HIGHLY SELECTIVE AND ULTRASENSITIVE METAL ION SENSOR

Abstract

The present application is directed to a sensor for detection and/or concentration determination of metal ions in a fluid comprising a complexing agent suitable for binding the metal ions to be detected, detection means and a linker moiety, wherein the detection means comprises a polymer membrane with nanopores. The sensor according to the present application can be used for fast, highly selective and ultrasensitive detection of metal ions in a fluid, in particular of Cu.sup.2+ ions. With such a sensor a qualitative and/or quantitative detection of metal ions can be achieved, which can be useful in the diagnosis and/or monitoring of diseases linked to abnormal metal ion concentrations such as for example Alzheimer's disease.

Claims

1. A sensor for detection and/or concentration determination of metal ions in a fluid comprising a complexing agent suitable for binding the metal ions to be detected, detection means and a linker moiety, wherein the detection means comprises a polymer membrane with nanopores.

2. The sensor according to claim 1, wherein the polymer membrane with nanopores is an ion-track etched polymer membrane.

3-15. (canceled)

16. The sensor according to claim 2, wherein the ion-track etched polymer membrane is an ion-track etched polyethylene terephthalate (PET) membrane.

17. The sensor according to claim 1, wherein the linker moiety comprises polyethylene glycol.

18. The sensor according to claim 17, wherein the linker moiety has the formula R.sub.1—[CH.sub.2CH.sub.2O].sub.a—R.sub.2, wherein a is from 2 to 10, R.sub.1 is —NH and R.sub.2 is CH.sub.2CH.sub.2COO—.

19. The sensor according to claim 1, wherein the linker moiety is bound to the polymer membrane with nanopores and to the complexing agent suitable for binding the metal ions to be detected.

20. The sensor according to claim 1, wherein the detection means further comprises a fluorophore.

21. The sensor according to claim 20, wherein the fluorophore is 5(6)-carboxyfluorescein.

22. The sensor according to claim 20, wherein the fluorophore is attached to the complexing agent suitable for binding the metal ions to be detected.

23. The sensor according to claim 1, wherein the complexing agent suitable for binding the metal ions to be detected is a peptide comprising 2 to 6 amino acids, modified amino acids, and/or amino acid mimics.

24. The sensor according to claim 1, wherein the sensor selectively detects Cu.sup.2+ ions in a fluid and/or determines a concentration of Cu.sup.2+ ions in a fluid.

25. The sensor according to claim 24, wherein the complexing agent suitable for binding Cu.sup.2+ ions comprises a copper-binding motif.

26. The sensor according to claim 25, wherein the copper-binding motif comprises 2,3-diaminopropionic acid(DAP)-βAla-X, wherein X is an amino acid or a modified amino acid.

27. The sensor according to claim 26, wherein X is histidine (His), alanine (Ala) or aspartic acid (Asp).

28. The sensor according to claim 1, wherein the sensor is used in diagnosing and/or monitoring diseases or for use in detecting metal ions and/or determining metal ion concentration in food products and in environmental water samples.

29. The sensor according to claim 28, wherein the sensor is used in monitoring diseases linked to abnormal Cu.sup.2+ ion concentrations and/or dysregulated Cu.sup.2+ ion metabolism.

30. The sensor according to claim 28, wherein the sensor is used in monitoring Alzheimer's disease, Wilson's disease, or Menke's disease.

31. A method for detecting metal ions and/or determining metal ion concentration in a fluid sample comprising the steps of bringing the sensor according to claim 1 into contact with the fluid sample, and determining a presence and/or concentration of metal ions in the fluid sample by measuring a change in current-voltage characteristics of the sensor and/or by measuring a change in fluorescence intensity of the sensor, and optionally further comprising a step of regenerating the sensor by subjecting the sensor to a further complexing agent.

32. The method according to claim 31, wherein the method is used for detection of Cu.sup.2+ ions and/or for determining Cu.sup.2+ ion concentration in a fluid sample, wherein the method further comprises the step of adjusting a pH of a fluid sample to be analyzed to 5.0 to 8.5 prior bringing the sensor into contact with the fluid sample.

33. The method according to claim 31, wherein the change in the current-voltage characteristics is measured, and/or wherein the metal ion concentration in the fluid sample is in a range of 1 fM to 60 μM.

34. The method according to claim 31, wherein the change in fluorescence intensity is measured, and/or wherein the metal ion concentration in the fluid sample is in a range of 1 nM to 60 μM.

35. A test strip for detecting metal ions and/or determining a concentration of metal ions in a fluid sample comprising the sensor according to claim 1.

Description

DESCRIPTION OF FIGURES

[0044] FIG. 1 shows the “on-off” characteristics of a detection method using the sensor of the present application comprising 5(6)-carboxyfluorescein as further detection means.

