IES20080900A2

IES20080900A2 - Chemical messenger sensor

Info

Publication number: IES20080900A2
Authority: IE
Inventors: John Colleran; Claire Harley; Bernadette Alcock; Carmel Breslin
Original assignee: Nat Univ Ireland Maynooth
Priority date: 2008-11-10
Filing date: 2008-11-10
Publication date: 2010-05-12
Also published as: WO2010052342A2; WO2010052342A3

Abstract

A sensor for the detection of chemical messengers is described herein. In particular, a sensor for the detection of serotonin is reported. Serotonin plays a pivotal role as a neurotransmitter in the modulation of a myriad of physiological responses including anger, aggression, mood, sleep, sexuality, and appetite. An electrode for detecting serotonin comprising a conducting or semiconducting substrate, and a polymer material on said substrate is disclosed. Said polymet conprises a conducting polymer doped with a cyclodextrin macrocycle. Suitable cyclodextrin macrocycles include anionic cyclodextrin macrocycles, for example sulfonated B-cyclodextrins (CDs). Also disclosed is a sensor capable of selectively detecting serotonin in the presence of ascorbic acid, epinephrine, norepinephrine and dopamine.

Description

Title Chemical Messenger Sensor Field of the Invention [0001] A sensor for the detection of chemical messengers is described herein In particular, a sensor for the detection of serotonin is reported. Methods of constructing sensors according to the present invention are also described, Suitable materials for the construction of such sensors are disclosed with a view to developing a sensor capable of real-time in-vivo serotonin monitoring.

Background to the Invention [0002] Within the central nervous system a number of chemical messengers serve to maintain and regulate healthy brain function. Amongst these, serotonin (1) plays a pivotal role as a neurotransmitter in the modulation of a myriad of physiological responses including anger, aggression, mood, sleep, sexuality, and appetite. ·* ·*?·..> ·.'- :.'.*«.-rs.»·····

[0003] Implication of serotonin in the regulation of such a wide variety of physiological response has led to its association in a corresponding high number of neurological diseases In particular, imbalances in levels of serotonin in the brain are believed responsible for several psychiatric disorders, including depression. Thus, the ability to reliably monitor serotonin concentrations in-vivo, and on a real time scale would provide further valuable insight into the role of serotonin in the pathophysiology of the aforementioned disorders.

[0004] However, reliable in-vivo monitoring of serotonin is particularly challenging because it co-exists with a number of interfering species capable of oxidising at similar potentials, lnterferant induced false positives ultimately lead to unreliable sensor readings In particular, ascorbic acid (4) represents one of the key target interferants. Other common interferants such as uric acid (5), DOPAC (2) and homovanillic acid (3) are also problematic. The exclusion of these last two species is particularly advantageous as these are metabolites of dopamine, which are known to poison other biosensors reducing their sensitivity As both dopamine and serotonin v neurotransmitters are active in the brain, there is a need to exclude cross metabolite contamination.

[0005] The English language abstract of Japanese Patent Publication number 9127056 describes a sensor for selectively detecting serotonin in the presence of NO and NO2. The sensor is comprised of three carbon fibre electrodes in a single assembly wherein an operational electrode is moved up and down upon a central axis. The English language abstract of Japanese Patent Publication number 3068858 discloses a simple arrangement forthe electrochemical detection of serotonin. This publication is silent to the detection of serotonin the presence of interferants such as ascorbic acid.

[0006] Notwithstanding the foregoing, it would still be desirable to provide a biosensor capable of reliable real-time in-situ, in-vivo measurement of serotonin in the presence of interfering molecules such as ascorbic acid. Further still, such a biosensor must be able to function in the presence of metabolites without suffering the aforementioned problems associated with metabolite poisoning.

Summary of the Invention [0007] In one aspect, the present invention provides for an electrode for detecting serotonin comprising: (i) a conducting or semi-conducting substrate; and (ii) a polymer material on said substrate, wherein said polymer comprises a conducting polymer doped with a cyclodextrin macrocycle.

