WO2010007451A1

WO2010007451A1 - Nanosensor for determining the concentrations of physiologically active inorganic ions on subcellular level

Info

Publication number: WO2010007451A1
Application number: PCT/HU2008/000109
Authority: WO
Inventors: László HÉJA; Julianna Kardos; Gyula Tolnai; Livia NAGYNÉ NASZÁLYI; Zsuzsanna Riedl; Roberta PALKÓ; Júlia BENÉNÉ VISY; Llona Fitos; Miklós PALKOVITS; Árpád DOBOLYI
Original assignee: Magyar Tudományos Akadémia Kémiai Kutatóközpont
Priority date: 2008-07-14
Filing date: 2008-09-29
Publication date: 2010-01-21
Also published as: HUP0800428A2

Abstract

The invention relates to a sensor of formula (I) for determining the concentrations of physiologically active inorganic ions on subcellular level wherein (H) is a silicon dioxide, noble metal or carbon carrier particle with an average diameter of 10-200 nm or a dendrimer with an average diameter of 10-200 nm optionally comprising dye molecules which are selectively sensitive to a physiologically active inorganic ion, A is a secondary antibody, X is the residue of a dye molecule selectively sensitive to a physiologically active inorganic ion, Y is the residue of a fluorescent marker molecule, mis 1-3, n is at least 10, but if (H) is a dendrimer, n may also be 0-9, p is zero or a positive number not exceeding n/10, L₁ is a linker of the structure -F₁-B-F₂-, L₂ is a linker of the structure -F₁B'-F₂, and L₃ is a linker of the structure -F'₁-B'-F'₂-- The invention also relates to methods of preparing and use of the sensor.

Description

NANOSENSOR FOR DETERMINING THE CONCENTRATIONS OF PHYSIOLOGICALLY ACTIVE INORGANIC IONS ON SUBCELLULAR LEVEL

The invention relates to sensors applicable to determine the concentrations of physiologically active inorganic ions on subcellular level. The invention also relates to methods for preparing these sensors and to the use of the sensors for the above purpose.

The functioning of all living organisms is based on the separation of the intracellular region from its environment and on a precise regulation of the intra- and extracellular concentrations of compounds which participate in cell functions. Physiologically active inorganic ions (further on: physiological ions) play a key role in the regulation of cell functions. Na⁺ K⁺, Ca²⁺, H⁺, Cl^" and HCO₃ ^" ions regulate or initiate several processes which are vital for the appropriate functioning of cells. Na⁺ ions participate in both central and peripheral signal transduction processes, in mediating neuronal functions, in controlling cellular energy maintenance and in the formation of pain sensation, furthermore they have a central role in regulating heart function and blood pressure. K⁺ ions, which are present in higher concentrations in the intracellular space, exert their regulating functions mostly in parallel with those of Na⁺ ions which predominate in the extracellular region. Cl^" ions have a central role in the inhibitory neural processes and, together with HCO₃ ^" ions, exert important functions in regulating cell volume. Ca²⁺ ions play a central role, among others, in the functioning of smooth muscles and in the regulation of cellular signalling pathways and myocardial processes. Changes in H⁺ ion concentrations (i.e. in pH) influence among others neural conduction processes and Ca²⁺ homeostasis, and participate in the proper functioning of bone cells.

