DE102004060742A1 - Micro-sensor for determining bodily fluid contents, comprises a conical shape and comprises a micro-electrode with fibers, one of which is a working electrode with a layer of an electro-active polymer - Google Patents

Abstract

A micro-sensor (1) has a conical shape and comprises a micro-electrode (10) with fibers (14) one of which is a working electrode (60) provided with a specific layer (80) made from a biologically acceptable electro-active polymer. Independent claims are also included for: (1) process for the production of a micro-sensor; and (2) process for analytically determining the contents of an organism.

Description

The The invention relates to a microsensor according to the preamble of claim 1, and a method for its production according to the preamble of claim 31. Besides comprises a method of determining at least one ingredient with the microsensor according to the invention according to the generic term of claim 35 and according to the preamble of claim 38 and a use thereof according to claim 39 and claim 40.

you uses microelectrodes in a variety of ways for determination of ingredients in solutions and body fluids. Thus, GB A 2 284 267 discloses a micro-wire electrode for the partial pressure of oxygen to determine in the blood. This consists of several electrode fibers, each surrounded by a borosilicate glass jacket. The thus obtained metal fiber filled Become a vitreous in a carrier embedded, made of a fluorinated ethylene / propylene copolymer consists. For this purpose, they are bundled through an extrusion bath with molten polymer and wind it up after solidification a coil. Each electrode fiber first with a glass jacket and However, surrounding all together with a polymer carrier is laborious and costly.

This circumvents the US PS 5,002,651 in that it is a carrier describes an electrochemically resistant polymer, in the at least one electrode fiber is introduced. However, it acts it is a metal thread running along its entire length a mixture of polymers and active molecules of variable percentages is coated. Also, such a designed microelectrode expensive and expensive to manufacture.

The DE A1 101 12 384 avoids these disadvantages and describes an ultramicro-electrode with a centrally embedded in a cylindrical body electrode fiber and at least three further fibers insulated surrounding this fiber. All fibers are made of a uniform material, such as metal and are directly in an existing example of quartz glass cylindrical body integrated. Because the ultramicroelectrode described but a flat plan or concave Sensor head, can be small openings on cells or membranes do not penetrate in any case. So it's not just any measurement environment unlimited reachable. Furthermore let yourself do not completely rule out that the revealed Microelectrode due to their geometry or their polymeric coating the chemical and physical situation of a test sample in one over the Measurement going way changed.

aim The invention is to overcome these and other disadvantages of the prior Overcome technology and to create a microsensor that is inexpensive and easy to manufacture is. He is supposed to be able to select different types of signals selectively and precise too Received side by side and a probe for quantitative determination be the most diverse substances. In addition, a long operational readiness also in changing measuring environments with consistently high measuring accuracy sought.

main features the invention are in the characterizing part of claims 1, 31, 35 and 38, and in the claims 39 and 40 indicated. Embodiments are the subject matter of claims 2 to 30, 32 to 34, 36 and 37.

at a microsensor for qualitative and quantitative determination at least one ingredient in a liquid medium consisting of a multifiber microelectrode with at least two electrode fibers, of which at least one is designed as a counter electrode and / or as a reference electrode and with a carrier, the one piece all electrode fibers at the same time integrally, across from the liquid Medium indifferent electrical insulator is in which the electrode fibers from each other embedded in isolation, each of them with the carrier cohesively directly is connected, the invention provides that the microsensor at least one partially conical shape and at least one of the fibers has a working electrode is, the meßseitig with a specific layer of a biocompatible, electroactive polymer is provided. Achieved by this conception one, that with the same microsensor electrochemical activity on cell networks as Whole, for example on neural networks, but also on single cells a cell network can be located and measured. In addition, let solid or liquid surfaces of different Size in one Step specifically to the presence of certain u.U. different Examine particles. Although in these measurements electrochemical Signals are very variable and different in severity, each of them succeeds precisely to record and forward. Even in continuous operation, it does not come to failures. The at least partially tapered and thus narrow microsensor let yourself in narrow-pore or membrane-enclosed environments, where the electroactive polymer due to its biocompatibility none affecting the measurement changes in an examination medium.