[0008] Desirably, the electrode of the present invention comprises a conducting substrate As will be appreciated by a person skilled in the art the conducting substrate may comprise a metal selected from the group consisting of Pt, Ag, Au, Ru, Rh, Pd, Re, Os, Ir, Ti, Indium tin oxide (ITO) coated glass and combinations thereof. Further still, the conducting substrate may comprise a non-metallic conductor such as carbon fibres, graphite, glassy carbon, diamond, carbon paste and pyrolithic carbon electrodes, or boron doped diamond. Desirably, the conducting substrate comprises Au [0009) Preferably, the conducting polymer is a biocompatible conducting polymer. As used herein the term biocompatible is a reference to materials that are non-toxic to biological tissues. [0010) The conducting polymer of the electrode of the present invention may be selected from the group consisting of polythiophenes, polypyrroles and combinations thereof Desirably, the conducting polymer comprises a polythiophene. Suitably, the conducting polymer comprises PEDOT [polyethylenedioxythiophene] (6) wherein n > 1 . [0011) Desirably, the cyclodextrin macrocycle comprises an anionic cyclodextrin macrocycle. Further desirably, the anionic cyclodextrin macrocycle comprises an anionic a-cyclodextrin, β-cyclodextrin, y-cyclodextrin and combinations thereof. In a preferred embodiment the anionic cyclodextrin macrocycle comprises an anionic βcyclodextrin.

[0012] It is advantageous that the cyclodextrin macrocycle of the electrode of the present invention is anionic (negatively charged). The negative charge associated with the macrocyclic cage promotes complexation of cationic (positively charged) species within the cage structure. At physiological pH metabolites known to poison prior art electrodes (for example DOPAC and homovanillic acid) are anionic species. As such the electrode construction of the present invenlion should not be affected by these metabolites to the same extent, as the anionic cyclodextrin should repel these anionic metabolites The size of the cavity of the macrocyclic cage, i.e whether α,β, or y, may also function as a size exclusion barrier or the like, allowing complexation of molecules below a certain molecular weight only.

[0013] The electrode works by forming an inclusion complex between the macrocyclic cage immobilised in the polymer and serotonin. The oxidative response of the film to ΙΕ ο 8 Ο Ϋ Ο ί) serotonin is catalytic. That is, it results in the oxidation of serotonin occurring at a different potential than at the bare electrode (bare = gold, platinum, glassy carbon, etc.) [0014] In a preferred embodiment the anionic cyclodextrin macrocycle comprises a sulfonated cyclodextrin macrocycle. Desirably, the sulfonated cyclodextrin macrocycle may comprise a sulfonated α-cyclodextrin, a sulfonated β-cyclodextrin, a sulfonated ycyclodextrin and combinations thereof. Further preferably, the anionic cyclodextrin macrocycle comprises a sulfonated β-cyclodextrin.

[0015] As used herein the term “sulfonated cyclodextrin macrocycle” refers to any cyclodextrin wherein one or more of the hydroxy groups of the glucopyranoside rings are sulfonated Within the art the term is sometimes used interchangeably with sulfated cyclodextrin. Within this specification the terms sulfated cyclodextrin and sulfonated cyclodextrin are to be interpreted as one in the same provided the definition above is satisfied, i.e. having one or more of the hydroxy groups of the glucopyranoside rings sulfonated. For example, sulfonated β-cyclodextrin (7) is commercially available from Sigma-Alrich® as sulfated β-cyclodextrin.