[0045] FIG. 2(a) shows UV-vis spectra of the Cu.sup.2+ complex at pH 8 (black line) and Ni.sup.2+ complex at pH 10.5 (grey line) with the peptide (cf. also FIG. 2e) attached to a linker moiety (PEG.sub.4), the insert showing a magnification at 350 to 650 nm. FIGS. 2(b-d) are UV-vis spectra showing the pH dependent formation of the Cu.sup.2+ complex (black square) in relation to the formation of the corresponding Ni.sup.2+ complex (grey dots) with the peptides b) Ac-DAP-βAla-His (Ac-ATCUN) (1 mM, water), c) Ac-DAP-βAla-Asp (Ac-ATCUN H3D) (1 mM, water) and d) Ac-DAP-βAla-Ala (Ac-ATCUN H3A) (1 mM, water), attached to a linker moiety (PEG.sub.4). Boltzman fit (black and grey lines) show the sigmoidal trend of the complex formation. FIG. 2(e) is a scheme of the complex between the copper-binding motifs DAP-βAla-His, DAP-βAla-Asp, DAP-βAla-Ala and the metal M.sup.2+ (Cu.sup.2+), wherein R is either 5(6)-carboxyfluorescein (FAM) or acetyl (Ac).

[0046] FIG. 3(a) is a fluorescence spectrum showing the decrease of fluorescence intensity of 5(6)-carboxyfluorescein-DAP-βAla-His-linkerPEG.sub.4 (1 μM, 2-(N-morpholino)ethanesulfonic acid (MES) buffer, pH 6.50) upon addition of CuSO.sub.4 from 0 to 25 μM in water. FIG. 3(b) shows the change in fluorescence intensity as a function of the molar ratio of Cu.sup.2+ to 5(6)-carboxyfluorescein-DAP-βAla-His-linkerPEG.sub.4 (termed “peptide” in the description of the x-axis). FIG. 3(c) is a fluorescence spectrum showing the decrease of fluorescence intensity of 5(6)-carboxyfluorescein-DAP-βAla-His-linkerPEG.sub.4 (1 μM, MES buffer, pH 6.50) upon addition of NiSO.sub.4 from 0 to 3 mM in water. FIG. 3(d) shows the change in fluorescence intensity as a function of the molar ratio of Ni.sup.2+ to 5(6)-carboxyfluorescein-DAP-βAla-His-linkerPEG.sub.4 (termed “peptide” in the description of the x-axis).

[0047] FIG. 4(a) is a fluorescence spectrum showing the decrease of fluorescence intensity of 5(6)-carboxyfluorescein-DAP-βAla-His-linkerPEG.sub.4 (0.1 μM, phosphate buffer, pH 8.0) upon addition of CuCl.sub.2 from 0 to 0.15 μM in water. FIG. 4(b) shows the change in fluorescence intensity as a function of the molar ratio of Cu.sup.2+ to 5(6)-carboxyfluorescein-DAP-βAla-His-linkerPEG.sub.4 (termed “peptide” in the description of the x-axis).

[0048] FIG. 5(a) is a fluorescence spectrum showing the decrease of fluorescence intensity of 5(6)-carboxyfluorescein-DAP-8Ala-His-linkerPEG.sub.4 (0.025 μM, CAPS buffer, pH 10.55) upon addition of Ni.sup.2+ from 0 to 0.038 μM in water. FIG. 5(b) shows the change in fluorescence intensity as a function of the molar ratio of Ni.sup.2+ to 5(6)-carboxyfluorescein-DAP-βAla-His-linkerPEG.sub.4 (termed “peptide” in the description of the x-axis).

[0049] FIG. 6(a) is an UV-Vis titration spectrum of Ac-DAP-βAla-His-linkerPEG.sub.4 (0.68 mM, MES buffer pH 6.50) upon addition of Cu.sup.2+ (0 to 0.7 mM) in water. FIG. 6(b) shows the absorption coefficient ε of the Cu.sup.2+ complex as a function of the molar ratio of Cu.sup.2+ to Ac-DAP-βAla-His-linkerPEG.sub.4 (termed “peptide” in the description of the x-axis).

[0050] FIG. 7(a) is an UV-Vis titration spectrum of Ac-DAP-βAla-His-linkerPEG.sub.4 (0.68 mM, MES buffer pH 6.50) upon addition of NiSO.sub.4 (0 to 16 mM) in water. FIG. 7(b) shows the change in absorption as a function of the molar ratio of Ni.sup.2+ to Ac-DAP-βAla-His-linkerPEG.sub.4 (termed “peptide” in the description of the x-axis).