These processes proceed at well defined regions of the intracellular space or of the cell surface which are spatially well separated from one another, thus the knowledge of the local ion concentrations on subcellular level provides important pieces of information on the status of the processes regulated by them. These data are of great diagnostic value. Just to mention as an example, numerous diseases, such as Alzheimer disease, sclerosis multiplex and ischaemia may be traced back to disturbances in Ca²⁺ homeostasis. Fluorescence measurements utilizing ion selective organic dyes have become increasingly widespread in determining physiological ion concentrations in biological systems [Meier et al: J. Neurosci. Methods 155, 251-259 (2006); Chatton et al: Proc. Natl. Acad. Sci. USA 100, 12456-12461 (2003); Lo et al: Biophys. J. 90, 357-365 (2006)]. These measurements are performed by adding an organic dye to the biological system which selectively binds the ion in question, and measuring the intensity of fluorescent light emitted by the dye molecules upon excitation. Upon binding the ions, the intensity of the light emitted by the dye molecule changes proportionally to the ion concentration, which enables the concentration to be calculated. Recently numerous ion selective dyes are commercially available [Minta et al: J. Biol. Chem. 264, 19449-19457 (1989); Baron et al: J, Am. Soc. Nephrol. ^6, 3490-3497 (2005); Fiacco et al: Neuron. 54, 611-626 (2007); Heimlich et al: J. Biol. Chem. 281, 2232-2241 (2006)], which enable the determination of physiological ions within wide concentration ranges.

In such measurements the ion selective dye, depending on its chemical and physical properties, enters either the extracellular or the intracellular space and distributes there almost evenly. Consequently, with ion selective dyes only the average ion concentration can be determined in the space concerned, and no information can be obtained on the ion concentration present at the region of the cell surface or of the intracellular space where the biological process actually takes place. Although these average values do have scientific and diagnostic value, much valuable pieces of information would be obtainable in the knowledge of the ion concentration prevailing at the actual place of the biological process.

In principle, dyes could be targeted with antibodies, because, depending on the dimension ratios, 3-6 dye molecules can be coupled to an antibody without affecting its targeting properties. This method has been successfully applied in targeting various dyes of high quantum yield which emit a very intense fluorescent light upon unit exciting energy, and in targeting quantum dots which produce even more intense signals [Kim et al: Anal. Chim. Acta 163-170 (2006); Kaul et al: Biochem. Cell. Biol. 133-140 (2007); Horwath et al: Proc. Natl. Acad. Sci. USA 7583-7588 (2005)]. Fluorescent markers targeted by this method are, however, insensitive to the changes which occur in their environment (among others, to the concentration of physiological ions and to its variation); their signal intensity depends solely on the intensity of the exciting light. Thus this method is well applicable to label specific cell parts e.g. for anatomic purposes, but it does not give information on the functioning of the labelled parts. This method is inapplicable for ion selective dyes, which are typical representatives of molecules with low quantum yield producing no detectable signal in a concentration which can be targeted with antibodies (3-6 molecules for one antibody).

Further difficulties arise from the fact that the fluorescent marker should always be bound to an antibody capable to attach to the cell part concerned, which means that no universally applicable reagent can be produced which could be rendered suitable for the examination of any desired cell part by simple subsequent treatment steps.

Our aim was to provide a sensor which enables the subcellular targeting of ion selective dyes in amounts which produce a detectable signal, and, upon a simple subsequent treatment, can be directed to any desired target. These aims can be fully met with a sensor wherein the molecules of the ion selective dye are bound to a carrier and/or incorporated into a carrier, and at least one secondary antibody and optionally one or more fluorescent marker(s) is(are) bound to the sensor.

Based on the above, the invention relates to a sensor for determining the concentrations of physiologically active inorganic ions on subcellular level. The sensor according to the invention corresponds to formula (I)

wherein

( H J is a silicon dioxide, noble metal or carbon carrier particle with an average diameter of 10-200 nm or a dendrimer with an average diameter of 10-200 nm optionally comprising dye molecules which are selectively sensitive to a physiologically active inorganic ion,

A is a secondary antibody,

X is the residue of a dye molecule selectively sensitive to a physiologically active inorganic ion,

Y is the residue of a fluorescent marker molecule, m is 1-3, n is at least 10, but if (R) is a dendrimer, n may also be 0-9, p is zero or a positive number not exceeding n/10,

L₁ is a linker of the structure -F₁-B-F₂-,

L₂ is a linker of the structure -FyB'-F^-, and

L₃ is a linker of the structure -F"_rB"-F"₂-, wherein

F₁, F¹., and F", represent residues of functional groups on the carrier and may be the same or different,

B, B' and B" represent 1-10 membered alkyl chains optionally having a chain complement on their part attached to F₁, F', or F", and may be the same or different,

F₂ is a functional group coupling dye molecule residue X to alkyl chain B, F'₂ is a functional group coupling secondary antibody A to alkyl chain B', and F"₂ is a functional group coupling fluorescent marker molecule residue Y to alkyl chain B".