According to claim 2, the multi-fiber microelectrode is connected to a micro-board, are soldered to the contact wires of a noble metal. This connection forms the interface between Components with micrometer dimensions and a much larger sized external electrical supply. Because the microplate is mounted in the immediate vicinity of the multifiber microelectrode as a rigid member, great mechanical deformation of the electrode fibers between the circuit board and the carrier is avoided, thereby increasing the useful life of the microsensor.

claim 3 discloses as an important feature of the microsensor according to the invention, that be carrier a round or oval quartz glass jacket is. By his rounded It can easily be inserted into a wide variety of cavities. Quartz as hard and very temperature resistant Material also makes the microsensor universally usable without that too fear remains, parts of the carrier could be under certain measuring conditions in solution go and thus distort analysis results.

As well it is essential for the microsensor that his Electrode fibers according to claim 4 made of a noble metal, for example made of platinum and / or a platinum / tungsten alloy and a diameter of 4 to 18 microns, in a preferred embodiment from 10 to 12 μm have. By the claimed precious metals or precious metal alloys it is ensured that the electrode fibers durable and not susceptible to oxidation. The chosen diameter on the one hand guarantee easy control, on the other hand however, so small that surface areas with micrometer dimensions can be easily covered and measured can.

From of special importance for The invention is also claim 5, according to which at least one electrode fiber covered with a layer and different electrode fibers with different layers and / or with different specific ones Layers are provided. Only by this feature education is it is possible Electrodes for certain Particles, ions or molecules to make selective. To obtain particularly stable measurement results, is located the layer and / or the specific layer according to claim 6 only on a part of the electrode fiber that is not adjacent to the carrier.

For the invention it is indispensable according to claim 7, that the electrode fibers on a side pointing in the direction of a contacting end, from the surrounding carrier exposed and soldered to the microplate. Without this exposure, the Tetrode can not be connected to the microplate and the microsensor do not forward the current to a meter. Claim 8 determines that the exposed side of the electrode fibers, the microplate and ends the contact wires embedded in an adhesive matrix and together the contacting end form. It makes sense to use these components of the microsensor in to fix an adhesive block to protect it from damage or demolition and protect to prevent loose contacts and short circuits in the microsensor.

In An embodiment of the invention according to claim 9, the microsensor n Elektodenfasern, where n is an integer value of 2 to 50, preferably from 3 to 13 and most preferably from 4 to 6 assumes and the individual fibers, for example, arranged equidistant from each other are. They are also grouped according to claim 10 to each other, that one Centrically arranged electrode fiber of n electrode fibers preferably concentric where n is an integer value of 1 to 49, preferably from 2 to 12, and most preferably from 3 to 6. Only with Sensors constructed in this way allow multiple particles to be simultaneously prove side by side in a defined measuring volume. This succeeds besides, especially good, if the individual fibers are connected according to claim 11, that she independent from each other electrically addressable micro-disk electrodes form with electrical shielding.

Around in microporous Structures and intercellular spaces to be able to penetrate the multifiber microelectrode according to claim 12 has a diameter of less than or equal to 250 μm. In addition, it has according to claim 13 at one end, the contacting end opposite a sensor head that is a circular or oval or at least partially conically tapered, smooth polished surface. With such a topography you get well reproducible and homogeneous measuring signals.

A compactly built and versatile form of multi-fiber microelectrode is a tetrode according to claim 14, wherein the three electrode fibers a fourth, central in the carrier Surrounding arranged electrode fiber concentrically. It concerns with you according to the claim 15 around a circular or oval fiber with a diameter of 50 to 110 μm and in a preferred embodiment from 70 to 90 μm. moreover Does she have a length? from 5 to 15 cm, and preferably from 10 to 12 cm.

These A hair comparable shape gives the tetrode elasticity and allows it, not only in solution but also on and in different cellular tissues for measurements to use. This can be due to their slim geometry, a shift or traumatization of individual cells in the studied target area be avoided as far as possible. Because the four are isolated from each other Fibers are located in the smallest space, can also be close to each other cover overlying areas and measure.

The claimed tetrode is moreover When used as an annular disk electrode, when concentric arranged electrode fibers interconnects to form a ring electrode and the centered electrode fiber is used as a second electrode of a measuring chain. This control of the four electrode fibers also ensures that individual signals due to the so-called "tetrode effect" can be filtered out of a neural summation signal.

