Nanozeolites and their Analytical Use as Chemosensors in Biorelevant Media

Abstract

The present invention relates to the use of monodisperse nanozeolites with a specific particle size distribution in analytical determination methods, methods for the qualitative and quantitative determination of one or more neutral, zwitterionic or positively charged bioanalytes in a sample, in particular in saline media, using such nanozeolites, and new chemosensors based on such nanozeolites with doping with functionalised dyes or indicators.

Claims

1-15. (canceled)

16. A method of determination of at least one of (i) neutral, (ii) zwitterionic and (iii) positively charged bioanalytes comprising (a) contacting at least one of (i) neutral, (ii) zwitterionic and (iii) positively charged bioanalytes with monodisperse nanozeolites having a particle size distribution in the range of 5 to 400 nm wherein the bioanalytes become chemically and/or sterically bound to the nanozeolites, to form a bioanalyte-nanozeolite complex, and (b) analyzing said bioanalyte-nanozeolite complex with UV-Vis or fluorescence spectroscopy

17. The method of claim 16, wherein the monodisperse nanozeolites have a particle size distribution in the range of 5 to 200 nm

18. The method of claim 16, wherein the monodisperse nanozeolites comprise Si and Al, and wherein the Si/Al ratio is within the range of 0.5 to 50.

19. The method of claim 17, wherein the monodisperse nanozeolites comprise Si and Al, and wherein the Si/Al ratio is within the range of 0.5 to 50.

20. The method of claim 16, wherein the monodisperse nanozeolites are comprised in an aqueous colloidal dispersion, or comprised in a layer of particles obtained by spray or aerosol printing methods from a colloidal dispersion in an aqueous medium onto a carrier.

21. The method of claim 17, wherein the monodisperse nanozeolites are comprised in an aqueous colloidal dispersion, or comprised in a layer of particles obtained by spray or aerosol printing methods from a colloidal dispersion in an aqueous medium onto a carrier.

22. The method of claim 16, wherein the bioanalytes to be determined are biogenic and bioactive molecules selected from the groups of hormones, lipids, metabolites, neurotransmitters and bioactive agents.

23. The method of claim 22, wherein the bioanalytes to be determined are selected from the group consisting of serotonin, dopamine, tryptamine, tyramine, epinephrine, norepinephrine, phenylephrine, octopamine, phenethylamine, histamine, nicotine, propanolol, L-DOPA, phenylalanine, tyrosine, histidine, tryptophan, TrpNH2, 5-HTP, tryptophan-glycine, indole, Indole-3-acetic acid, melatonin, adenosine, estradiol, propanil, catechol, paracetamol, acetylcholine, glycine, D-serine, aspartate, glutamate, GABA, cadaverine, ethanolamine and glucose.

24. The method of claim 16, for the determination of neutral, zwitterionic and/or positively charged bioanalytes in at least one selected from the group consisting of physiological media, endogenous fluids, secretions, PBS, urine, saliva, blood, sweat, semen, amniotic fluid, tear fluid or cerebrospinal fluid.

25. The method of claim 18, wherein (i) the bioanalytes to be determined are selected from the group of positively charged bioanalytes and the monodisperse nanozeolites have an Si/Al ratio of from 0.5 to 50, or (ii) the bioanalytes to be determined are selected from the group of neutral or zwitterionic bioanalytes and the monodisperse nanozeolites have an Si/Al ratio of from 10 to 20.

26. The method of claim 16, wherein the monodisperse nanozeolites are doped with at least one of functionalised dyes and indicator molecules.

27. The method of claim 17, wherein the monodisperse nanozeolites are doped with at least one of functionalised dyes and indicator molecules.

28. The method of claim 26, wherein the functionalised dyes or indicator molecules are sterically anchored in the cavities of the monodisperse nanozeolites.

29. The method of claim 16, wherein (a) the at least one of (i) neutral, (ii) zwitterionic and (iii) positively charged bioanalytes with monodisperse nanozeolites is comprised in an aqueous dispersion, (b) a layer of the dispersion is prepared by spraying or aerosol printing on a carrier; and wherein (c) bioanalytes in the dispersion are determined by UV-vis or fluorescence spectroscopy.