Thus, in the sensor according to the invention the ion selective dye molecules are bound by covalent bonds to the surface of the carrier when the carrier is silicon dioxide, a noble metal or carbon, or are bound by covalent bonds to the surface of the carrier and/or are physically bound in the hollows of the carrier when the carrier is a dendrimer [for the preparation of dendrimers see e.g. Grayson et al: Chem. Rev. 101, 3819-3867 (2001 ); Crespo et al: Chem. Rev. ^05, 1663- 1681 (2005); Hwang et al: JACS 128, 7505-7509 (2006)]. The carrier holds at least 10 ion selective dye molecules (n is at least 10). The upper limit of the number of ion selective dye molecules bound to or incorporated into the carrier is not decisive and depends practically on the particle size of the carrier and on the number of functional groups on the carrier. It is preferred to attach as many ion selective dye molecule to a carrier nanoparticle as possible, because in this way the targeted sensor would produce a more intense signal.

The particle size of the carrier is preferably 20-150 nm, more preferably 50- 100 nm.

Upon an appropriate combination of the building blocks of linker L₁ any desired ion selective dye molecule can be attached to the silicon dioxide, noble metal, carbon or dendrimer carrier. The formation of linker L₁ is described in more detail when discussing the preparation of the sensor.

The term "ion selective dye molecule" or "dye molecule selective to a physiological ion" encompasses not only dye molecules which react to a single ion type (e.g. only to Na⁺ or only to K⁺ ions) with a concentration-dependent change in fluorescence, but also covers those which give such a reaction with more than one ion type but react to one of the ion types with a fluorescence change of at least threefold intensity. Obviously, it is preferred to use dye molecules with the highest possible selectivity. A broad choice of such dye molecules is known from the literature. Examples of Na⁺ selective dye molecules are those sold under the trade names SBFI, Sodium Green and CoroNa Green.

A second important characteristic of the sensor according to the invention is that it has 1-3 (preferably only 1) secondary antibody bound to the carrier with a covalent bond. In harmony with the literature, the term "secondary antibody" refers to an antibody fragment which recognizes the conservative region of all of the antibodies of a given species (human, monkey, rat, etc.). These secondary antibodies are known; of them the rat-specific Fab' antibody is mentioned as an example. The universal applicability of the sensor according to the invention is provided just by the fact that it contains a secondary antibody and not an antibody itself which performs targeting (further on: primary antibody). Due to the presence of the secondary antibody the once produced and marketed sensor can be coupled before use to any primary antibody which corresponds to the cell region to be examined, and the sensor is targeted by this latter primary antibody. The architecture of the nanosensor according to the invention wherein all of the ion selective dye molecules are bound to the surface of the carrier and the way of targeting it to a particular site is shown in Fig. 1.

In some instances it is advisable to couple a fluorescent marker molecule to the carrier in order to ascertain before starting the determination of the ion concentration whether the sensor has reached the target. Fluorescent marker molecules already mentioned at the description of the state of art which produce a fluorescent signal with an intensity independent of the concentration of the surrounding ions can be used for this purpose. These fluorescent marker molecules are also termed as marker dyes. It is preferred to use marker dyes which have a greater quantum yield than the ion selective dye. In order to make the signal of the marking dye clearly distinguishable from the signal of the ion selective dye it is preferred to use a marking dye/ion selective dye pair which emit fluorescent light at different wavelengths.

If the marker dye is available as one bound to the appropriate primary antibody one can also proceed so that the sensor is treated before use with this marker dye/primary antibody complex instead of the targeting primary antibody. In this instance the presence of a marker dye in the sensor according to the invention may be unnecessary.