The stability of the microsensor increases when the multifiber microelectrode according to the continuation in Claim 16, for example by means of an epoxy or acrylic resin-based adhesive in a cannula is glued. The latter consists according to claim 17 of a glass material or stainless steel and has an outer diameter of 250 microns to 350 microns, preferably of 280 μm up to 305 μm. Such sized cannulas cause almost no effect on a target tissue to be examined Injuries and are biologically well tolerated. They also shield the interior of the microsensor against external electrical or electrochemical influences from.

In an advantageous extension according to claim 18 are the tetrode and the cannula at the contacting end, for example by an adhesive bond fixed. This ensures a fast forwarding of signals to the microplate and power Shorts, which could arise when slipping tetrode against microplate, impossible.

Want If you make the microsensor even more robust, you fix it in a practical embodiment of the invention according to claim 19 in a capillary, for example by mixing it with acrylic or epoxy resin sticking in her. This consists according to claim 20 expediently made of glass and / or of a flexible plastic. Both materials are mechanically stressable and biologically compatible.

In An advantageous embodiment according to claim 21, it is particularly Cheap, two of the electrode fibers over to connect a contact wire as a counter electrode, because this increases one within a measuring environment a reference measuring surface detected by the counter electrode. Should this area in the immediate vicinity of all other electrode fibers be arranged, it is convenient if in the sense of claim 22 a centric and a concentric arranged electrode fiber interconnected to form the counter electrode. Such an arrangement is always particularly desirable if a concentration of an ingredient to determine itself in small areas a sample quickly changes.

Should designed a miniaturized Zweistabmeßkette which is to manage without external electrodes is the expanding one execution according to claim 23 essential. These demands that the layer is present on the reference electrode and of deposited silver which is chemically silver chloride on its surface implemented.

From There you get through a promising expansion to a special three-meter chain, if in the microsensor in the sense of claim 24, the specific layer for at least an ingredient is selective and made of an electrically conductive polymer consists. By this development according to the invention it is possible in a confined space, a classic three-electrode arrangement of working, Counter and reference electrode to realize. Can with her targeted ingredients, such as free radical nitrogen monoxide (NO) or oxidizable food ingredients in a liquid medium become.

Consists the conductive one A polymer according to claim 25 of conjugated heterocyclic Aromatics, thus preventing the resulting electrochemically active, good on the precious metal surface adherent polymer layer effectively an electrode fouling, ie the adsorption of analyte particles on the metal surface of the Working electrode. This remains so long usable and shows reproducible measuring currents.

A for the Invention very significant development is that the conductive polymer after Claim 26 is a polycarbazole and / or a polythiophene. Both Heterocyclic classes of compounds are each suitable individually, as well as in interaction with each other very well as electroactive Electrode coatings. They can be neurotransmitters or convert vitamin C quasi reversible. This shows for dopamine, Serotonin and ascorbate striking current yields. Polythiophene forms In addition, a very well-structured and homogeneous layer on the working electrode out.

In a continuing The microsensor type according to claim 27 is the conductive polymer Polypyrrole. Neurotransmitters can be attached to one of this polymer Also selectively convert reacted coating. polypyrrole shows a particularly pronounced surface adhesion on a platinum electrode fiber and a regular surface texture. Furthermore a layer of polypyrrole is suitable for heat-induced enrichment of carbon on the electrode fiber.

The invention undergoes substantial extension according to claim 28, because the ingredient-specific layer is dopable or doped For example, with compounds that have a steric and / or charge-altering influence on them. This feature increases the longevity of a polymer layer deposited on a working electrode. In addition, their particle-selective properties are further improved. It is particularly economical if the specific layer according to claim 29 is doped with special ions, for example with metal or halide ions or with ions from quaternary ammonium salts, boron halides, phosphorus halides or perchlorates.

For some Applications, it is also necessary, the invention according to claim 30 continue. For this purpose, the uncoated working electrode is roughened, for example by means of sulfuric acid. Bad adherent polymer layers require such pretreatment or Primer to then permanently fixable on the working electrode Being a prerequisite for reproducibly stable measurement results is.