30. The method of claim 29, wherein (c) bioanalytes in the dispersion are determined by UV-vis spectroscopy.

31. The method of claim 30, wherein prior to step (a) the monodisperse nanozeolites are prepared by particulating a zeolite material to a monodisperse particle size distribution in the range of 5 to 400 nm by means of sonication with an acoustic intensity having an operating frequency ≥30 kHz and an energy density ≥300 W cm.sup.2.

32. The method of claim 30, wherein the monodisperse nanozeolites are prepared by sterile high-pressure filtration.

33. The method of claim 31, further comprising the step of doping the nanozeolites with one or more functionalised dyes or indicators, wherein the dye or indicator doping may occur before or after the high acoustic intensity sonication.

34. A monodisperse nanozeolite having a particle size distribution in the range of 5 to 400 nm doped with one or more functionalised dyes or indicators.

35. The monodisperse nanozeolite of claim 34, wherein the functionalised dye or indicator molecules are sterically anchored in the cavities of the monodisperse nanozeolites.

Description

DESCRIPTION OF THE FIGURES

[0083] FIG. 1 (a) Sedimentation process of a commercial zeolite (zeolite Y.sub.15-Si/Al ratio=15) by means of dye detection and liquid phase spectroscopy under determination of the intensity decrease of the dye in the course of time by sedimentation of the zeolite in which the dye molecules are embedded. [0084] (b) Enzyme kinetics using a sedimenting chemosensor: signal of the chemosensor without enzyme addition in the presence of a non-binding guest (upper curve), signal of the chemosensor with enzyme addition and thereby conversion of the non-binding guest to a binding guest (lower curve).

[0085] FIG. 2 (a) Comparison of the particle size distribution of a nanozeolite according to the invention with that of a commercially available zeolite (zeolite Y.sub.15) [0086] (b) Enzyme kinetics using a chemosensor according to the invention: signal of the chemosensor without enzyme in the presence of a non-binding guest (upper curve), signal of the chemosensor with enzyme and thereby conversion of the non-binding guest to a binding guest (lower curve).

[0087] FIG. 3 (a) Comparison of the particle size of a commercial zeolite L.sub.3.0 with the particle size of a nanozeolite produced from this commercial zeolite by sonication with high sound intensity (operating frequency ≥30 kHz, energy density ≥300 W cm.sup.2) and high pressure filtration. [0088] (b) Zoom-In

[0089] FIG. 4 Absorption spectra of chemosensors according to the invention, which show a signal change in the UV-Vis spectra when positively charged analytes (guests) are added. [0090] (a) analyte: serotonin [0091] (b) analyte: dopamine [0092] (c) Detection and differentiation of various dopamine-serotonin mixtures by UV-vis spectroscopy.

[0093] FIG. 5 Schematic representation of the cation exchange reaction that leads to the decomposition of conventional chemosensors in biological (relevant) media such as PBS or urine. The dye is forced out of the carrier material by the high salt concentration and the signal-generating binding pockets are no longer available for the detection of the analytes.

[0094] FIG. 6 Schematic representation of a chemosensor according to the invention based on a nanozeolite according to the invention doped with functional dyes, wherein the dye molecules are functionalised with functional groups, linked or polymerised with one another by bonding or polymerisation after loading of the zeolite and thus sterically or mechanically anchored in the zeolite cavities.

[0095] FIG. 7 Investigation of the influence of high salt concentrations in the test medium on the signal strength when using conventional chemosensors compared to the new chemosensors according to the invention. The signal increase after the addition of salt signals the escape of the dye molecules and thus the cancellation of the slight quenching by the zeolite cavities. The chemosensor is no longer functional (top). The signal decrease of the new carrier material after salt addition confirms the interaction of the derivatised dyes with the added ions. The binding pockets nevertheless remain (bottom).