O S-ONa or H II o

[0016] In a further aspect, the invention extends to a method of preparing an electrode for detecting serotonin comprising: (i) providing a conducting or semi-conducting substrate; (ii) providing an aqueous solution of monomeric precursor to a conducting polymer and an anionic cyclodextrin; (iii) contacting said substrate and said aqueous solution; and (iv) applying an electrical potential to provide an anionic cyclodextrin doped conducting polymer film on said substrate. ΙΕ ο 8 ο s ο ο [0017] Desirably, the monomeric precursor to a conducting polymer comprises a monomeric precursor to a polythiophene, a monomeric precursor to a polypyrrole and combinations thereof. Further desirably, the monomeric precursor to a conducting polymer comprises a monomeric precursor to a polythiophene. Preferably, the monomeric precursor to a conducting polymer comprises ethylenedioxythiophene (EDOT), a precursor to polyethylenedioxythiophene (PEDOT). (0018] The anionic cyclodextrin is incorporated into the PEDOT during polymerisation as a counter ion in order to neutralise the positive charge formed on the PEDOT chain during the oxidation of the monomer, with one cyclodextrin incorporated for every four EDOT units.

[0019] Desirably, in the method of the present invention the substrate comprises a conducting substrate. As will be appreciated by a person skilled in the art the conducting substrate may comprise a metal selected from the group consisting of Pt, Ag, Au, Ru, Rh, Pd, Re, Os, Ir, Ti, Indium tin oxide (ITO) coated glass and combinations thereof. Further still, the conducting substrate may comprise a nonmetallic conductor such as carbon fibres, graphite, glassy carbon, diamond, carbon paste and pyrolithic carbon electrodes, or boron doped diamond. Desirably, the conducting substrate comprises Au.

[0020] Desirably, the cyclodextrin macrocycle comprises an anionic cyclodextrin macrocycle. Further desirably, the anionic cyclodextrin macrocycle comprises an anionic a-cyclodextrin, β-cyclodextrin, γ-cyclodextrin and combinations thereof. In one embodiment the anionic cyclodextrin macrocycle comprises an anionic β-cyclodextrin [0021] In a preferred embodiment of the method of the present invention the anionic cyclodextrin macrocycle comprises a sulfonated cyclodextrin macrocycle. Desirably, the sulfonated cyclodextrin macrocycle may comprise a sulfonated α-cyclodextrin, a sulfonated β-cyclodextrin, a sulfonated γ-cyclodextrin and combinations thereof Further preferably, the anionic cyclodextrin macrocycle comprises a sulfonated βcyclodextrin.

[0022] The present invention further provides for a sensor for selective detection of serotonin in the presence of dopamine, epinephrine, norepinephrine, ascorbic acid and combinations thereof comprising; an electrode comprising: (i) a conducting or semi-conducting substrate; and (ii) a polymer material on said substrate, wherein said polymer comprises a conducting polymer doped with a sulfonated β-cyclodextrin macrocycle. ΙΕ Ο Β ο Γ ο ο [0023] As used herein ascorbic acid comprises neutral ascorbic acid and the anionic derivative ascorbate.

[0024] Desirably, the sensor of the present invention comprises a conducting substrate. As will be appreciated by a person skilled in the art the conducting substrate may comprise a metal selected from the group consisting of Pt, Ag, Au, Ru, Rh, Pd, Re, Os, Ir, Ti, Indium tin oxide (ITO) coated glass and combinations thereof. Further still, the conducting substrate may comprise a non-metallic conductor such as carbon fibres, graphite, glassy carbon, diamond, carbon paste and pyrolithic carbon electrodes, or boron doped diamond. Desirably, the conducting substrate comprises Au.

[0025] Preferably, the conducting polymer is a biocompatible conducting polymer. As used herein the term biocompatible is a reference to materials that are non-toxic to biological tissues.

[0026] The conducting polymer of the sensor of the present invention may be selected from the group consisting of polythiophene materials, polypyrrole materials and combinations thereof. Desirably, the conducting polymer comprises a polythiophene material. Suitably, the conducting polymer comprises PEDOT [polyethylenedioxythiophene].

[0027] The electrode and sensor of the present invention provide for detecting serotonin in solution. As used herein the term solution comprises bodily fluids such as plasma, blood, extra-cellular fluid, etc. having serotonin dissolved therein.

[0028] Advantageously, the electrodes utilised in detecting serotonin levels have the potential to be miniaturised and conveniently placed in the living organism to give invivo data at the sub-second timescale.