[0051] FIG. 8(a) shows fluorescence emission images obtained with confocal laser scanning microscopy (CLSM) after addition of different amounts of NiSO.sub.4 (top). It also shows the median of fluorescence intensity in relation to different amounts of NiSO.sub.4 (bottom). FIG. 8(b) shows a fluorescence image obtained with CLSM after addition of 100 mM ZnSO.sub.4 (top). It also shows the median of fluorescence intensity after addition of 100 mM ZnSO.sub.4 in relation to reference “washed out” nanopores fluorescence intensities with 100 mM EDTA (bottom).

[0052] FIG. 9(a) is a fluorescence spectrum showing fluorescence titration results of 5(6)-carboxyfluorescein-DAP-βAla-His-linkerPEG.sub.4 (1 μM, 100 mM MES buffer, pH 6.50) upon addition of ZnSO.sub.4 from 0 to 3 mM in water. FIG. 9(b) shows an UV-vis titration spectrum of Ac-DAP-βAla-His-linkerPEG.sub.4 (1 mM, 100 mM MES buffer pH 6.50) upon addition of ZnSO.sub.4 (0 to 2 mM) in water.

[0053] FIG. 10(a) shows the surface of an ion-track etched PET-membrane with nanopores upon which 5(6)-carboxyfluorescein-DAP-βAla-His-linkerPEG.sub.4 was immobilized (via linkerPEG.sub.4 as the linker moiety) (reflected light). FIG. 10(b) shows confocal laser scanning microscopy (CLSM) images of a sensor comprising 5(6)-carboxyfluorescein-DAP-βAla-His-linkerPEG.sub.4 linked (via linkerPEG.sub.4) to an ion-track etched PET-membrane with nanopores. The area on the right hand side represents nanopores which have not been etched and thus no 5(6)-carboxyfluorescein-DAP-βAla-His-linkerPEG.sub.4 was immobilized thereon. FIG. 10(c) shows the linear dependency of decrease in median fluorescence intensity with increase in Cu.sup.2+ ion concentration. FIG. 10(d) shows CLSM fluorescence emission images of the “on-off” characteristics the sensor, in particular the re-usability of the sensor after regeneration with 1 mM EDTA and washing with water and MES buffer, pH 6.50.

[0054] FIG. 11 shows a plot of F.sub.0−F in relation to Cu.sup.2+ ion concentration for determining the limit of detection (LOD) of the sensor comprising 5(6)-carboxyfluorescein-DAP-βAla-His-linkerPEG.sub.4 (1 μM, 100 mM MES buffer pH 6.50; addition of CuSO.sub.4 (0 to 60 nM) in water), wherein F.sub.0 is the fluorescence intensity of the solution without analyte, i.e. without Cu.sup.2+ ions and F is the fluorescence intensity of the solution with Cu.sup.2+ ions.

[0055] FIG. 12 shows the current-voltage (I-V) characteristics of the ion-track etched PET-membrane with nanopores without any sensor immobilized thereon (as-prepared), the ion-track etched PET-membrane with nanopores modified with EDA (EDA modified) and the ion-track etched PET-membrane with nanopores to which 5(6)-carboxyfluorescein-DAP-βAla-His-linkerPEG.sub.4 was immobilized (peptide modified).

[0056] FIG. 13(a) shows the current-voltage (I-V) characteristics of the sensor comprising the ion-track etched PET-membrane with nanopores to which 5(6)-carboxyfluorescein-DAP-βAla-His-linkerPEG.sub.4 was immobilized upon application of different concentrations of Cu.sup.2+ ions. FIG. 13(b) shows the conductance depending on the Cu.sup.2+ ion concentration. FIG. 13(c) shows the reversible complexation/decomplexation behavior of Cu.sup.2+ ions when using EDTA as the further complexing agent.

[0057] FIG. 14 shows the current-voltage (I-V) characteristics of the sensor comprising the ion-track etched PET-membrane with nanopores to which 5(6)-carboxyfluorescein-DAP-βAla-His-linkerPEG.sub.4 was immobilized upon application of 1 nM concentration of Cu.sup.2+, Ni.sup.2+ and Zn.sup.2+ ions, in buffer (MES/KCl 100 mM, pH 6.5).