For the invention is the independent one Claim 31 very significant. It discloses a method of manufacture a microsensor for the determination of at least one ingredient in a liquid medium, in the invention at least two Precious metal wires in a quartz tube given and deformed in the heat under tension to Taylor becomes; the deformed quartz tube is removed on one side, so that the metal wires as Electrode fibers are exposed; an exposed electrode fibers opposed measuring range the multifiber microelectrode to a sensor head flat smooth is polished, and at least one of the electrode fibers on the sensor head with an electroactive layer and / or with an electroactive specific Layer is provided.

at Such a procedure can be mass produced microsensors consistent quality obtained, which are mechanically stable and on the sensor head even after many measurements show no signs of erosion. Also the difficulty a metal on or in a quartz body without the use of additional Permanently attaching adhesion promoter, is overcome elegantly with this method. After all Such a coated electrode is suitable for selective detection selected Particles.

To Claim 32, the sensor head is polished so that it has a conical shape or a partial cone without apex. This shaping facilitates the introduction of the microsensor into narrow cavities and porous wells considerably. In accordance with claim 33, it is possible as an electroactive layer a conductive one Electrochemically or chemically deposit polymer. This procedure extends the range of possible ones to be used chemical compounds. To a transition from the multifiber electrode to create a measuring instrument, this is in the embodiment of claim 34 to the exposed electrode fibers connected to a microplate. This ensures that recorded Signals unadulterated to a measuring device Will you pass the subject of the invention for measurements use, defines the independent method claim 35 the process steps required for this purpose. For the determination at least an ingredient in a liquid Medium with a microsensor with sensor head becomes said sensor connected to a high sensitivity potentiostat; with the Sensor head ahead in the liquid Medium, for example, introduced within a tissue compartment and recorded by means of cyclic voltammetry current-voltage curves. This procedure allows ingredients down to a few μmol / l in to prove a study environment. In addition, you can so a so-called Multi-unit activity, for example, the neuronal activity of many neurons, electrochemically investigate and at the same time single cell activities from this so-called Measure total signal isolated. Similarly, when scanning a Cell culture with a scanning electrochemical microscope (= SECM) at a constant potential only one active ingredient selectively implemented and its local propagation through the resulting three-dimensional Electricity profile spatially be followed.

The inventive method provides fast information about Ingredients of the liquid Medium, if one according to claim 36 from the current-voltage curve for one measurement peak an associated one Read voltage value and this one basis by comparison values Ingredient assigns. Furthermore learns one in the embodiment according to claim 37 in a straightforward way, how concentrated an ingredient is in the liquid medium. For this you have to from the current-voltage curve for one measurement peak an associated one Read current value and this on the basis of a characteristic of an ingredient Current / quantity calibration line one allocate appropriate quantity.

• – bei dem in einen Schädel ein kreisrundes Loch gebohrt wird, durch das ein aus Futter und Dorn bestehender Mandrin bis in eine Zielregion des Gehirns eingeführt wird;
• – der Dorn herausgezogen und der erfindungsgemäße Mikrosensor eingeschoben und bis an die Zielregion herangeführt wird;
• – mittels zyklischer Voltammetrie Strom- Spannungskurven aufgenommen werden;
• – auf einem Bildschirm oder an einem Meßgerät die erhaltenen Kurven übereinanderlegt sowie Spannungswerte abgelesen werden und durch Zuordnung zu Eichwerten ein Inhaltsstoff identifiziert wird;
• – und Stromwerte abgelesen werden, die man auf eine für den Inhaltsstoff spezifische Eichgerade abträgt und dadurch den Inhaltsstoff mengenmäßig bestimmt.

Claim 38 describes a particular embodiment of the invention and should therefore enjoy individual protection. It is a method for the analytical determination of at least one in an organism, for example within the brain existing ingredient with a microsensor;

• In which a circular hole is drilled in a skull, through which a mandrin consisting of food and thorn is introduced into a target region of the brain;
• - The mandrel pulled out and the erfindungsge inserted microsensor and is brought up to the target region;
• - recorded by means of cyclic voltammetry current-voltage curves;
• - superimposed on a screen or on a meter, the curves obtained and voltage values are read and identified by assignment to calibration values, an ingredient;
• And current values are read off, which are removed to a specific calibration line for the ingredient and thereby determines the ingredient in terms of quantity.