[0029] Advantageously, the electrode and sensor of the present invention provide for real-time measurement of serotonin, both in-vivo and in-vitro. Further still, the electrode and sensor of the present invention for detecting serotonin has the potential for in-situ monitoring.

[0030] Potential applications of the electrode and sensor of the present invention include the evaluation of test compounds on serotonin concentrations in the brain, and the resulting neurological response.

[0031] The relative simplicity with which these materials can be prepared, coupled with the excellent selectivity, high biocompatibility and ease of preparation shows that these novel materials have real potential in the sensing of Serotonin and are a significant improvement on the existing technologies. The materials utilised in the electrode are ΙΕ Ο 8 Ο ί, Ο Ο highly biocompatible (PEDOT is used in tissue engineering applications and cyclodextrins are used In drug delivery) and easy to prepare (10-min preparation time) Brief Description of the Drawings [0032] Additional features and advantages of the present invention are described in, and will be apparent from, the detailed description of the invention and from the drawings in which:

[0033] Figure 1 illustrates electropolymerisation of PEDOT/cydodextrin film at a Gold electrode according to the present invention. Potential cycled from -0 5 to +1 06 V vs SCE [0034] Figure 2 illustrates the cyclic voltammetric response of the PEDOT/sulfonated β-CD film on a gold electrode according to the present invention to 5x10'5 Μ 5-HT and a mixture of 5x1 O'5 M 5-HT & 5x104 Μ AA.

[0035] Figure 3 depicts the cyclic voltammetric response of the PEDOT/sulfonated βCD film on a gold electrode according to the present invention to 5x105 M 5-HT & DA, and a mixture of 5x10‘6 M 5-HT, DA & AA.

[0036] Figure 4 depicts the cyclic voltammetric response of the PEDOT/sulfonated βCD film on a gold electrode according to the present invention to a mixture of 5-HT, AA, DA, EP and norEP Detailed Description of the Invention Preparation of the Electrode [0037] The poor solubility of the 3,4-ethylene dioxythiophene (EDOT) monomer in aqueous solution, has led to the electropolymerisation of this monomer being predominantly performed in organic media.1,2 Surfactants, such as sodium dodecyl sulphate (SDS), have been reported to improve the solubility of EDOT in aqueous and organic media? In general, it has been communicated that a critical micellar concentration (cmc) of surfactant is required in solution if polymerisation of EDOT is to occur Cyclodextrins have been used in place of surfactants?·5 owing to the ability of cyclodextrins to form a host guest interaction with EDOT, thus increasing the solubility of the EDOT monomer in water In the example disclosed herein, sulfonated βcyclodextrin (β-CD) was utilised as the dopant anion necessary for film formation to occur.

[0038] The films were electropolymerised onto gold electrodes from an aqueous solution of 0.1 M ethylenedioxythiophene and 0.01 M sulfonated β-cyclodextrin, sodium IE 0 8 0 9 0 0 salt Polymerisation was carried out by cycling the potential between -0.5 and 1.06 V/SCE at a scan rate of 50 mV s'1 for a total of three cycles.

[0039] The PEDOT/sulfonated β-cyclodextrin film properties vary depending on: a) the EDOT;sulfonated β-CD solution concentrations (and ratio); and b) the polymerisation technique utilised - when cyclic voltammetry is utilised the following parameters can be modified to vary the PEDOT/sulfonated βcyclodextrin film properties; i) the upper (anodic) potential of the voltammetric sweep used when fabricating the polymer film. This upper potential is important for system optimisation, and ii) the sweep rate.

[0040] The conditions resulting in optimal serotonin sensing comprise: • a polymerisation solution of 0.1M EDOT:0.01M sulfonated β-CD; this 10:1 ratio is important; • cyclic voltammetry (CV) is important to ensure that a homogeneous thin film is formed. Films grown using this technique exhibit enhanced serotonin signals when compared to potentiostatic growth films; • sweeping from -0.5 to +1.06 V vs SCE at a scan rate of 50 mV s’; and • three electropolymerisation cycles to form a thin but a homogeneous film.