EXAMPLES

Solid Phase Peptide Synthesis (SPPS) and Characterization of Copper-Binding Motifs used as Complexing Agents

[0058] 5(6)-carboxyfluorescein-DAP-βAla-His-linkerPEG.sub.4 (1), Acetyl(Ac)-DAP-βAla-His-linkerPEG.sub.4 (2), Acetyl(Ac)-DAP-6Ala-Asp-linkerPEG.sub.4 (3) and Acetyl(Ac)-DAP-βAla-Ala-linkerPEG.sub.4 (4) were synthesized following the standard Fmoc-SPPS using chlorotrityl chloride resin (0.966 mmol/g). After linkerPEG.sub.4 coupling the resin was capped with 8.5:1:0.5 dichloromethane (DCM)/methanol/N-ethyl-N-(propan-2-yl)propan-2-amine (DIEA) followed by coupling of Fmoc-His(Trt)-OH, Fmoc-Asp(Trt)-OH or Fmoc-Ala(Trt)-OH, Fmoc-βAla-OH, Fmoc-Dap(Boc) and 5(6)-carboxyfluorescein (fluorophore only for (1)). correspondingly. Deprotection was obtained by 20% piperidine in N,N-dimethylformamide (DMF) and the coupling efficiency was monitored by UV-Vis-spectrometry at 301 nm. Copper-binding motifs (2, 3 and 4) were acetylated at the N-terminus with acetic anhydride. The final cleavage of copper-binding motifs (1) to (4) from the resin was carried out in 2/2/48/48 triisopropylsilane (TIPS)/water/trifluoroacetic acid (TFA)/DCM and agitated for 1 h at room temperature.

[0059] RP-HPLC purification was performed on a C18 column (MultoKrom 100-5. 250×20 mm, 100 Å pore diameter, 5.0 μm particle size) using a linear gradient of 5 to 40% of eluent B (eluent A: water (0.1% TFA) and eluent. B: acetonitrile (0.1% TFA)) in 60 min. Copper-binding motifs (2-4) were purified following an isocratic method of 5% eluent B for 12 minutes followed by a linear gradient to 30% eluent B within a total of 60 minutes. The molecular mass was confirmed by ESI-MS ((1): m/z: [M+H].sup.+ calculated for C44H51N7O15 918.92, found 918.35; (2): m/z: [M+H].sup.+calculated for C25H43N7O10 602.31, found 602.31), (3): m/z: [M+H].sup.+ calculated for C23H41N5O15 580.28, found 580.28: (4): m/z: [M+H].sup.+ calculated for C22H41N5O10 536.26, found 536.29).

[0060] Collected fractions were combined, freeze-dried and stored at −28° C. Purity of the collected fractions was confirmed by analytical RP-HPLC on a Waters XC e2695 system (Waters, Milford, Mass., USA) employing a Waters PDA 2998 diode array detector equipped with ISAspher 100-3 C18 (C18, 3.0 μm particle size, 100 Å pore size, 50×4.6 mm, Isera GmbH, Duren, Germany). The copper-binding motif (1) was eluted with a gradient of 0%-30% eluent B in 10 min at a flow rate of 2 mL/min. (2), (3) and (4) were eluted using a isocratic method of 0% eluent B for 4 minutes followed by a linear gradient to 30% eluent B in 20 minutes total run time. Chromatograms were extracted at 214 nm. The molecular weight of the purified copper-binding motifs as well as complexation of (1) to (4) with Cu(II), Ni(II) was confirmed by ESI mass spectrometry on a TOF-Q impact II spectrometer (Bruker Daltonik GmbH, Bremen, Germany) and calibrated using Bruker's ESI-Tune-Mix.

Preparation of a Sensor Comprising a Copper-Binding Motif, a Fluorophore, a Linker Moiety and an Ion-Track Etched PET-Membrane with Nanopores

[0061] Ion-track etched PET-membranes with conical nanopores were fabricated through asymmetric chemical etching of latent ion tracks as has been described in Nucl Instrum Methods Phys Res, Sect B 2001, 184, 337-346. PET foils or membranes were first irradiated with single swift heavy ions (Au) of kinetic energy 11.4 MeV/nucleon at the linear accelerator UNILAC (GSI Helmholtz Centre for Heavy Ion Research, Darmstadt, Germany). Then, the latent ion tracks in the polymer foils or membranes were sensitized with soft UV light. The chemical track-etching process was performed in a conductivity cell. The ion tracked foils or membranes were fixed in between two chambers of the cell. An etching solution (9 M NaOH) was filled in one chamber and a stopping solution (1 M KCl+1 M HCOOH) was filled in the other chamber. The etching process was carried out at room temperature. The etching process was monitored by applying a potential of −1 V across the foil or membrane. The etching process was stopped when the current reached a certain defined value after the breakthrough point. Then, the etched foil or membrane was washed with stopping solution and dipped in deionized water overnight to remove residual salts.