This Method not only allows pathophysiological mechanisms in particular neurodegenerative malformations and diseases postoperatively to explore. It also allows to be recorded from intraoperatively Data errors a population signal of basal ganglia nuclei and downstream motor centers in system-physiological approaches to analyze more accurately. there For example, a tissue can be electrophysiologically "scanned" by means of the microsensor.

The The design of the microsensor predestines it for one in claim 39 formulated use for qualitative and quantitative Detection of Neurotransmitters and / or Antioxidants in Solutions and on or in biological material. Wear in the same way its geometry and its small design. Also his particle selective Polymer coating is important. This also applies to the in Claim 40 formulated use of the microsensor for displacement of equilibria between oxidized and reduced forms of a Neurotransmitters or a hormone within cell tissue associations and intercellular Interspaces.

Further Features, details and advantages of the invention will become apparent the wording of the claims as well as from the following description of exemplary embodiments with reference to FIG Drawings. Show it:

1a a section-wise schematic view of a microsensor with a tetrode as a multi-fiber microelectrode,

1b a schematic oblique view of the front part of another microsensor with a tetrode as a multi-fiber microelectrode,

2 a photographic plan view of a sensor head of the microsensor 1 .

3 a schematic plan view of another design of the sensor head,

4 current / voltage curves obtained with a polythiophene-coated microsensor in phosphate-buffered solution for various dopamine concentrations,

5 a section enlargement of 4 .

6 a current / concentration curve for dopamine obtained with a polythiophene-coated microsensor,

7 current / voltage curves obtained with a polythiophene-coated microsensor in phosphate-buffered solution for a) dissolved dopamine, b) dissolved serotonin and c) a mixture of equal proportions of dissolved dopamine and serotonin,

8th Structural formulas of a) dopamine, b) serotonin and c) norepinephrine,

Under multifiber microelectrode 10 is every microelectrode with a carrier 11 and at least two electrode fibers 14 Thus, a three-fiber triode, a five-fiber pentode or generally to understand an n-fibrous microelectrode, where n can take any integer value greater than or equal to 2. The microsensor described below 1 contains as a multifiber microelectrode 10 a four-fiber electrode. All statements made for this purpose also apply, if possible, to microsensors 1 with n electrode fibers 14 ,

An in 1 mapped microsensor 1 consists of a tetrode 15 with a sensor head on one side 12 owns and on the other side in a contacting end 20 passes. Under tetrode 15 one understands a carrier 11 , in the four electrode fibers 14 are embedded without touching each other. They end at the sensor head 12 where they form punctiform areas that are in direct contact with a medium to be examined during a measurement. If an electrochemical event takes place in this medium, this leads to a local shift of charges. This charge transfer is transferred to the sensor head 12 on the electrode fibers 14 the tetrode 15 and there is an electrical signal at the contacting end 20 to a microplate 30 and from there to a control and / or measuring instrument, for example to a highly sensitive potentiostat, is passed.

The tetrode 15 has dimensions that are comparable to those of a hair. It is between 5 cm and 15 cm, ideally 10 cm long and has a diameter of 50 microns to 110 microns, if possible between 70 microns and 90 microns. Your sensor head 12 takes one in one embodiment Angle of 90 ° to the tetrode longitudinal axis and forms a dipping into the liquid medium Tetrodenstirnfläche. This is round or oval and polished smooth. Their diameter corresponds to the tetrode in a cylindrical tetrode shape 15 , In a conical design, however, their sensor head diameter is smaller and even almost zero, provided the tetrode 15 on the measuring side, as in 1b shown, a tip trains. In such a pointed shape, the punctiform regions are arranged on a cone sheath.

Much of the tetrode 15 consists of an electrically insulating carrier 11 or coat. In addition to various high-melting glass materials such as borosilicate glass pure silica in the form of quartz glass is particularly suitable for this purpose. Thermoset polymers which can be subjected to tensile stress can also be used for the electrode fibers 14 encasing insulating.