[0096] FIG. 8 Detection of different analytes (guests) in physiological or biological media [0097] (a) Detection of serotonin in 1×PBS corresponding to a physiological salt concentration of sodium chloride of 137 mM. [0098] (b) discrimination of different analytes (guests) in human urine from volunteers

[0099] FIG. 9 Detection of different analytes (guests) in physiological or biological media [0100] (a) Detection of serotonin in artificial CSF [0101] (b) Recording the enzymatic conversion of non-binding L-tyrosine to binding tyramine using the new chemosensor according to the invention in artificial cerebrospinal fluid.

[0102] FIG. 10 (a) Detection of serotonin in blood serum (human serum, HS, diluted 1:2 with 50 mM HEPES). [0103] (b) Detection of serotonin in blood serum (human serum, HS, diluted 1:2 with 50 mM HEPES, top) or in the presence of the protein human albumin (human serum albumin, HSA, bottom).

[0104] FIG. 11 Schematic representation of the non-binding of neutral analytes (guests) to conventional zeolite-based chemosensors and the binding to the new chemosensors according to the invention with a balance between hydrophobicity and hydrophilicity.

[0105] FIG. 12 (a) Detection of the neutral analyte indole by means of a new chemosensor according to the invention with binding of the indole molecules in the binding pockets formed by the dye molecules with signal reduction due to interaction; [0106] (b) Detection of the neutral analyte indole by means of a new chemosensor according to the invention with binding of the indole molecules in 1×PBS, corresponding to a physiological salt concentration of sodium chloride of 137 mM. [0107] (c) Detection of the zwitterionic analyte tryptophan (top) and the neutral analyte indole (bottom) by means of the new chemosensor according to the invention with signal reduction through interaction with the dye. Plot related to a selected wavelength for the determination of binding affinity

[0108] FIG. 13 Detection of the neutral analyte indole using a new chemosensor according to the invention and UV-vis spectroscopy

[0109] The present invention is described in more detail by the following examples, but without being limited thereto.

EXAMPLES

Example 1—Sedimentation Studies on Commercially Available Zeolites

[0110] a) Dye Detection

[0111] In a commercially available zeolite (zeolite Y.sub.15-Si/Al ratio=15; particle size distribution 400-1700 nm according to DLS (description of the method under example 2), twofold positively charged, diazapyrene-based dye molecules were deposited in the cavities according to conventional procedures and the decrease in intensity of the dye due to sedimentation of the zeolite was detected by determining the dye intensity after 0 h, 5 h and 8 h using liquid phase spectroscopy.

[0112] The measurement was carried out under the following conditions: The loading of the zeolite materials used with dye was always chosen in a range of 0.23-2.3 wt % dye relative to the zeolite material. In the present example, the dye loading is 0.23 wt % relative to the zeolite material and the concentration of the chemosensor in the dispersion is 250 μg/ml. For preparation, zeolite and dye were mixed, centrifuged (8000 rpm, 5 min) and the material was washed three times with 10 mL MilliQ water. By measuring and dye concentration determination of the wash solutions and under known initial concentration, the dye loading could be accurately determined. Measurements were performed on a Jasco FP-8300 fluorescence spectrometer using a 450 W xenon lamp with a Platereader attachment. The storage of the dispersion-filled plates between the measurements was carried out under exclusion of light at room temperature. Excitation was at 371 nm, detection at 424 nm.

[0113] FIG. 1a clearly shows that the intensity of the dye decreases over time due to sedimentation of the zeolite in which the dye molecules are embedded.

[0114] b) Enzyme Kinetics

[0115] By adding an enzyme (tyrosine decarboxylase, TDC) to a dispersion of a commercial zeolite loaded with unbranched, positively charged dye molecules (as described in Example 1 a) and a solution of non-binding L-tyrosine, the formation of an analyte that quenches the sensor signal was stimulated. The signal intensity over time was measured.

[0116] The measurement was carried out under the following conditions: The chemosensor was prepared using the procedure described in 1 a (dye loading 2.3 wt %) and then diluted with 10 mM HEPES buffer (pH 6.2) to a chemosensor concentration of 550 μg/ml. The zeolite used was L.sub.3.0, and the dyes were the doubly positively charged diazapyrene-based molecules. In addition, an enzyme cofactor (pyridoxal-5-phosphate, PRP) and 500 μM L-tyrosine as a non-binding analyte were added to this dispersion. After incubation at 37° C. for 30 minutes, the dispersion was dispersed again and then measured on a Jasco FP-8300 in disposable cuvettes (top) or after the addition of the enzyme tyrosine decarboxylase (TDC, 33 μg) in disposable cuvettes (bottom). TDC catalyses the conversion of the non-binding L-tyrosine to the binding tyramine. Excitation took place at 300 nm, emission at 500 nm.

