute solution of an alkanethiol in ethanol for a period of between about 1 to 24 hours results in assembly of a monolayer of alkanethiolates on the surface of the substrate. A disordered monolayer is initially formed, within about a few minutes, with the thickness of the monolayer reaching 80 to 90% of its final value. Van der Waals forces on the carbon chains then aid the long alkanethiol chains to pack into a well-ordered crystalline layer. During this process contaminants are replaced, solvents are expelled from the monolayer, and defects are reduced while packing is enhanced by lateral diffusion of the alkanethiols. The alkanethiolates often assemble in a hexagonal-packing arrangement.

An electrochemical sensor detects the production or loss of electrons during a chemical or biological process. Electrochemical sensors can be conductometric, potentiometric or amperometric. A conductometric sensor measures the change in conductance between a pair of metal electrodes. A potentiometric sensor measures the potential difference between a working electrode and a reference electrode. An amperometric sensor measures current-voltage characteristics resulting from the electrochemical oxidation or reduction of an electroactive species in the presence of a redox active group. This redox active group can be in solution or immobilized to an electrode.

The basis of an amperometric measurement can be illustrated using a simple system comprising a metal electrode immersed in an inert electrolyte containing a redox active group. Transfer of electrons can occur via electron tunnelling from the electrode to the redox active group or vice versa. The driving force is the potential gradient at the interface. When a potential is applied to the metal, charges are built up.

As a consequence, oppositely charged ions and dipoles in solution are electrostatically attracted to its surface, and an electrical double layer is formed. Formal potential is the minimum potential that one has to apply in order to oxidize or reduce a species.

The shift in the formal potential of a redox active group embedded in a SAM immobilized on a metal electrode can be explained by electrical effects at the double layer.

The aim of the present application is to provide improved sensors, in particular electrochemical sensors comprising an electrode having thereon a self-assembled monolayer for monitoring, interrogating and detecting biological interactions, and especially detecting target analytes.

Thus, in a first aspect, the present invention provides an electrode for use in an electrochemical sensor for detecting a target analyte in a solution comprising a solid conducting substrate having thereon a self-assembled monolayer which comprises: a first amphipathic molecule which comprises a recognition element specific to the target analyte and which is accessible within the self-assembled monolayer to bind to the target analyte; and a second amphipathic molecule which comprises a redox-active group, and whereby the formal potential of the redox active group is altered upon binding of the target analyte to the recognition element.

The self assembled monolayer may comprise any amphipathic molecule and solid conducting substrate suitable for providing a SAM of the first aspect. The solution is preferably an electrolyte solution.

As used herein, the term accessible' refers to the availability of the recognition element to the target analyte in solution to enable the recognition element to bind the target analyte. This may be provided by the recognition element being present at the outer surface of the SAM.

Amphipathic molecules suitable for providing the SAM include alkyithiols, alkylsilanes and ailcylcarboxylates. The structure of the amphipathic molecules may depend on the desired properties of the SAM. The amphipathic molecules are preferably based on n-alkyl chains, i.e. linear alkyl chains. Long chain ailcanethiols (> C4) form well-organized structures on solid conducting substrates, with dielectric constants of around 2.3 to 2.6 for a SAM on a gold substrate. On the other hand, short carbon chains ( C4) provide a less well-organized structure with defects, allowing permeation of the electrolyte through the SAMs and causing electrochemistry to occur at the electrode. The length of carbon chain length is preferably between about C5 to about C30, more preferably between about C5 to C20, and most preferably between about C10 to C15. For example, amphipathic molecules may have a linear alkly chain of carbon chain length of C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19 or C20. A SAM may comprise amphipathic molecules having linear alkyl chains of differing carbon chain length. For example, the SAM may comprise a first amphipathic molecule of carbon chain length Cio, a second amphipathic molecule of carbon chain length Cii, and a third amphipathic molecule of carbon chain length C6.

The amphipathic molecules are preferably n-alkanethiolates (i.e. obtained by adsorption of an n-alkanethiol to a solid conducting substrate).

The recognition element can be either charged (positive or negative) or uncharged.

The recognition element is complementary to and binds specifically the target analyte.

The target analyte is a biological material such as a microorganism (cell or spore), or a biological molecule such as an antibody, a carbohydrate or a polynucleotide. The recognition element may, for example, be a protein, a carbohydrate, an aptamer, an oligonucleotide or a polynucleotide.

As used herein, a redox-active group is a molecule or component of a molecule that is capable of being oxidized or reduced by the application of a suitable voltage.