[0062] The carboxylic acid groups on the polymer foil or membrane surface, in particular on the surface of the nanopores originated from the chemical etching. These groups were first activated through standard carbodiimide coupling chemistry. The track-etched foil or membrane was exposed to an ethanol solution containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (100 mM) and pentafluorophenyl (PFP) (200 mM) at room temperature for 1 h. After washing with ethanol several times, the activated polymer foil or membrane was treated with ethylenediamine (EDA, 50 mM) solution overnight. During this reaction period, amine-reactive PFP-esters were covalently coupled with amine group of the EDA. Subsequently, the modified polymer foil or membrane was washed thoroughly with ethanol followed by careful rinsing with deionized water.

[0063] Then, the EDA-modified foil was used for the immobilization of the sensor having carboxylic acid groups at the terminus of the linker moiety (FIG. 2(c)). Carboxylic acid groups of the linker were activated with 1-[bis(dimethylamino)methylene]-1H-1-1,2,3-triazolo[4,5]pyridinium-3-oxide hexa-fluorophosphate (HATU)/DIEA and reacted with (1) to (4) (0.5 mM) in DMF, and left to react overnight at room temperature.

Characterization of the Cu.SUP.2+ and Ni.SUP.2+ Complex with the Copper-Binding Motif as Complexing Agent of the Sensor and Selectivity of the Sensor of the Present Application for Cu.SUP.2+ Ions over Ni.SUP.2+ and Zn.SUP.2+ Ions

[0064] Fluorescence studies performed with (1) at pH 8.0 at which pH optimal formation of the Cu-complex was observed, show that when adding one equivalent of Cu.sup.2+ ions, fluorescence intensity is quenched to 89% (11% remained intensity) by formation of a non-fluorescent complex between the Cu.sup.2+ ions and the copper binding motif (FIGS. 4(a),(b)). For Ni.sup.2+ ions pH 10.55 was evaluated as the lowest pH at which complete formation of the Ni-complex was achieved, and upon addition of 1 equivalent Ni.sup.2+ ions a remaining fluorescence of 29% is observed (FIGS. 5(a),(b)). To obtain a distinguished selectivity of the sensor towards Cu.sup.2+ ions, a pH of 6.50 was determined for copper-binding motif DAP-βAla-His. This can be seen in the pH titration study shown in FIG. 2(b). A similar selectivity towards Cu.sup.2+ ions at pH 6.5 can be achieved with DAP-βAla-Asp (Ac-ATCUN H3D) (FIG. 2(c)) and with DAP-βAla-Ala (Ac-ATCUN H3A) (FIG. 2(d)). Moreover, Ac-ATCUN H3D has an even broader pH range (approx. pH 5.5 to 7.2) in which it is highly selective for Cu.sup.2+ (FIG. 2(c)). pH Titration studies were performed using a 1 mM sensor solution in deionized water. One equivalent of the according metal ion, Cu.sup.2+ or Ni.sup.2+, was added and the pH lowered to pH <3 using 0.1 mM HCl. Adding small aliquots of 0.1 mM NaOH the pH was increased and with each step a spectrum recorded.

[0065] At pH of 6.50 nickel ion complexation is insignificant whereas for copper this pH represents the pH.50. UV-Vis titration using different Cu.sup.2+ ion concentrations shows the anticipated behavior at pH.sub.50 illustrating a saturation after addition of around 0.5 equivalents Cu.sup.2+ ions (FIGS. 6(a),(b)).

[0066] The fluorescence spectrum in FIG. 3(a) shows the decrease of fluorescence intensity of 5(6)-carboxyfluorescein-DAP-βAla-His-linkerPEG.sub.4 (1) (1 μM, MES buffer, pH 6.50) upon addition of CuSO.sub.4 from 0 to 25 μM. Emission (A) was determined to be 518 nm. Fluorescence titration upon addition of 20 equivalents of Cu.sup.2+ at pH 6.50 showed quenching of the fluorescence intensity to 88% (12% remaining fluorescence intensity) (FIG. 3(b)). In comparison thereto, fluorescence titration upon addition of nickel at pH 6.50 showed quenching of the fluorescence intensity after addition of 1364 equivalents of nickel to only 43% (57% remaining fluorescence intensity) (FIGS. 3(c),(d)) which can be explained by the insufficient formation of the nickel complex at pH 6.50.

[0067] Further, an experiment with Ni.sup.2+ and Ac-DAP-βAla-His-linkerPEG.sub.4 (2) at a pH of 6.50 did not show the required maximum absorption at 438 nm which indicates the formation of the planar coordination of Ni.sup.2+ to the complexing agent, i.e. the copper-binding motif, but rather showed a maximum absorption at 394 nm indicating that the complex between the copper-binding motif and the Ni.sup.2+ ions is not assembled but rather a non-specific octahedral binding is achieved (FIGS. 7(a),(b)). Thus, it was confirmed that when adjusting the pH to the claimed range, a selectivity of the sensor according to the present application is guaranteed for Cu.sup.2+ over Ni.sup.2+ and the almost exclusive formation of the copper-binding complex is confirmed. This also applies to (3) and (4).