The sensors according to the invention are prepared as follows: (a) when the carrier is silicon dioxide, a noble metal or carbon, fragment -F₁-B-Z of linker L₁, fragment -FyB'-Z' of linker L₂ and optionally fragment -F"_rB"-Z" of linker L₃ (in these formulae Z, Z' and Z" represent functional groups which are precursors of the F₂, F'₂ and F"₂ moieties, respectively, and the other symbols are as defined above) are built up on the functional groups of the carrier by methods known per se, and the resulting product is reacted with a compound of formula R-X, with a compound of formula R'-A and optionally with a compound of formula R"-Y (in these formulae R is a functional group which, when reacted with Z, forms the F₂ moiety, R' is a functional group which, when reacted with Z¹, forms the F₂ moiety, and R" is a functional group which, when reacted with Z", forms the F"₂ moiety and the other symbols are as defined above); or (b) when the carrier is a dendrimer, fragment -FVB'-Z¹ of linker L₂, optionally fragment -F₁-B-Z of linker L₁, and optionally fragment -F"_rB"-Z" of linker L₃ (in these formulae Z, Z¹ and Z" represent functional groups which are precursors of the F₂, F₂ and F"₂ moieties, respectively, and the other symbols are as defined above) are built up by methods known per se on the functional groups of the carrier which optionally comprises dye molecules selective to physiological ions, and the resulting product is reacted with a compound of formula R'-A, optionally with a compound of formula R-X and optionally with a compound of formula R"-Y (in these formulae R is a functional group which, when reacted with Z, forms the F₂ moiety, R' is a functional group which, when reacted with Z', forms the F₂ moiety, and R" is a functional group which, when reacted with Z", forms the F"₂ moiety and the other symbols are as defined above).

The so-called Stober synthesis is a well known method for producing silicon dioxide carriers, which enables one to produce particles of various size over a broad scale of the nanometer range (between 20 nm and 200 nm) as stable sols in aqueous or alcoholic media. This method is particularly preferable also because the size of the resulting particles is easy to control and the surface of the particles can be easily reacted with reactants capable of forming the required functional groups [Kazakevich et al: Langmuir 18, 3117-3122 (2002)].

The surface functional groups of colloidal silicon dioxide particles are the -OH groups bound to silicon atoms (for this instance the F₁, F^ and F^ moieties in linkers L₁, L₂ and L₃ are the same). These hydroxy groups can be silanylated very easily with compounds of the formula Si(OEt₃)-B-Z, such as with 3-amino-propyl- triethoxysilane (Et = ethyl, B = propylene, Z = NH₂) or with cyanoethyl-triethoxy- silane (Et = ethyl, B = ethylene, Z = CN) [see e.g. Plueddmann et al: Silane coupling agents 2nd ed.; Plenum Press, New York, 1991]. In this reaction, depending on whether a single reactant or more than one reactants were used, side chains of the formula -O-Si-B-Z (and optionally -O-Si-B'-Z' and/or -O-Si-B"-Z") are formed on the surface of the carrier; thus in this instance the B moieties (and the B' and/or B" moieties if present) are coupled to the residues of the functional groups on the carrier [F₁, FY F\; for this instance -O-] through chain complement -Si-. As it has been mentioned before, alkyl chains B, B¹ and B" are 1-10 membered alkyl chains, which means that 1-10 carbon atoms are located between moiety F₁ (plus the chain complement) and moiety F₂. The alkyl chains may be optionally branched. Alkyl chain B, which is a member of the linker to the ion selective dye molecule, is preferably a 2-6 membered one; alkyl chain B", which is a member of the linker to the marker dye (if present), may be of similar size. Alkyl chain B¹, which is a member of the linker to the secondary antibody, is preferably longer than alkyl chain B and/or B".