Examples of redox-active molecules include ferrocene, ferricyanide, hexammineruthenium, metallic proteins such as azurin, DNA and DNA aptamers. In a preferred embodiment of the electrode of the first aspect the redox-active group is ferrocene. The second amphipathic molecule may for example be 6-ferrocenyl-1-hcxanethiolate or 11 -ferrocenyl-1 -undecanethiolatc.

The solid conducting substrate may be a metal such as gold, silver, copper, palladium, chromium and platinum, a metal oxide, glassy carbon, a semiconductor such as silicon, silicon dioxide or silicon nitride, a semiconductor oxide or a semiconductor nitride. The solid conducting substrate is preferably gold.

To enable predictable and accurate detection of a target analyte by the recognition element the SAM should have properties that prevent or at least discourage non-specific adsorption of analytes other than the target analyte. The monolayer should also present the recognition element in a structurally well defined manner that minimizes the influence of the surface of the monolayer. A self assembled monolayer environment that discourages or prevents non-specific adsorption may comprise amphipathic molecules presenting neutral and/or hydrophilic terminal groups, such as alcohols, e.g. 6-hydroxy-1-hexanethiolate. Thus, the self assembled monolayer preferably further comprises a third amphipathic molecule having a neutral (i.e. non-charged) terminal group. Non-specific adsorption can also be discouraged or avoided by using alkanethiols terminated with oligo-or polyethylene glycol. Alkanethiols terminated with polyethylene glycol form a dense, ordered monolayer on a gold substrate with the same molecular conformation found for n-alkanethiols.

As used herein, the term terminal group' refers to a chemical group at the opposite end of the amphipathic molecule (and lipophilic tail group) to the hydrophilic head group, for example 6-hydroxy-1-hexanethiolate has a hydroxyl group at the opposite of the hexane tail to the hydrophilic thiol group.

The concentration ratio of first amphipathic molecule: second amphipathic molecule third amphipathic molecule in the SAM preferably ranges from about 1:1:1 to 10:1:10, including the ratios 1:1:5, 1:1:10, 2.5:1:5, 5:1:5, 4:1:5 and 10:1:10. Solid conducting substrates comprising a SAM with these ratios function particularly well as electrodes in an electrochemical cell. Approximation of the preferable concentration ratios is due to the fact that the concentration ratio finally adsorbed to the substrate surface reflects, but is not necessarily the same as, the concentration ratio of amphipathic molecules in the solution prior to chemisorptionlself assembly.

In a second aspect, the present invention provides an electrode comprising a solid conducting substrate having thereon a self-assembled monolayer which comprises: a first amphipathic molecule which comprises a functional group providing for covalent attachment of a recognition element; and a second amphipathic molecule which comprises a redox-active group; and whereby covalent attachment of the recognition element to the functional group results in an electrode according to the first aspect of the present invention.

The functional group enables the SAM to be derivatised by a recognition element specific to a target analyte. Typical functional groups for attachment of a recognition element include amine, carboxylic acid, alcohol, ester, azide and alkyne. For example a carboxyl functional group can easily couple to recognition elements such as biotin, a protein (such as an antibody) or peptide, a carbohydrate, an aptamer, an oligonucleotide or a polynucleotide, via well known coupling chemistries such as those using 1 -Ethyl-3 -[3 -dimethylaminopropyl]carbodiimide (EDC) and N-hydroxysuccinimide (NH S).

The first amphipathic molecule is preferably an n-alkanethiolate comprising a functional group, and the second amphipathic molecule an n-alkanethiolate comprising a redox-active group. The first amphipathic molecule may for example be 11-amino-i -undecanethiolate or 1 0-carboxy-1 -decanethiolate and the second amphipathic molecule may for example be 6-ferrocenyl-i-hexanethiolate or 11-ferrocenyl-1 -undecanethio late.

In a preferred embodiment, the SAM further comprises a third amphipathic molecule having a neutral terminal group to discourage or prevent non-specific adsorption, such as 6-hydroxy-1 -hexanethiolate.

In a third aspect, the present invention provides an electrochemical sensor for detecting a target analyte in a solution which comprises an electrochemical cell having an electrode of the first aspect, a counter electrode and a reference electrode; means for applying a potential to the electrode; and means for measuring current between the electrode and the counter electrode.

The electrochemical sensor may comprise a plurality or microarray of electrochemical cells.