[0041] All scans were completed on a Solartron 1285 potentiostat. The data provided herein and in the figures were obtained using cyclic voltammetry.

[0042] For example, in Figure 1 we see a cyclic voltammagram of three electropolymerisation cycles of a solution of 0.1M EDOT:0.01M sulfonated β-CD, Irreversible oxidation, i.e. electropolymerisation of the monomer occurs at approximately 0.8V and leads to film formation (101). No peak in the reverse sweep direction indicates that this is essentially an irreversible process.

Serotonin (5-HT) Detection Detection at the PEDOT/CD film:

[0043] The cyclic voltammetric response of the PEDOT/sulfonated β-CD film on a gold electrode to 5x1 O'5 M 5-HT (trace 201) and a mixture of 5x10'5 M 5-HT & 5x10 Μ AA (trace 202) is shown in shown in Figure 2. The main 5-HT oxidation peak is observed at -0 48V in the 5-HT only trace 201. In trace 202 the 5-HT peak is measured in the presence of AA. In the presence of AA the 5-HT peak is shifted slightly to *0.5V.

Thus, the electrode array of the present invention comprising the PEDOT/sulfonated βCD film can selectively detect 5-HT in the presence of AA, Interference of Dopamine (DA) with the Serotonin (5-HT) oxidation signal:

[0044] As dopaminergic and serotoninergic neurons are active within close proximity in the animal/human brain desirably the electrode array of the present invention should be capable of detecting 5-HT in the presence of DA. Figure 3 shows the cyclic voltammetric response of the PEDOT/sulfonated β-CD film on a gold electrode according to the present invention to 5x105 M 5-HT & DA (lower trace 301), and a mixture of 5x1O'5 M 5-HT. DA & AA (upper trace 302). In 301, notwithstanding an overlap between the two peaks, we observe separate signals for DA 303 and 5-HT 304 The oxidation peaks are at -0.41V and -0 48V respectively. Trace 302 in the presence of AA, DA and 5-HT illustrates separate discernable peaks for AA 305, DA 306 and 5-HT 307.

[0045] Gratifyingly, the elecfrode comprising the PEDOT/sulfonated β-CD film according to the present invention was capable of selectively detecting all three species as independent peaks.

Selective Detection of Serotonin in the presence of Ascorbic Acid (AA), Dopamine (DA), Epinephrine (EP) and Norepinephrine (norEP):

[0046] Similarly, selective detection of 5-HT in the presence of AA, DA, EP and norEP was evaluated. Figure 4 shows the cyclic vottammetric response of the PEDOT/sulfonated β-CD film on a gold electrode according to the present invention to a mixture of 1.67x10’5 M 5-HT, 2x10’4 AA, 2x10’5 DA, 1x10 s EP and 6.67x10 6 norEP. The signals labelled 401 at -0.035V, -0.15V & -0.5V correspond to 5-HT. Signal 402 at -0.2V is from ascorbic acid, and signal 403 at -0.42V is a mixture of DA, EP and norEP.

[0047] Figure 4 clearly illustrates that the gold electrode comprising the PEDOT/sulfonated β-CD film according to the present invention is capable of selectively detecting 5-HT in the presence of AA, DA, EP and norEP. [004&] The words “comprises/comprising and the words “having/including when used herein with reference to the present invention are used to specify the presence of stated features, integers, steps or components but do not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. ΙΕ Ο 8 o S G ο [0049] It is appreciated that certain features ofthe invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination References 1. V. Noel, H. Randriamahazaka, C, Chevrot, J. Electroana!. Chem., 542 (2003) 33. 2. J Bobacka, A. Lewenstam, A. Ivaska, J. Eiectroanal. Chem. 489 (2000) 17. 3. N. Sakmeche, J. J. Aaron, M. Fall, S Aeiyach. M Jouini, J. C. Lacroix, P. C Lacaze, Chem. Commun., (1996), 2723. 4. C. Lagrost, J. C. Lacroix, S. Aeiyach, M. Jouini, K. I. Chane-Ching, P C Lacaze, Chem. Commun, (1998), 489.