If the terminal group of the thus-formed side chain is not yet suitable for direct reaction with the secondary antibody and/or with the various dye molecules or with the reactive derivatives of these substances, the terminal group is converted into a functional group capable for such reactions by methods known per se. Thus e.g. a terminal -CN group can be converted into a carboxy group by hydrolysis, or a terminal amino group can be converted into a maleimide group for reaction with the secondary antibody.

The side chains can be formed on the surface of the carrier in a single step or in more consecutive steps. The consecutive reactions can also be performed as a one-pot synthesis. Thereafter the carrier with the desired side chains is reacted with a reactive derivative of the ion selective dye, with a reactive derivative of the secondary antibody, and optionally with a reactive derivative of the marker dye. These reactions can also be performed in a single step or in more steps.

The term "reactive derivative" as used in the previous paragraph refers to either the entities to be coupled (dye molecules, secondary antibody) if they do contain functional groups, or to reactive compounds formed therefrom by previous functionalization. The secondary antibody can be reacted directly with certain functional groups (e.g. maleimide or thio groups). However, certain dye molecules should be functionalized first to introduce groups R and R" capable of reacting with groups Z and Z" of the side chains on the carrier As an example, SBFI of formula (II) (a Na⁺-selective dye) can be subjected to partial hydrolysis to convert one of the acetyl groups into hydroxy group which is now capable to react with the carboxy terminal groups of the side chain on the carrier. Similarly, the carboxy group of Alexa Fluor 488 (a marker dye) can be converted into acyl chloride group in order to increase its reactivity for reactions with amino, hydroxy or carboxy terminal groups.

It is to be mentioned here that although the carrier-side members of linkers L₁, L₂ and L₃ (i.e. moieties F₁, P₁ and F",) are usually the same, it is not excluded that these members differ from one another. The various methods applicable for preparing the carrier (particularly when the carrier is a noble metal to be described in more detail later on) may give rise to the formation of such starting substances. In such instances the different side chains are formed on the carrier in separate steps.

It is also to be mentioned that linkers L₁, L₂ and L₃, furthermore side chains -F₁-B-Z, -FyB'-Z' and -F"_rB"-Z" leading to their formation, may be the same or different. When all the three side chains and/or linkers (or two of them) are the same, carefully selected reagent ratios and accurately controlled reaction conditions are required in order to produce particles comprising moieties X, A (and optionally Y) in the desired ratio. Therefore it is more appropriate to form side chains wherein at least terminal groups Z and Z' differ from one another (consequently in the resulting sensor L₁ and L₂ will also be different).

Numerous methods are available for the preparation of noble metal nano- particles applicable as carriers. According to a frequently used method trivalent gold compounds (such as HAuCI₄) can be reduced in a citrate-containing aqueous solution to form carriers with a particle size of 16-147 nm, the actual particle size being dependent on the reducing agent/stabilizing agent concentration ratio [Turkevitch et al: Discuss. Faraday Soc. IJ., 55-75 (1951 ); Frens: Nature Phys. Sci. 241 , 20-22 (1973)]. Production of mercapto-stabilized gold particles is also rather widespread; in this instance the particle size depends on the gold/stabilizing ligand concentration ratio [Yonezawa et al: Physico-chem. Eng. Asp. 149, 193-199 (1999)]. Over or instead of mercapto ligands numerous amphiphilic molecules have also been studied for stabilizing gold particles [Daniel et al: Chem. Rev. 104, 293- 346 (2004)]. The ligands of the nanoparticle, such as thiolates when gold particle is stabilized with an alkane thiolate, can be replaced by other tiols. The rate of this replacement process depends on the chain length [Hostetler et al: Langmuir 1.5, 3782-3789 (1999)], on the types of the exiting and entering thiols [Montalti et al: Langmuir 19, 5171-5174 (2003)], on the charge of the nanoparticle [Song et al: J. Am. Chem. Soc. 124, 7096-7102 (2002)] and on the oxidation processes [Pasquato et al: Chem. Commun. 2253-2254 (2000)]. The last step is a change in the functional terminal groups of the stabilizing agent for the nanoparticle. Peptid bond formation or nucleophilic substitution reactions may proceed on the surface [Pasquato et al. loc. cit.; Templeton et al. loc. cit.]. A characteristic feature of the reactions proceeding on nanoparticles is that the reactivity of the thin thiol layer which covers the strongly curved surface differs from that appearing on a plane surface. This effect can be utilized to perform organic reactions (such as SN₂ type reactions) on the surface of the nanoparticle which cannot proceed on a plane surface due to steric hindrance.