In a fourth aspect, the present invention provides a method for monitoring an electrochemical parameter of an electrode of the first aspect in an electrochemical sensor of the third aspect comprising contacting the electrochemical cell with a solution, applying a variable potential to the electrode and measuring the current between the electrode and the counter electrode as a function of the potential, and extrapolating the electrochemical parameter from the relationship of the current to the potential.

The technique of measuring the current between the electrode and the counter electrode as a function of potential is generally known as voltammetry. The class of voltammetry is dependent on the manner in which the potential is varied, but includes cyclic voltammetry, square wave voltammetry and AC voltammetry. Examples of suitable electrochemical parameters are known to those skilled in the art. The electrochemical parameter is altered upon binding of the target analyte to the recognition element. The relationship of the current to the potential can be plotted graphically (a voltammogram), and the electrochemical parameter extrapolated from the graph. The electrochemical parameter of the electrode is preferably the formal potential of the redox-active group.

Thus, in a fifth aspect the present invention provides a method for detecting a target analyte in a solution comprising monitoring an electrochemical parameter of an electrode according to the method of the fourth aspect both in the presence and absence of the target analyte in the solution, and determining the presence of the target analyte through a difference in the electrochemical parameter.

The solution is preferably an electrolyte solution, and the electrochemical parameter of the electrode is preferably the formal potential of the redox-active group.

A sixth aspect of the present invention provides a method for producing an electrode for use in an electrochemical sensor for detecting a target analyte in a solution comprising chemisorption of a mixture of amphipathic molecules to a solid conducting substrate to create a self assembled monolayer on a surface of the solid conducting substrate, wherein the mixture of amphipathic molecules comprises a first amphipathic molecule which comprises a recognition element specific to the target analyte and a second amphipathic molecule which comprises a redox active group.

The mixture of amphipathic molecules may comprise a first amphipathic molecule comprising a recognition element and a second amphipathic molecule comprising a redox-active group, creating an electrode of the first aspect. However, in alternative embodiments, the mixture of amphipathic molecules may create a self-assembled monolayer comprising one or more functional groups capable of being derivatised with a redox-active group and/or a recognition element. The method may then subsequently comprise derivatisation of the SAM with a redox-active group and/or a recognition element to create an electrode of the first aspect.

Amphipathic molecules suitable for providing the SAM include alkylthiols, alkylsilanes and ailcylcarboxylates. The structure of the amphipathic molecules may depend on the desired properties of the SAM. The amphipathic molecules are preferably based on n-alkyl chains, i.e. linear alkyl chains. Long chain ailcanethiols (> C4) form well-organized structures on solid conducting substrates, with dielectric constants of around 2.3 to 2.6 for a SAM on a gold substrate. On the other hand, short carbon chains ( C4) provide a less well-organized structure with defects, allowing permeation of the electrolyte through the SAMs and causing electrochemistry to occur at the electrode. The length of carbon chain length is preferably between about C5 to about C30, more preferably between about C5 to C20, and most preferably between about C10 to C15. For example, amphipathic molecules may have a linear alkly chain of carbon chain length of C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19 or C20. A SAM may comprise amphipathic molecules having linear alkyl chains of differing carbon chain length. For example, the SAM may be produced from a solution containing a first amphipathic molecule of carbon chain length C10, a second amphipathic molecule of carbon chain length C11, and a third amphipathic molecule of carbon chain length C6. The amphipathic molecules are preferably n-alkanethiols.

Examples of redox-active molecules include ferrocene, ferricyanide, hexammineruthenium, metallic proteins such as azurin, DNA and DNA aptamers. In a preferred embodiment of the electrode of the first aspect the redox-active group is ferrocene. The second amphipathic molecule may for example be 6-ferrocenyl-1-hexanethiolate or 1 1-ferrocenyl-1-undecanethiolate.

The properties of a SAM are determined by the selection and concentration of amphipathic molecules. The mixture of amphipathic molecules may comprise a third amphipathic molecule having a neutral terminal group to discourage non-specific binding to the SAM, such as 6-hydroxy-1-hexanethiol. Additional amphipathic molecules in the mixture may also enable control over the concentration ratio of the redox-active group and/or the recognition element in the SAM. The concentration ratio of the different amphipathic molecules in the solution reflects, but is not necessarily the same as, the concentration ratio finally adsorbed on the substrate surface. Suitable concentration ratios for first amphipathic molecule: second amphipathic molecule: third amphipathic molecule in the mixture are between 1:1:1 and 10:1:10, and include the ratios 1:1:5, 1:1:10, 5:1:5, 4:1:5 and 10:1:10. The mixture of amphipathic molecules is preferably in solution.