V. S. Vasantha, K. L. N. Phani, J. Eiectroanal. Chem., 520 (2002), 79. ΙΕ ο 8 Ο 3 ο ο

Claims

1. An electrode for detecting serotonin comprising: (i) a conducting or semi-conducting substrate; and (ii) a polymer material on said substrate, wherein said polymer comprises a conducting polymer doped with a cyclodextrin macrocycle.

2. An electrode according to Claim 1 wherein the substrate comprises a conducting substrate.

3. An electrode according to Claim 1 or 2 wherein the conducting substrate comprises Au.

4. An electrode according to any preceding Claim wherein the conducting polymer is selected from the group consisting of polythiophenes, polypyrroles and combinations thereof.

5. An electrode according to Claim 4 wherein the conducting polymer comprises a polythiophene.

6. An electrode according to Claim 5, wherein the conducting polymer comprises polyethylenedioxythiophene.

7. An electrode according to any preceding Claim wherein the cyclodextrin comprises an anionic cyclodextrin.

8. An electrode according to Claim 7, wherein the anionic cyclodextrin comprises an amonic a-cyclodextrin, β-cyclodextrin, γ-cyclodextrin and combinations thereof

9. An electrode according to Claim 7 or 8, wherein the anionic cyclodextrin comprises an anionic β-cyclodextrin.

10. An electrode according to Claim 9 wherein the anionic β-cyclodextrin comprises a sulfonated^-cyclodextrin. IE Ο 8 Ο 9 Ο Ο

11. A sensor for selective detection of serotonin in the presence of dopamine, epinephrine, norepinephrine, ascorbic acid and combinations thereof comprising: an electrode comprising: (i) a conducting or semi-conducting substrate; and (ii) a polymer material on said substrate, wherein said polymer comprises a conducting polymer doped with a sulfonated β-cyclodextrin macrocycle

12. A sensor according to Claim 11 comprising a conducting substrate

13. A sensor according to Claim 12 wherein the conducting substrate comprises Au.

14. A sensor according to any preceding Claim wherein the conducting polymer is selected from the group consisting of polythiophene materials, polypyrrole materials and combinations thereof.

15. A sensor according to Claim 14 wherein the conducting polymer comprises a polythiophene material. 15 A sensor according to Claim 15, wherein the conducting polymer comprises polyethy lened ioxythiophene.

16. 17. A method of preparing an electrode for detecting serotonin comprising: (i) providing a conducting or semi-conducting substrate; (ii) providing an aqueous solution of a monomeric precursor to a conducting polymer and an anionic cyclodextrin; (iii) contacting said substrate and said aqueous solution; and (iv) applying an electrical potential to provide an anionic cyclodextrin doped conducting polymer film on said substrate.

17. 18 A method according to Claim 17 wherein the monomeric precursor to a conducting polymer comprises a monomeric precursor to a polythiophene, a monomeric precursor to a poly pyrrole and combinations thereof

18. 19. A method according to Claim 18 wherein the monomeric precursor to a conducting polymer comprises a monomeric precursor to a polythiophene. IE

19. 20. A method according to Claims 17 Io 19 wherein the monomeric precursor to a conducting polymer comprises ethylenedioxythiophene.

20. 21. A method according to any preceding Claim comprising providing a conducting substrate

21. 22. A method according to Claim 21 wherein the conducting substrate comprises Au.

22. 23 A method according to any preceding Claim wherein the anionic cyclodextrin comprises an anionic a-cyciodextrin, β-cyclodextrin, γ-cyclodextrin and combinations thereof.

23. 24 A method according to Claim 23 wherein the anionic cyclodextrin comprises an anionic β-cyclodextrin.

24.

25. A method according to Claim 24 wherein the anionic β-cyclodextrin comprises a sulfonated^-cyclodextrin.