As it appears from the above, the side chains formed on a noble metal carrier comprise most frequently thiol groups as F₁, Z, Z' and/or Z" moieties, and the side chains differ from one another most frequently in the number of the members of alkyl chains B, B' and B". Thiol groups can be reacted usually directly with secondary antibodies, whereas the dye molecules comprising groups X and Y are usually to be be converted into reactive forms by appropriate derivatization steps.

Carbon nanotubes are characteristic representatives of carbon carriers. Direct oxidation of the carbon atoms on their outer surface is the simplest way of their chemical modification. The surface functional groups can be converted easily into other groups, e.g. they can be activated with thionyl chloride and esterified then with octadecanol [Hammon et al: Appl. Phys. A, Materials Science & Processing 74, 333 (2002)]. These reactions can be utilized to advantage to bind dye molecules and secondary antibodies to carbon carriers.

Methods for preparing dendrimers, e.g. those referred to above, can be easily completed at an appropriate stage with the introduction of dye molecules selective to physiological ions. Side chains for coupling secondary antibodies and optionally further (ion selective and/or marker) dye molecules can be formed on the surfaces of the resulting dendrimers by methods discussed above. It should be noted here that dendrimers comprising no ion selective dye molecules in their hollows can also be utilized as carriers. For these carriers n must be at least 10.

The sensor according to the invention is utilized for determining the concentrations of physiological ions in biological samples by conventional fluorescent measurement techniques (measurement of the starting fluorescence, adding to a biological sample, measurement of the final fluorescence) with the difference that a primary antibody which targets the sensor according to the invention to the cell region to be examined is also added to the biological sample. As it has been discussed above, primary antibodies carrying fluorescent marker dye molecules can also be used for this purpose. The sensor according to the invention is treated with the primary antibody preferably before adding it to the biological sample.

Further details of the invention are presented below by the aid of an example which describes the preparation and use of a sensor comprising silicon dioxide as carrier.

EXAMPLE

(a) Synthesis of SiO₂ nanoparticles with a diameter of 20 nm: 5 cm of 25 % aqueous ammonia are admixed with 250 cm of 99.7 % ethanol. The mixture is stirred at room temperature for 15 minutes, thereafter 10 cm³ of 98 % tetraethyl orthosilicate are added, and the resulting mixture is stirred at room temperature for 8 hours. The excess of ammonia is removed by distillation (completion of removal is checked by pH measurement). A stable colloidal system is obtained, which is subjected to evaporation under mild conditions avoiding boiling. In this way 2.50 g of colloidal SiO₂ (a so-called colloidal dry substance) are obtained.

(b) Formation of side chains with amino and cyano terminal groups on the surface of the nanoparticles:

2.50 g of dry colloidal SiO₂ are dispersed at room temperature in 250 cm³ of 98 % acetonitrile. A solution of 560 μl of 98 % 3-aminopropyl-triethoxysilane and 580 μl of 96 % 2-cyanoethyl-triethoxysilane in 10 cm³ of acetonitrile is added to the colloidal system at room temperature. The mixture is warmed to 50⁰C and stirred at this temperature for one hour. At the end of the reaction the mixture is evaporated under mild conditions to obtain the colloidal SiO₂ modified with two types of side chains [-O-Si-(CH₂)₃-NH₂ and -O-Si-(CH₂)₂-CN] as a solid substance.