In one embodiment, a mixture of alkanethiols in solution is chemisorbed to a gold substrate. A high concentration of alkanethiols (approx. 1 mM) can assemble at a gold surface in less than 1 hour, with 80% -90% coverage obtained after a few minutes, though a well defined SAM will often take more than 12 hours to form. The structure of a SAM can continue to evolve over 7 to 10 days. The coverage of the surface increases with extended chemisorption times. The typical time allowed for chemisorption is 12 to 18 hours. Experimental conditions can bias the relative ratio of the components constituting the SAM, for example the choice of solvent can modify the relative mole fraction of amphipathic molecules adsorbed. Similarly, mixtures of alkanethiols with different chain lengths will form SAMs with a composition enriched with the longer alkanethiol; this bias increases over time. Although alkanethiols can displace non-specific or weakly adsorbed contaminants and impurities on the substrate, the cleanliness of the substrate is very important prior to immersion of alkanethiols to have reproducible results, good rate of formation and less defects.

Ethanol is the solvent most widely used for preparing SAMs, though other solvents such as the non-polar heptane or hexane, and polar solvents such as water or DMSO may be used.

Forming SAMs at temperatures above 25°C can improve the kinetics of formation, increase the rate of desorption of non-specifically physisorbed materials and solvent molecules, and thereby reduce the number of defects present in the SAM.

The present invention will now be described with reference to the following non-limiting examples and drawings in which Figure 1 is a typical cyclic voltammogram for a redox species immobilized on a surface; Figure 2 depicts formation of a C6Fc SAM on a gold working electrode; Figure 3 is a cyclic voltammogram produced for a C6Fc SAM in 5 mM phosphate buffer pH7; Figure 4 illustrates the dimensions of a) a pure C6Fc SAM and b) a more compact SAM comprising C6Fc and HHT; Figure 5 is a cyclic voltammogram produced for a HHT SAM in 5 mM phosphate buffer pH7; Figure 6 is a depiction of the gold electrode surface indicating the surface area occupied by the ailcanethiol chains; Figure 7 is a cyclic voltammogram produced for a mixed C6Fc/HHT (1:5) SAM in 5mM phosphate buffer pH 7; Figure 8 is a depiction of a mixed C6Fc/HHT/AUT SAM; Figure 9 is a cyclic voltammogram produced for a mixed C6Fc/HHT SAM (0.2 niIVl/1 mM) that has subsequently been incubated with AUT (1 mlvi), inS mlvi phosphate buffer pH 7; Figure 10 is a cyclic voltammogram produced for a mixed C6Fc/AUT SAM that has subsequently been incubated with HHT, in 5 mM phosphate buffer pH 7.0; Figure 11 is a cyclic voltammogram produced for a mixed C6Fc/HHT/AUT SAM (1:8:8) in 5 mIVI phosphate buffer pH 7.0; Figure 12 is a comparison of a cyclic voltammogram produced for a SAM containing positively charged amine groups with a cyclic voltammogram produced for the neutralized SAM, in 5 mM phosphate buffer pH 7; Figure 13 is a depiction of a mixed C6Fc/HHT/CDT SAM; Figure 14 is a cyclic voltammogram produced for a mixed C6Fc/HHT/CDT SAM (1:5:4) in 5 mIVI phosphate bufferpH 7; Figure 15 is a comparison of a cyclic voltammogram produced for a SAM containing negatively charged carboxyl groups with a cyclic voltammogram produced for the neutralized SAM, in 5 mIVI phosphate buffer pH 7; Figure 16 is the structure of the thiolated biotin molecule; Figure 17 is a square wave voltammogram produced for a biotin containing SAM, both before and after interaction with streptavidin, in 5 mM phosphate buffer pH 7.

Cyclic Voltammetry Cyclic voltammetry (CV) is an amperometric measurement. The instrumentation contains a cell having three electrodes: a gold working electrode (WE), a reference electrode (REF) and a counter electrode (CE), surrounded by an electrolyte solution.

A potential is applied to the working electrode with respect to the reference electrode, resulting in a current between the working electrode and the counter electrode. The clectrochemical surface properties of the gold electrode are revealed by measuring the current. Having regard to Figure 1 during the forward scan, the species are being oxidized, and in the reverse scan, the species are being reduced. The formal potential is the mid-point between the potentials at which the peak currents due to oxidation 1 and reduction 2 occur. Therefore cyclic voltammetry can be used to detect the change of formal potential due to specific bio-interactions.