[0068] In FIG. 8(a) it is demonstrated that there is no decrease in fluorescence intensity visible for Ni.sup.2+ ions in a range of 0 to 200 μM in MES buffer, pH 6.50. Only upon addition of 1 mM (1000 μM) NiSO.sub.4 in MES buffer a reduction in fluorescence intensity is observed. With regard to Zn (FIG. 8(b)) it can be seen that even addition of 100 mM ZnSO.sub.4 in MES buffer, pH 6.50 does not affect the fluorescence intensity. The titration experiments were performed at pH 6.50 to demonstrate selectivity towards Cu.sup.2+ ions.

[0069] The binding constants for and Ni.sup.2+ to the complexing agent, i.e. the copper-binding motif according to the present application were determined by UV-Vis spectroscopy at pH 6.50 using the Benesi-Hildebrand method. The obtained binding constants were similar to the binding constants determined with fluorescence spectroscopy using the Stern-Volmer plot at pH 6.50. Binding constants are as follows logK.sub.Cu=6.8 for 5(6)-carboxyfluorescein-DAP-βAla-His-linkerPEG.sub.4 (1) with K.sub.b=6.27×10.sup.6 M.sup.−1 and logK.sub.Cu=6.56 for Ac-DAP-βAla-His-linkerPEG.sub.4 (2) with K.sub.b=4.00×10.sup.6 M.sup.−1 showing a slightly better binding to the moiety comprising the fluorophore. At pH 6.50 the binding constants towards nickel were determined to be logK.sub.Ni=3.26 with K.sub.b=1.80×10.sup.3 M.sup.−1 for (1) and logK.sub.Ni=2.76 with with K.sub.b=5.80×10.sup.2 M.sup.−1 for (2) demonstrating again the outstanding selectivity towards copper. By optimizing the pH value a superior copper binding of the copper-binding motif comprised by the sensor of the present application was achieved, leading to a selective formation of the Cu-complex at the respective pH value.

[0070] No binding of Zn.sup.2+ ions was detected for 5(6)-carboxyfluorescein-DAP-βAla-His-linkerPEG.sub.4 (1) or Ac-DAP-βAla-His-linkerPEG.sub.4 (2) (FIGS. 9(a),(b)).

Measurement of Fluorescence Intensity of the Sensor Comprising a Copper-Binding Motif, 5(6)-Carboxyfluorescein as Detection Means, a Linker Moiety and an Ion-Track Etched PET-Membrane with Nanopores

[0071] When using fluorescence intensity as parameter for determining the presence and/or concentration of Cu.sup.2+ ions, in particular when using 5(6)-carboxyfluorescein as fluorophore a decrease in fluorescence intensity indicates the presence of Cu.sup.2+ ions. The higher the Cu.sup.2° concentration the lower the fluorescence intensity. This has been shown in FIGS. 10(a)-(d). FIGS. 10(a)-(d) show 5(6)-carboxyfluorescein-DAP-βAla-His-linkerPEG.sub.4 (1) attached to an ion-track etched PET-membrane with nanopores (via the linker moiety linkerPEG.sub.4) which was subjected to confocal laser scanning microscopy (CLSM) measurements.

[0072] 5(6)-carboxyfluorescein-DAP-βAla-His-linkerPEG.sub.4 (1) is almost exclusively present on the surface of the nanopores of the ion-track etched PET-membrane or foil as can be seen in FIG. 10(a) and displays a green color when excited by the laser at 488 nm in the CLSM image (FIG. 10(b)). FIG. 10(d) shows a titration experiment showing the dependency of decrease in fluorescence with the increase in Cu.sup.2+ ion concentration as well as the re-usability of the sensor of the present application. CuSO.sub.4 was added in concentrations from 0 to 100 μM in MES buffer solution, pH 6.50. In particular, the “on-off” characteristics of the sensor of the present application showing re-usability after regeneration with 1 mM EDTA and washing with water and MES buffer, pH 6.50 can be seen in said figure. FIG. 10(c) shows clearly the linear dependency of decrease in fluorescence with the increase in Cu.sup.2+ ion concentration. This experiment was performed by measuring the decrease in median fluorescence intensity after addition of CuSO.sub.4 from 0 to 60 μM in MES buffer, pH 6.5.

[0073] As can be seen in FIGS. 10(c) and (d) fluorescence intensity decreases with addition of Cu.sup.2+ with a linear dependency. Furthermore, when EDTA was used as complexing agent it was possible to regenerate the Cu.sup.2+ sensitivity of the sensor. As can be seen in FIG. 10(d) the sensing towards Cu.sup.2+ could be revived for at least seven times. Further, a shelf life of the sensor of at least six months was confirmed.