(c) Conversion of cyano terminal groups into carboxy terminal groups: The resulting colloidal SiO₂ with modified surface is dispersed in 100 cm³ of distilled water and 100 cm³ of 10 % aqueous sulfuric acid solution are added. The mixture is boiled for 1 hour and then evaporated at 70⁰C. 2.0 g of colloidal SiO₂, modified with two types of side chains [-O-Si-(CH₂)₃-NH₂ and -O-Si-(CH₂)₂-COOH], are obtained.

(d) Conversion of amino terminal groups into maleimide terminal groups: A mixture of 0.2 g of colloidal SiO₂ with modified surface, prepared as described above, 0.3 g (3 mmoles) of maleic anhydride and 10 cm³ of chloroform is refluxed for 20 hours. Thereafter the mixture is filtered, the solid is washed with chloroform and dried in vacuo. 10 cm³ of acetic anhydride and 40 mg of sodium acetate are added to the resulting solid, and the suspension is intensely stirred for 30 minutes. The mixture is cooled to 4⁰C, stirred for 4 hours, thereafter the solid product is filtered off and dried in vacuo. 0.22 g of a solid product are obtained, wherein the amino terminal groups of the side chains are replaced by maleimide terminal groups.

(e) Conversion of a Na⁺ selective dye into its reactive derivative: 0.25 cm³ of 0.1 N aqueous potassium hydroxide solution are added drpowise to a solution of 0.107 g (0.1 mmole) of SBFI dye of formula (II) in 10 cm³ of dioxane, and the resulting mixture is stirred at room temperature for 4 hours. Thereafter the mixture is neutralized to pH 7 with 1 N aqueous hydrochloric acid and evaporated in vacuo. 0.1 g of activated SBFI dye carrying hydroxy functional groups is obtained.

(f) Coupling of the dye molecules to the carrier:

The product obtained in step (e) is coupled to the -COOH terminal groups of the side chains of the product obtained in step (d) as follows: 0.5 cm³ of isobutyl chloroformate are added dropwise at O⁰C to a stirred suspension of 0.2 g of the product obtained in step (d) in 10 cm³ of dimethyl formamide, and the mixture is allowed to warm to room temperature. After 2 hours of stirring a solution of 0.1 g of the product obtained in step (e) in 5 cm³ of dimethyl formamide is introduced. The reaction mixture is stirred overnight and the solvent is evaporated then in vacuo. The residue is suspended three times in 10 cm³ of distilled water, each, the liquid is decanted, ahd the solid is sedimented by centrifuging. 0.3 g of a solid product are obtained, wherein the -O-Si-(CH₂)₂-COOH side chains of the carrier are replaced by -O-Si-(CH₂)₂-COO-SBFI side chains.

(g) Coupling of the secondary antibody:

The secondary antibody is coupled to the carrier through the maleimide terminal groups of the side chains of the product obtained in step (f) as follows: 30 nanomoles of a product obtained in step (f) are solubilized in 1 cm³ of an aqueous buffer comprising 150 mmoles of NaCI, 20 mmoles of Na₂PO₄ and 1 mmole of EDTA (pH 6.5), and then 0.2 mg of Fab¹ secondary antibody are added. After 1-16 hours of incubation the product is separated by gel filtration.

(h) Treatment of the sensor with a primary antibody: 1 μg of the antibody specific to ankyringG protein localized on the axon hillock of rat brain [Zhou et al: J. Cell. Biol. 143, 1295-1304 (1998)] is dissolved in 5 μl of PBS, and this solution is added to the sensor according to the invention obtained in step (g). The mixture is maintaned at room temperature for 10 minutes, thereafter 10 μl of a blocking reagent is added, and the antibody complex of the sensor according to the invention is separated from the non-bound components by column chromatography.

(i) Determination of the concentration of /Va⁺ ions in rat brain slice: 300 μm thick hippocampal slices are excised from brains of 9-15 days old male rats stored at 4⁰C in an artificial cerebrospinal fluid (ACSF). The slices are incubated for 1 hour at 37⁰C under carbogenic atmosphere in an interface type chamber comprising ACSF buffer. Thereafter 200 μl of ACSF buffer comprising 30 μg of the product obtained in step (h) are added to the slices, and incubation is continued for an additional hour.