Cleaning of Bulk Gold Electrodes The gold working electrode is cleaned by sonication in 3% Decon for 5 minutes, rinsing with water, followed by polishing with 0.03 tm alumina powder for 5 minutes. The electrode is then rinsed with a strong flow of tap water followed by deionised (Millipore) water, sonicated in deionised water for 5 minutes, and polished with blank polishing pad to remove any residue of particles resulting from the polishing. Finally, the electrode is rinsed with deionised water followed by sonication in deionised water for 5 minutes. The electrode is mechanically polished followed by electrochemical cleaning is situ with 0.5 M H2S04. The potential is swept from -0.05 V to 1.1 V, when compared to a Hg/Hg2SO4 reference electrode, for 60 cycles to remove non-specifically adsorbed molecules on the gold surface.

Examples

Ferrocene SAM The SAM was prepared by soaking the gold working electrode in 0.2 mM of 6-ferrocenyl-1 -hexanethiol (C6Fc) in ethanol.

Having regard to Figure 3 the cyclic voltammogram differs from the theoretical curve of adsorbed species in that the anodic and cathodic peaks are not symmetric. At a potential of over 0.1 V, when compared to the reference electrode, the charging current is large in comparison to the redox current suggesting that the SAM does not produce a compact arrangement on the gold surface, and consequently that water molecules are able to penetrate the SAM and contact the gold surface.

Having regard to Figure 4a and Figure 4b, for a well arranged SAM the distance between adjacent sulphur atoms is 0.5 nm, while the lateral width of the Fe is 0.64 nm. Therefore a well organized SAM will not be achieved with C6Fc SAMs alone (Figure 4 a). In order to make a better insulating and compact SAM, a space filler molecule such as 6-hydroxy-1-hexanethiol may be used to passivate the gold electrode (Figure 4b).

6-hydroxy-1-hexanethiol SAM The SAM was prepared by soaking the gold working electrode in 1 mM of 6-hydroxy-1-hexanethiol (HHT) in ethanol. Cyclic voltammetry was used to investigate the passivation ability of HHT. Having regard to Figure 5 the double layer capacitance varies non-linearly with the applied potential. However within the potential range of our experiment the relationship can be considered as linear.

Therefore base line correction of current due to the double layer capacitance was approximately linear with potential. Furthermore HHT gave a better passivation of the gold since the current obtained was of the order 107A as opposed to 106 A for the C6Fc SAM.

Mixed ferrocene and 6-hydroxy-1-hexanethiol SAM The SAM was prepared by soaking the gold working electrode in a mixture of the two molecular species as detailed: Concentration Ratio Concentration 1mM (C6Fc:HHT) C6Fc HHT 1:5 0.2 1 1:10 0.1 1 A well defined SAM was produced, as evident from electrochemical analysis. Having regard to Figure 4b as expected good passivation of the gold surface was achieved. A comparison of the formal potential between the 1:5 SAM and the 1:10 SAM indicated that the higher the coverage of C6Fc, the more positive the formal potential.

To predict the optimum ratio of molecular species within a mixed C6Fc and HHT SAM an analysis at the atomic level of the gold electrode is required. Having regard to Figure 6 sulphur atoms 3 are situated at the 3-fold hollows of the gold atoms 4, with each possible binding site for sulfur being separated by a distance of 5 A. The dashed lines 5 indicate the projected surface area occupied by the alkane chains. The size of ferrocene is 6.4 A and thus taking into account the spatial arrangement of atoms it is only possible for 1 in every 6 sulfur binding sites to be occupied by C6Fc.

Two factors primarily determine the chemisorption rate of alkanethiols on gold, the length of methylene chain and the choice of the terminal functional group. The longer the methylene chain, the faster the chemisorption occurs. The hydroxyl head group of HHT can form hydrogen bonds which stabilize the monolayer as it forms, while the ferrocene can participate in a hydrophobic interaction with gold. Assuming the binding kinetics for C6Fc and HHT on gold is similar, since they have an identical number of methylene groups (six carbons), the coverage for each molecular species can be estimated from the concentration ratio of C6Fc to HHT in solution.

Different concentration ratios of C6Fc to HHT in solution were prepared to achieve the optimum coverage of C6Fc in a compact SAM. Having regard to Figure 7, a comparison of cyclic voltammograms produced for a ratio of 1:10 (6) and 1:5 (7) C6Fc:HHT, it was clear that a ratio of 1:5 provided the best SAM in terms of coverage. Also a lower charging current was observed indicating a better arrangement of the SAM.