[0117] FIG. 1b shows that already over a period of 10 minutes there is a clear decrease in intensity due to sedimentation. Slower enzymatic reactions would therefore be masked by the baseline drift and thus not analysable. The signal curve with enzyme also shows undesired noise, which means that no Michaelis-Menten kinetics can be fitted. Furthermore, the measurement was limited to HEPES buffers as analysis medium, since the biologically relevant sodium phosphate buffers led to disintegration of the chemosensor (in this case conventional zeolite L.sub.3.0 without prior treatment by a rod sonicator/filtration).

Example 2—Particle Size Distribution and Sedimentation Studies on Zeolites According to the Invention

[0118] a) Particle Size Distribution

[0119] A comparison of the particle size distribution of a nanozeolite according to the invention (nanozeolite shown here with an Si/Al ratio of 1.76) with that of a commercially available zeolite (zeolite Y.sub.15; Si/Al=15) is shown in FIG. 2a.

[0120] The particle size distribution was determined using dynamic light scattering (DLS) on a Malvern ZetaSizer Nano ZS from Malvern Panalytics in acrylic disposable cuvettes in water. For this purpose, the dispersions were prepared analogously to the procedure described under 1 a.

[0121] b) Enzyme Kinetics

[0122] As in Example 1 b, by adding an enzyme (tyrosine decarboxylase, TDC) to a chemosensor according to the invention based on the nanozeolite according to the invention used in Example 3b, the formation of an analyte quenching the sensor signal was simulated and the intensity over time was measured by means of enzyme kinetics. Zeolite L.sub.3.0 was used as the carrier material, which was homogenised after dispersion by using a rod sonicator with very high acoustic intensity (operating frequency ≥30 kHz, energy density ≥300 W cm.sup.2) and subsequent high-pressure filtration. Dye loading was performed as described in Example 1, although this can be carried out either before or after homogenisation (in the example shown, this was done before homogenisation). Further conditions of the enzyme monitoring can be taken from example 1 b.

[0123] The comparison of FIG. 2b with FIG. 1b shows that the chemosensors according to the invention can detect kinetic curves with low noise and good fit.

Example 3—Preparation of a Nanozeolite According to the Invention and a Chemosensor According to the Invention

[0124] a) Preparation of a Nanozeolite According to the Invention by Sonication with High Acoustic Intensity

[0125] A nanozeolite according to the invention with a particle size distribution of 80-500 with agglomerated particles with sizes up to 6500 nm was prepared from a commercially available zeolite (zeolite L.sub.3.0) with a particle size distribution of 80-500 with agglomerated particles with sizes up to 6500 nm by the method according to the invention using sonication with high intensity (working frequency ≥30 kHz, energy density ≥300 W cm.sup.2) and subsequent sterile high pressure filtration.

[0126] The preparation was carried out under the following conditions: The commercial zeolite material was dispersed in water and measured using dynamic light scattering (DLS) on a Malvern ZetaSizer Nano ZS from Malvern Panalytics. Subsequently, the dispersion was sonicated for 15 min at high intensity using a rod sonicator and the remaining large particles were separated using high-pressure filtration. These process steps can also be applied after the introduction of the dye. For this purpose, the dispersions are prepared analogously to the procedure described under 1 a.

[0127] The comparison result is shown in FIGS. 3a and 3b.