[0074] Also the limit of detection (LOD) for Cu.sup.2+ ions using the fluorescence signal was determined. In order to determine the sensitivity of the sensor according to the present application the limit of detection of the sensor comprising 5(6)-carboxyfluorescein-DAP-βAla-His-linkerPEG.sub.4 (1) towards Cu.sup.2+ ions in solution from fluorescence intensity titration studies is defined to be 12 nM. This value is extremely important for application of the sensor according to the present invention for detection of trace amounts of copper ions in food/water control or detection of copper ions for the early diagnosis of Alzheimer's disease in urine.

[0075] The LOD was calculated using the following equation: LOD=(3×σ) slope, wherein σ=standard deviation of blank solution. 1 μL of a blank solution (100 mM MES buffer pH 6.50) was pipetted to a 1 μM peptide solution in 100 mM MES buffer and the difference between fluorescence intensity F.sub.0−F relating to the starting intensity was determined (FIG. 11). From a six fold titration study the standard deviation of the change in fluorescence intensity with the addition of blank solution is determined and used as the a value (1.28) (Eur J Biochem 2002, 269, 1323-1331)

Measurement of the Current-Voltage (I-V) Characteristics of the Sensor Comprising a Copper-Binding Motif, a Fluorophore, a Linker Moiety and an Ion-Track Etched PET-Membrane with Nanopores as Detection Means

[0076] In the following the measurement of the current-voltage (I-V) characteristics of the sensor according to the present application is outlined. The sensor comprises a copper-binding motif, a fluorophore and a linker moiety, i.e. 5(6)-carboxyfluorescein-DAP-βAla-His-linkerPEG.sub.4 whereby the copper-binding motif with the fluorophore is attached to the ion-track etched PET-membrane with nanopores by means of the linker. The PET-membrane with nanopores behaves as an ohmic resistor before Cu.sup.2+ complexation meaning that the net surface charge on the nanopore surface is zero and no flux from the narrow cone opening to the wide opening takes place. The ion-track etched PET-membrane with conical nanopores having carboxyl groups on the surface with no immobilized copper-binding motif (as-prepared) shows a flux from the narrow cone opening to the wide opening because of the negative carboxyl groups (FIG. 12). After modification with ethylene diamine (EDA) the surface charge switches from negative to positive resulting in an anion selectivity and the concomitant inversion of the rectification behavior of the nanopore (EDA modified). Immobilization of the copper-binding motif with the fluorophore via the linker to the surface of the nanopores of the ion-track etched PET-membrane results in change of the nanopore transport behavior from a rectifying to a non-rectifying due to the loss of nanopore surface charges. The PET-membrane with the immobilized copper-binding motif behaves as an ohmic resistor, i.e. the net surface charge on the nanopore walls is zero.

[0077] Upon complexation of Cu.sup.2+ by the copper-binding motif, a positive charge is generated on the nanopore surface, which concomitantly changes the ion transport behavior of the nanopore. The nanopore becomes ion selective as evidenced from the successive increase in positive current by increasing the Cu.sup.2+ concentration ranging from fM to nM concentrations as shown in FIGS. 13(a) and (b). Thus, the presence of the generated Cu complex on the surface of the nanopores switches the nanopore transport behavior from a non-conducting “off” state to a conducting “on” state. As can be further seen in FIGS. 13(a) and (b) the sensor according to the present application is able to recognize Cu.sup.2+ ions even at concentrations as low as 1 fM and this recognition process can be transduced in an electronic signal originating from the transport behavior of the nanopore. From the data given in FIG. 13 it can be seen that the sensor exhibits a remarkable interaction ability towards Cu.sup.2+ due to the particular copper-binding motif used in the sensor according to the present application. Further, the ion selectivity of the sensor (PET immobilized FAM-ATCUN-PEG.sub.4, cf. FIG. 2e) for different metal ions such as Cu, Ni and Zn at pH 6.5 is shown in FIG. 14 clearly demonstrating a strong Cu selectivity.

[0078] The reproducibility of the conductance measurements over at least six cycles is shown in FIG. 13(c).

[0079] The present application further elate to the following items.

[0080] Item 1. A sensor for detection and/or concentration determination of Cu.sup.2+ ions in a fluid according to the invention, wherein the complexing agent is the copper-binding motif 2,3-diaminopropionic acid(DAP)-βAla-X, wherein X is an amino acid or a modified amino acid, in particular wherein X is histidine (His), alanine (Ala) or aspartic acid (Asp).

[0081] Item 2. The sensor according to item 1 comprising the copper-binding motif 2,3-diaminopropionic acid(DAP)-βAla-His, DAP-βAla-Asp, or DAP-βAla-Ala, wherein DAP is preferably L-DAP.