The brain slice treated with the sensor according to the invention is placed into a measuring chamber equipped with an optical microscope and ACSF is perfused above it at a rate of 4 cm³/minute. The SBFI dye is excited with a light 340 nm in wavelength, and the emitted light is detected at 525 nm with a high-speed CCD camera or with a scanning confocal laser microscope.

With this method the activation of Na⁺ ion channels, which are concentrated in an extremely high number at the axon hillock, can be well detected.

Claims

What we claim is:

1. A sensor of formula (I) for determining the concentration of physiologically active inorganic ions at subcellular levels

wherein

(HJ is a silicon dioxide, noble metal or carbon carrier particle with an average diameter of 10-200 nm or a dendrimer with an average diameter of 10-200 nm optionally comprising dye molecules which are selectively sensitive to a physiologically active inorganic ion,

A is a secondary antibody,

Y is the residue of a fluorescent marker molecule, m is 1-3, n is at least 10, but if (hi) is a dendrimer, n may also be 0-9, p is zero or a positive number not exceeding n/10,

L₁ is a linker of the structure -F₁-B-F₂-,

L₂ is a linker of the structure -F'_rB'-F'₂-, and

L₃ is a linker of the structure -FVB"-F"₂-, wherein

F₁, F'_Ϊ and F\ represent residues of functional groups on the carrier and may be the same or different,

B, B' and B" represent 1-10 membered alkyl chains optionally having a chain complement on their part attached to F₁, F^ or P₁ and may be the same or different,

F₂ is a functional group coupling dye molecule residue X to alkyl chain B, F₂ is a functional group coupling secondary antibody A to alkyl chain B', and F"₂ is a functional group coupling fluorescent marker molecule residue Y to alkyl chain B".

2. A method for preparing a sensor according to claim 1 , characterized in that

(a) when the carrier is silicon dioxide, a noble metal or carbon, fragment -F₁-B-Z of linker L₁, fragment -F'_rB'-Z' of linker L₂ and optionally fragment -F"_rB"-Z" of linker L₃ (in these formulae Z, Z' and Z" represent functional groups which are precursors of the F₂, F'₂ and F"₂ moieties, respectively, and the other symbols are as defined in claim 1) are built up on the functional groups of the carrier by methods known per se, and the resulting product is reacted with a compound of formula R-X, with a compound of formula R'-A and optionally with a compound of formula R"-Y (in these formulae R is a functional group which, when reacted with Z, forms the F₂ moiety, R' is a functional group which, when reacted with Z', forms the F'₂ moiety, and R" is a functional group which, when reacted with Z", forms the F"₂ moiety and the other symbols are as defined in claim 1); or

(b) when the carrier is a dendrimer, fragment -FyB'-Z' of linker L₂, optionally fragment -F₁-B-Z of linker L₁, and optionally fragment -F"_rB"-Z" of linker L₃ (in these formulae Z, Z' and Z" represent functional groups which are precursors of the F₂, F'₂ and F"₂ moieties, respectively, and the other symbols are as defined in claim 1 ) are built up by methods known per se on the functional groups of the carrier which optionally comprises dye molecules selective to physiological ions, and the resulting product is reacted with a compound of formula R'-A, optionally with a compound of formula R-X and optionally with a compound of formula R"-Y (in these formulae R is a functional group which, when reacted with Z, forms the F₂ moiety, R' is a functional group which, when reacted with Z', forms the F'₂ moiety, and R" is a functional group which, when reacted with Z", forms the F"₂ moiety and the other symbols are as defined in claim 1 ).

3. Use of a sensor according to claim 1 to determine the concentration of physiologically active inorganic ions at subcellular levels, wherein a sensor according to claim 1 is added to the biological sample in combination with a primary antibody which is specific to the subcellular cell region to be examined, and then the concentration of the inorganic ion in question at the subcellular cell region is determined by measuring the intensity changes in fluorescence.