One uncertainty about mixed SAMs is phase separation between the different molecular species, especially in a mixed SAM comprising hydrophilic and hydrophobic species: similar species may cluster together. This may be avoided, and passivation of the gold may be improved, by using molecular species with similar chemical properties.

Mixed Ferrocene, 6-hydroxy-1-hexanethiol and 11-amino-1-undecanethiol Three adsorption strategies were investigated for producing the mixed SAM: 1) co-adsorption of C6Fc and HHT, then incubated with the 11-amino-1-undecanethiol (AUT); 2) co-adsorption C6Fc and AUT, then incubated with HHT; 3) co-adsorption of all three. Having regard to Figure 8, the amine group in a mixed SAM will be protonated and thus positively charged in the measuring buffer of 5mM phosphate buffer pH 7.

1) Concentrations of C6Fc and HHT were 0.2 mM and 1 mM, respectively. The electrode was rinsed with ethanol after immersion in the C6Fc/HHT solution for two days. It was then transferred to a solution containing 1 mM of AUT overnight. Having regard to Figure 9, a comparison of the SAM comprising C6Fc and HHT 8 with the SAM after incubation with AUT 9, subsequent incubation of the SAM with AUT causes an increase in charging current due to distortion of the well arranged C6Fc:HHT SAM. A decrease in peak current is also observed, resulting in reduction of the area under the peak. This would suggest a loss of C6Fc from the SAM, probably due to displacement of C6Fc by AUT.

2) Two concentration ratios of C6Fc and AUT were evaluated: 0.2mM: imiVi and 0.lrnM:lrnM. The electrode was rinsed with ethanol after immersion in the C6Fc/AUT solution for one day. It was then transferred to a solution containing 1 mM HHT for 2 hours. Having regard to Figure 10, a ferrocene redox peak was not observed for either ratio, suggesting that no C6Fc adsorbed to the gold surface. This may be explained by the dependency of chemisorption rate on the length of methylene chain: the longer the methylene chain, the faster chemisorption. In this case, AUT with 11 carbons has a faster chemisorption rate than C6Fc with just 6 carbons.

Moreover, even if C6Fc did initially adsorb it would be gradually replaced by AUT over the one day period of immersion. This immobilization strategy was thus not appropnate.

3) Co-adsorption of all three species may result in a similar phenomenon to that seen for adsorption strategy 2, i.e. no or minimal adsorption of C6Fc. Two possible solutions to this problem were to use a much shorter immersion time, to avoid replacement of C6Fc by AUT, or conversely a much longer immersion time, to produce a better arrangement of SAM, hopefully comprising C6Fc. Three different concentration ratios of C6Fc:HHT:AUT were investigated, with immersion for between 2 to 5 days.

Concentration Ratio Concentration /mM (C6Fc:HHT:AUT) C6Fc HHT AUT 1:5:5 0.2 1 1 1:8:8 0.125 1 1 1:10:10 0.1 1 1 Having regard to Figure 11, a typical cyclic voltammogram was obtained for a SAM produced with the ratio of 1:8:8. Both 1:8:8 and 1:5:5 gave better defined redox peaks than 1:10:10.

Neutralization of Amine SAM In the mixed Ferrocene, 6-hydroxy-1 -hexanethiol and 11-amino-i -undecanethiol SAM (1:5:5), the amino head group was positively charged inS mlvi phosphate buffer pH 7. Neutralisation of the positive NH3 was performed by immersing the electrode in a solution containing 1 mM 3-(Maleimide)propionic acid N-hydroxysuccinimide (NHS) ester in DMSO.