[0128] b) Manufacture of a Chemosensor According to the Invention

[0129] The preparation of a chemosensor based on the nanozeolites of the invention with a doping of functionalised dyes or indicators is described below: Previously used dye molecules did not have any linking/polymerisation possibilities. As an example, 2,7-dimethyldiazapyrenium dibromide can be mentioned here. By adding linker molecules, as explained in more detail in Example 5 and FIG. 6, the possibility of linking within the cavities is given. However, the original zeolite loading is the same for all dye molecules: the dye is placed in aqueous solution at a known concentration, and the zeolite material used is dispersed in water. The dye loading was always chosen in a range of 0.23-2.3 wt % dye related to the zeolite material and accordingly dispersion and dye solution were mixed, centrifuged (8000 rpm, 5 min) and the material was washed three times with 10 mL MilliQ water and centrifuged again. By measuring the wash solutions and under known initial concentration, the dye loading could be accurately determined. The measurements were performed on a Jasco FP-8300 fluorescence spectrometer using a 450 W xenon lamp with a Platereader attachment. The resulting chemosensor material can be stored in solution or as a solid. In order to trigger polymerisation within the zeolite cavities, an external influence must be changed (at least to achieve wide branching). For example, polymerisation via disulphide bonding (see FIG. 6a) is triggered by the supply of oxygen, while the thiol-maleimide reaction, for example, is favoured by the supply of temperature.

Example 4—UV-Vis Determination of Serotonin and Dopamine with the New Chemosensors According to the Invention

[0130] Using a new chemosensor prepared according to Example 3 using the method of the invention, the positively charged bioanalytes serotonin and dopamine were determined by UV-Vis detection.

[0131] The measurement was carried out under the following conditions: The chemosensors were prepared according to the procedure described in 3b and 2000 μL of the prepared dispersions were measured in disposable cuvettes. Subsequently, 1 mM stock solutions of the analytes to be determined were titrated in 1-10 μL steps under stirring and the corresponding UV-Vis spectra were recorded. All experiments were carried out at 25° C.

[0132] FIGS. 4a, 4b and 4c show that the process according to the invention and the nanozeolites obtainable therefrom allow the new chemosensors according to the invention to be produced with a lower scattering effect, thus enabling the UV-Vis determination of bioanalytes.

Example 5—Influence of Salt Concentration on Zeolite-Based Chemosensors

[0133] To demonstrate the influence of high salt concentrations in the test medium on the signal strength, a conventional chemosensor based on zeolite L.sub.3.0 with a doubly positively charged, diazapyrene-based dye without linkages and a new chemosensor according to the invention based on zeolite L.sub.3.0 with a doubly positively charged, diazapyrene-based dye with linkages of the individual dye units by means of polymerisation according to example 3b were compared. The change in signal intensity due to the release of the incorporated dye after the addition of salt to the test medium was measured.

[0134] The measurement was carried out under the following conditions: In both cases, the chemosensors were prepared according to the steps mentioned in 3b. In both cases the dye loading of the zeolite was 2.3 wt %. FIG. 6 shows the synthesis pathway of one of the functionalised dyes—the polymerisation was carried out as described in example 3b. After equilibration, the stability of the fluorescence signal (excitation at 371 nm, emission at 455 nm) was detected by means of kinetics measurement and then a highly concentrated PBS solution was added so that the concentration of 1×PBS (137 mM NaCl) was reached in the cuvette.

[0135] FIGS. 5 and 6 schematically show the underlying principle of the cation exchange reaction in conventional chemosensors compared to the new chemosensors according to the invention.

[0136] FIG. 7 shows a clear increase in fluorescence intensity after the addition of salt to conventional chemosensors, which signals the escape of the dye molecules and thus the elimination of the slight quenching by the zeolite cavities. The chemosensor is thus no longer functional (FIG. 7, upper curve). The reduction of the signal intensity after the addition of salt when using a new chemosensor according to the invention with dye molecules mechanically anchored therein confirms the interaction of the derivatised dyes with the added ions. However, the chemosensor remains intact due to the mechanical anchoring and the dye molecules cannot be displaced.

Example 6—Determination of Positively Charged Analytes in Physiological Media with a New Chemosensor According to the Invention

[0137] The new chemosensor according to the invention in Example 3b was used for the determination of positively charged analytes in various physiological media with high salt concentration.