[0082] Item 3. The sensor according to items 1 and 2, wherein the detection means further comprises a fluorophore, in particular 5(6)-carboxyfluorescein or 6-carboxyfluorescein.

[0083] Item 4. The sensor according to item 3, wherein the fluorophore is attached to the N-terminus of the DAP.

[0084] Item 5. The sensor according to items 1 to 4 wherein the linker moiety is attached to the C-terminus of the X.

[0085] Item 6. The sensor according to any one of items 1 to 5, wherein the ion-track etched PET membrane with nanopores is a membrane with functional groups such as carboxylate and/or amino groups on the surface of the membrane and on the surface of the nanopores.

[0086] Item 7. The sensor according to any one of items 1 to 6, wherein the copper-binding motif to which a fluorophore is attached, is bound via the linker moiety to the surface of the ion-track etched polymer membrane and to the surface of the nanopores thereof.

[0087] Item 8. The sensor according to any one of items 1 to 7, wherein the linker moiety comprises polyethylene glycol, in particular wherein the linker moiety has the formula R.sub.1—[CH.sub.2CH.sub.2O].sub.a—R.sub.2, wherein a is preferably 4, R.sub.1 is preferably —NH and/or R.sub.2 is preferably COO(H)—, wherein a linker moiety having the structure —HN—[CH.sub.2CH.sub.2O].sub.4—CH.sub.2CH.sub.2COOH (linkerPEG.sub.4) or —HN—[CH.sub.2CH.sub.2O].sub.4—CH.sub.2CH.sub.2COO.sup.− is most preferred.

[0088] Item 9. The sensor according to any one of items 1 to 8 for selectively detecting Cu.sup.2+ ions in a fluid in the presence of Ni.sup.2+ ions and/or Zn.sup.2+ ions.

[0089] Item 10. The sensor according to any one of items 1 to 9 for use in the diagnosis and/or monitoring of diseases, preferably of diseases linked to abnormal Cu.sup.2+ ion concentrations and/or dysregulated Cu.sup.2+ ion metabolism, in particular Alzheimer's disease, Wilson's disease and Menke's disease.

[0090] Item 11. The sensor according to any one of items 1 to 9 for use in determining Cu.sup.2+ ion concentration ex vivo in the urine of individuals to diagnose and/or monitor diseases linked to abnormal Cu.sup.2+ ion concentrations and/or dysregulated Cu.sup.2+ ion metabolism, in particular Alzheimer's disease, Wilson's disease and Menke's disease.

[0091] Item 12. The sensor according to any one of items 10 or 11 for use, wherein an abnormal Cu.sup.2+ ion concentration relates to a Cu.sup.2+ ion concentration in a body fluid such as urine and/or serum which is higher or lower than in a healthy individual.

[0092] Item 13. The sensor according to any one of items 1 to 9 for use in detecting Cu.sup.2+ ions and/or determining Cu.sup.2+ ion concentration in food products or fluids, in particular in environmental water samples.

[0093] Item 14. A method for detection of Cu.sup.2+ ions and/or for determining Cu.sup.2+ ion concentration in a fluid sample comprising the steps of [0094] adjusting the pH of a fluid sample to be analyzed to 5.0 to 8.5, in particular to a pH of 6.3 to 7.0. [0095] contacting the sensor according to any one of items 1-9 with the fluid sample, and [0096] determining the presence and/or concentration of Cu.sup.2+ ions in the fluid sample by measuring a change in the parameter of the detection means, and [0097] optionally further comprising a step of regenerating the sensor by subjecting the sensor to a further complexing agent.

[0098] Item 15. The method according to item 14, wherein the further complexing agent comprises EDTA.

[0099] Item 16. The method according to item 14, wherein the change in fluorescence intensity is measured, and/or wherein the Cu.sup.2+ ion concentration in the fluid sample is in a range of 1 nM to 60 μM, in particular in a range of 1 nM to 200 nM.

[0100] Item 17. The method according to item 14, wherein the change in the current-voltage characteristics is measured, and/or wherein the Cu.sup.2+ ion concentration in the fluid sample is in a range of 1 fM to 60 μM, in particular in a range of 1 fM to 200 nM.

[0101] Item 18. Test strip for the detection of Cu.sup.2+ ions in a fluid sample comprising the sensor according to any one of items 1 to 9.

[0102] Item 19. A method for manufacturing the sensor according to items 1 to 9 comprising the steps of [0103] synthesizing a compound including the complexing agent, the linker moiety and a fluorophore by solid phase peptide synthesis, [0104] providing a polymer membrane, foil or sheet, and [0105] coupling the compound via the linker moiety to the surface of the polymer membrane.