Having regard to Figure 12, the calculated formal potential from the cyclic voltammogram for the SAM comprising positively charged NH3 groups 10 was -156 mV, whereas after neutralization the formal potential from the cyclic voltammogram 11 was -i 8 i mV, and thus a negative shift of -25 mV was observed upon neutralisation. When the positively charged head group was neutralized, the potential at the plane of electron transfer PET was reduced, and therefore the term IPET -i1s decreased, causing a negative shift in the formal potential E0', according to: Eapp -E° +(IPET _s)+1n[j = E0?+1n[Q_1 (1) Mixed Ferrocene, 6-hydroxy-1-hexanethiol and 1 O-carboxy-1-decanethiol (CDT) Having regard to Figure 13 SAMs were produced from solutions of varying concentration ratios of C6Fc:HHT: CDT: Concentration Ratio Concentration /mM (C6Fc:HHT:CDT) C6Fc HHT CDT 1:4:4 0.25 1 1 1:5:4 0.125 1 1 1:5:5 0.2 1 1 1:8:8 0.125 1 1 The carboxylic group in the SAM will be deprotonated and thus negatively charged in the measuring buffer 5 mM phosphate buffer pH 7.0. Adsorption was achieved using a solution comprising all three alkane thiols. The SAM was produced by soaking the gold electrode in an alkanethiol containing solution. The solvent was ethanol. The electrode was removed from the adsorption solution after a period of 2 days to 1 week, and washed sequentially with the solvent and the buffer which is to be used during analysis. The electrode is stored in this buffer for 15 minutes for stabilization before performing electrochemical measurements. Having regard to Figure 14, the SAM produced from ratio 1:5:4 was observed to give the best redox peak.

Neutralization of Carboxylic SAM In the mixed Ferrocene, 6-hydroxy-1-hexanethiol and 1O-carboxy-1-decanethiol SAM the carboxylic acid head groups were negatively charged in the measuring buffer (5 mM phosphate buffer pH 7). The neutralization protocol for the negative charged C00 head groups was as follows: EDC was first coupled to the carboxylic group to form a good leaving group. To selectively form the amide, sulfo-NHS is added followed by ethanolamine. The concentrations of EDC, sulfoNHS and ethanolamine were 4OmIVl, 10mM and 20mM, respectively.

Having regard to Figure 15, it was observed that the formal potential shifted upon neutralisation, as calculated from the cyclic voltammogram for the SAM comprising the negatively charged C00 groups 12 and the cyclic voltammogram for the neutralised SAM 13, from -95.5 mV to -37.5 mV, thus providing a positive shift of + 58 mV. Upon neutralisation, the potential at the plane of electron transfer iIJPET increased, and therefore the term (PET -s) increased, causing a positive shift in formal potential E0.

Detection of Biotin-Streptavidin Interaction at a Mixed SAM A SAM was produced by incubating a solution containing C11Fc (second amphipathic molecule), HHT (third amphipathic molecule), and thiolated biotin (first amphipathic molecule) in the proportions 1:5:2.5, respectively, at the gold electrode surface. The structure of the thiolated biotin is depicted in Figure 16. The interaction with streptavidin was then monitored. The streptavidin has a p1 ranging from 6.10 to 6.35.

Having regard to Figure 17, at pH 7 a negative shift in formal potential of between 30 to 60 mV was observed (in four different experiments) as expected from the increase in net negative charge brought about by the streptavidin at pH 7. A reduction of the peak current was also observed, confirming that the interaction occurred. Control experiments with an antibody, and with 1 M phosphate buffer were also performed, for which no or negligible shift in the formal potential was observed.

Cyclic voltammetry was used to study the neutralization of positively and negatively charged SAMs. Upon chemical neutralization of NH3 groups (AUT containing SAM) a negative shift in the formal potential was observed. This was due to the reduction in cjpET after neutralization. On the other hand, a positive shift on the formal potential was observed upon neutralization of C00 groups (CDT containing SAM).

In both cases a decrease in the peak current was also observed. A negative shift of the formal potential was observed upon interaction of streptavidin with a biotin containing SAM. The change of charge at the plane of electron transfer can therefore cause a shift in the formal potential in either direction (i.e. both negative and positive shifts possible).

Both the shift in formal potential and the reduction in the peak current that occur upon biomolecular interaction can be used as a sensor signal. This technique which benefits from the flexibility of the SAM can be expanded to other biological systems. The flexibility arises from the incorporation of both a molecular species comprising a functional group and a molecular species comprising a redox active group in the SAM. The functional group can be modified to include any recognition element such as DNA or a protein. The bi-directional shift in formal potential depending on the charge is especially promising for the protein sensor, since by varying the pH of the measuring buffer, the net charge of the protein can be manipulated. Another advantage is, instead of looking at the formal potential, open circuit voltage can also reveal the perturbation of the equilibrium upon specific interaction. Field-effect devices can then be used as the transducer, exploiting the well established integrated circuit technology.

This will lead to a new potential technology in the form of multi-target and most of all point-of-care biosensors.