These fibers 14 span the tetrode 15 in full length and are materially bonded to the carrier 11 connected. They are made of a wire or thread of a re-solidified noble metal. Gold, silver, palladium, copper and platinum are particularly suitable. Alloys of these precious metals with representatives of transition metals from groups three to twelve of the periodic table (first to eighth subgroup) are also used, for example platinum-associated tungsten. The electrode fibers 14 each have a diameter of 4 to 18 microns, but cross-sections of 10 to 12 microns are particularly favorable.

You can choose any of the fibers 14 individually electronically drive so that in a tetrode 15 four independent micro-disc electrodes are placed. Conveniently, as in 2 see each of them coaxially around the one in the carrier 11 centrally located fourth fiber 14 grouped. This makes it possible for the tetrode 15 To operate as a ring-disk electrode, on the one hand, the centrally arranged fiber 14 and on the other hand, the concentrically arranged, now connected together as a ring electrodes 14 each generate a single electrical signal.

Of the four electrode fibers 14 Only two can be controlled or connected together. This will cause the sensor head 12 covering a surface area above the medium to be examined, which lies either only on a circular area forming the concentric ring or else both a part of this circular area and one in the center of the sensor head 12 arranged part surface detected. Are the electrode fibers 14 , as in 3 in the form of a rectangle in the carrier 11 localized, can a larger area of the sensor head 12 be detected homogeneously.

The at the sensor head 12 ending point-shaped areas of the electrode fibers 14 have no cohesive contact with the carrier 11 but only point into the medium to be examined. In a preferred embodiment, they have a different, electrochemical activity in the manner of a miniaturized three-bar measuring chain. For this purpose, they are modified or occupied in various ways and then form a counter electrode 40 , a reference electrode 50 and a working electrode 60 , At least one of these electrodes 40 . 50 . 60 has a homogeneous smooth structure and / or surface caused by brightener.

The counter electrode 40 consists of at least one unchanged electrode fiber 14 , In order to obtain the largest possible reference measuring surface within a measuring environment of about 300 μm ² to 9000 μm ² , two electrode fibers are formed 14 to a counter electrode 40 connected together. This covers within the measurement environment a reference measuring area of about 15 microns ² to 520 microns ² , whereas a single electrode thread 14 only about 7 microns ² to 260 microns ² detected.

The reference electrode 50 registered together with the counter electrode 40 all occurring in the measurement environment of the medium to be examined electrochemical changes, so it is not specific for only caused by certain ions or molecules state change. It consists for example of one with a silver layer 70 coated electrode fiber 14 where the silver layer 70 has a silver chloride coating. The metal chloride layer 70 has a thickness of about 1 to 2 microns, whereas the underlying amorphous metal is about 2 to 5 microns thick.

In order to specifically filter out electrochemical signals of certain ions or molecules as well as certain families of particles in the medium to be examined, each working electrode has 60 a specific layer 80 , which is electrically conductive and adheres particularly well, if one uses the uncoated electrode thread 14 , in particular its punctiform area previously roughened, for example by means of sulfuric acid.

One on this layer 80 The flow rate measured per analyte particle is the higher, the thicker it is. To determine the concentration of hormonal neurotransmitters such as dopamine and serotonin and to detect antioxidants such as ascorbic acid is the layer of a polymer 80 between 30 nm and 3 μm thick. It is insensitive to air and moisture and is also not oxidized anodically, provided that an applied potential measured against a calomel electrode does not exceed a maximum value of 0.6 V.

The layer 80 consists of a polycarbazole or a polythiophene or of a special polypyrrole. Polycarbazole or polythiophene and polypyrrole are also polymers which are obtained from derivatives of carbazole, thiophene or pyrrole by electropolymerization. Thus, the group of polythiophenes also includes polythiophene-3-carboxylic acid or poly-3-methylthiophene and di- or trimers of these compounds, such as poly-terthiophenes and poly-bithiophenes. These polythiophenes are obtained from thiophene-3-carboxylic acid or 3-methylthiophene and from di- or trimeric compounds, such as terthiophenes or bithiophenes.