[0138] The measurements were carried out under the following conditions: The chemosensors used were prepared in water according to the steps described under 3b and then mixed with the medium in which the investigation was to take place. It is possible that the analyte to be examined is already present in the medium (this is the case, for example, with urine from volunteers) or that the analyte to be examined is first added (this is the case, for example, with artificial cerebrospinal fluid). If the analyte is already present in the medium, a stock solution of serotonin was further added until all dye molecules within the chemosensor were quenched (concentration of these is known) and the difference between the concentration of the dye molecules and the concentration of the added analyte then corresponds to the original serotonin concentration in the medium.

[0139] The measurement was carried out under the following conditions: FIG. 8

[0140] (a) The chemosensor based on zeolite L.sub.3.0 and with a dye loading/polymerisation as described in examples 3b and 5 was dispersed in water and then mixed with 1.5×PBS (final concentration 1×PBS). The addition of the non-binding or binding guest was carried out as explained in example 4. Detection was carried out in disposable cuvettes (PP) using a fluorescence spectrometer.

[0141] (b) The chemosensor based on zeolite L.sub.3.0 and with a dye loading/polymerisation as described in FIG. 6 was dispersed in 50 mM HEPES buffer (pH 6.2) and then mixed 4:1 with the urine of the volunteers and measured by Platereader assay. The addition of the non-binding or binding guest was performed as described in example 4.

[0142] FIG. 9

[0143] (a) The chemosensor based on zeolite L.sub.3.0 and with a dye loading/polymerisation as described in examples 3b and 5 was originally prepared in water as medium, dried and then dispersed in artificial CSF. The artificial CSF used does not contain detectable neurotransmitters. The addition of the non-binding or binding guest was carried out as explained in Example 4. Detection was carried out in disposable cuvettes (PP) using a fluorescence spectrometer.

[0144] (b) The chemosensor was prepared as described in example 9a and the enzyme reaction was carried out as explained in example 1b. Detection was performed in disposable cuvettes (PP) using a fluorescence spectrometer.

[0145] FIG. 10

[0146] (a) The chemosensor based on zeolite L.sub.3.0 was prepared with dye loading/polymerisation as described in examples 3b and 5 in 50 mM HEPES as medium. The human serum was diluted 1:2 with 50 mM HEPES (final concentration approx. 500 μM HS) and mixed with a chemosensor dispersion (final concentration 250 μg/ml). Subsequently, serotonin was titrated as explained in example 4. Excitation at 420 nm, detection at 522 nm. Detection was carried out in disposable cuvettes (PP) using a fluorescence spectrometer.

[0147] (b) The chemosensor based on zeolite L.sub.3.0 was prepared with dye loading/polymerisation as described in examples 3b and 5 in 50 mM HEPES as medium. Human serum albumin was filtered (22 mM syringe filter, PP) and mixed with chemosensor dispersion (final concentration 250 μg/ml). Serotonin was then titrated as explained in example 4. Detection was carried out in disposable cuvettes (PP) using a fluorescence spectrometer. Excitation at 420 nm, detection at 522 nm.

[0148] The result is shown in FIGS. 8, 9 and 10 for different media and different analytes. The new chemosensors according to the invention can thus be used in biological media such as PBS or artificial cerebrospinal fluid, but also directly in endogenous fluids with a high salt concentration, such as urine or blood serum.

Example 7—Determination of Neutral and Zwitterionic Analytes with a New Chemosensor According to the Invention

[0149] The new chemosensor according to the invention based on homogenised zeolite Y.sub.15 according to example 3b was used for the determination of neutral and zwitterionic analytes.

[0150] The measurements were carried out under the following conditions: The chemosensors were prepared based on the procedure described in 3b. Then chemosensor concentrations of 250 μg/ml with a relative dye loading of 2.3 wt % were adjusted in water and the neutral or zwitterionic analyte to be determined (1 mM stock solution in each case) was titrated. Analogously, the experiment could also be carried out in 1×PBS. Detection was carried out in disposable cuvettes (PP) using a fluorescence spectrometer.

[0151] FIG. 11 schematically shows the underlying principle of binding in chemosensors with different Si/Al ratios. The result of the determinations is shown in FIGS. 12a, 12b, 12c and 13.