Claims

CLAIMS1. An electrode for use in an electrochemical sensor for detecting a target analyte in a solution comprising a solid conducting substrate having thereon a self-assembled monolayer which comprises: a first amphipathic molecule which comprises a recognition element specific to the target analyte and which is accessible within the self-assembled monolayer to bind to the target analyte; and a second amphipathic molecule which comprises a redox-active group, and whereby the formal potential of the redox active group is altered upon binding of the target analyte to the recognition element.
2. An electrode according to Claim 1, wherein the recognition element is a protein, a carbohydrate, an aptamer, an oligonucleotide or a polynucleotide.
3. An electrode according to Claim 1 or Claim 2, further comprising a third amphipathic molecule having a neutral terminal group.
4. An electrode according to Claims 3, wherein the first, second and third amphipathic molecules are n-alkanethiolates.
5. An electrode according to Claim 4, wherein the n-alkanethiolates have a carbon chain length of between C5 to C30.
6. An electrode according to Claims 1 to 5, wherein the redox active group is ferrocene, ferricyanide, hexaammineruthenium, a metallic protein, a nucleic acid or a DNA aptamer.
7. An electrode according to Claim 6, wherein the redox active group is ferrocene.
8. An electrode according to Claims 1 to 7, wherein the solid conducting substrate is a metal, metal oxide, semiconductor, semiconductor oxide or semiconductor nitride.
9. An electrode according to Claim 8, wherein the solid conducting substrate is gold, silver, copper, palladium, platinum or chromium.
10. An electrode comprising a solid conducting substrate having thereon a self-assembled monolayer which comprises: a first amphipathic molecule which comprises a functional group providing for covalent attachment of a recognition element; and a second amphipathic molecule which comprises a redox-active group; and whereby covalent attachment of the recognition element to the functional group results in an electrode according to Claims 1 to 9.
11. An electrode according to Claim 10, wherein the functional group is selected from the group consisting of carboxylic acid, amine, ester and alcohol.
12. An electrode according to Claim 10 or Claim 11, further comprising a third amphipathic molecule having a neutral terminal group.
13. An electrode according to Claim 12, wherein the first amphipathic molecule is 11-amino-i -undecanethiolate or 1 0-carboxy-1 -decanethiolate, the second amphipathic molecule is 6-ferrocenyl-i-hexanethiolate or 1 1-ferrocenyl-i-undecanethiolate, and the third amphipathic molecule is 6-hydroxy-1 -hexanethiolate.
14. An electrochemical sensor for detecting a target analyte in a solution which comprises an electrochemical cell having an electrode according to Claims 1 to 9, a counter electrode and a reference electrode; means for applying a potential to the electrode; and means for measuring current between the electrode and the counter electrode.
15. A method for monitoring an electrochemical parameter of an electrode according to Claims 1 to 9 in an electrochemical sensor according to Claim 14 comprising contacting the electrochemical cell with a solution, applying a variable potential to the electrode and measuring the current between the electrode and the counter electrode as a function of the potential, and extrapolating the electrochemical parameter from the relationship of the current to the potential.
16. A method for detecting a target analyte in a solution comprising monitoring an clcctrochemical paramctcr of an electrode according to the method of Claim 15 both in the presence and absence of the target analyte in the solution, and determining the presence of the target analyte through a difference in the electrochemical parameter.
17. A method according to Claim 15 or Claim 16, wherein the electrochemical parameter is the formal potential of the redox active group.
18. A method for producing an electrode for use in an electrochemical sensor for detecting a target analyte in a solution comprising chemisorption of a mixture of amphipathic molecules to a solid conducting substrate to create a self assembled monolayer on a surface of the solid conducting substrate, wherein the mixture of amphipathic molecules comprises a first amphipathic molecule which comprises a recognition element specific to the target analyte and a second amphipathic molecule which comprises a redox active group.
19. A method according to Claim 18, wherein the mixture of amphipathic molecules further comprises a third amphipathic molecule having a neutral terminal group.
20. A method according to Claim 18 or Claim 19, wherein the mixture of amphipathic molecules is in solution.
21. A method according to Claim 19 or Claim 20, wherein the first, second and third amphipathic molecules are n-alkanethiols.
22. A method according to Claims 19 to 21, wherein the concentration ratio of first amphipathic molecule: second amphipathic molecule: third amphipathic molecule is between 1:1:1 to 10:1:10.
23. An electrode for use in an electrochemical sensor for detecting a target analyte in a solution comprising a solid conducting substrate having thereon a self-assembled monolayer which comprises: a first amphipathic molecule which comprises a recognition element specific to the target analyte and which is accessible within the self-assembled monolayer to bind to the target analyte; and a second amphipathic molecule which comprises a redox-active group.