Electropolymerization means the specific polymer layer 80 galvanostatic, potentiostatic or potentiodynamic from a solution containing at least one monomer on the working electrode 60 deposit. In the case of a galvanostatic coating, the applied current is kept constant during the electropolymerization and a time-varying voltage is measured, while in the case of a potentiostatic coating the voltage is kept constant and the time-varying current is measured. In a potentiodynamic mode of operation, an applied voltage is varied and the changing current is measured. In all cases, the monomers used combine and separate as a specific layer 80 on each electrically contacted working electrode 60 from.

To the quality of the working electrode 60 to improve is their particle-specific layer 80 also chemically or electrochemically doped or dopable. This increases their stability and serves to electrochemically convert even more specifically selected molecules. By choosing a suitable dopant, the conductivity behavior of polycarbazole can be more than doubled.

A distinction is made between an n-type doping and a p-type doping. The latter arises when a completely deposited polymer layer 80 in a saline solution is partially oxidized by a positive potential. This results in p-holes, positively charged areas extending along the layer 80 move through delocalization. Anions from the salt solution penetrate into these holes and stabilize the polymer layer 80 , N-doped is a particle-selective polymer layer 80 when cathodically reduced in a saline solution. Now, negatively charged regions, called n-holes, form and delocalize along the conductive layer 80 , For reasons of charge neutrality, cations of the salt solution diffuse into the holes formed and thus generate the n-type doping.

Preference is given to doping in polar aprotic solvents, such as acetonitrile (ACN), dimethyl sulfoxide (DMSO), Dimetyhlformamid (DMF), dioxane, tetrahydrofuran (THF) and other alicyclic or aliphatic ethers and in very oxidation-resistant, cyclic ketones by. Suitable dopants are all common salts; they are called conductive salts. For the invention, those of camphorsulfonic acid (CSA) and polyvinylsulfonic acid (PVS) and iodides, bromides, chlorides, fluorides, sulfates and hydrogen sulfates can be used. Also various metal ions are usable. Also useful are nitrates, phosphorus halides, boron halides, perchlorates and quaternary ammonium salts. Also, salts with large cations such as tetra-n-butylammonium hexafluorophosphate (TBAPF ₆ ), tetra-n-butylammonium perchlorate (TBACIO ₄ ) and tetrabutylammonium tetrafluoroborate (TBABF ₄ ) are particularly useful.

In addition to doping, in the preparation of the conductive polymer, the electrochemical deposition method, selected potential range, applied rate of potential change, set temperature, monomer or oligomer concentration, solvent used, and catalytic bases employed, also affect the quality of the polymer coated working electrode 60 ,

In the turnover of neuronal messengers or other cyclic analytes on a polycarbazole coated working electrode 60 one obtains only pronounced current maxima, if the specific polymer coating 80 was produced in advance under certain conditions. Thus, the conductive polymer must be obtained by potentiodynamic, potentiostatic or galvanostatic electropolymerization of a dissolved monomerene carbazole with the addition of a sterically hindered hardly oxidizable nitrogen base and be doped during the formation process immediately.

When sterically hindered bases one uses five or six-ring heterocycles, which are alkylated several times. These include several tert-butyl and isopropyl or methyl pyrroles, pyrazoles and Imidazoles, pyridines, pyrimidines and indoles. Examples are 3,5-methyl-pyrazole, 2,4-lutidine and 2,4-di-tert-butylpyridine, 2,4,6-collidine, 2,4,6 Tri-tert-butylpyridine, 2,6-di-tert-butyl-4-methyl-pyridine and 1,2,2,6,6-pentamethyl-4-hydroxypiperidine. 1,4-Diazabicyclo [2.2.2] octane (DABCO) can also be used.

If the point-shaped end region of an electrode fiber is coated 14 Potentiodynamic in a narrow potential range of 0.8 V to 1.4 with polycarbazole, one obtains thin polymer layers 80 , These are good for low detection Analyte concentrations. On the other hand, with a potentiostatically deposited polycarbazole, a thick polymer layer is formed 80 , which can be used to determine high neurotransmitter or ascorbic acid concentrations.

The shows polythiophene obtained from a mono-, bi- or terthiophene in the conversion of said analytes also pronounced current maxima. However, too here the potentiodynamic, potentiostatic or galvanostatic Electropolymerization and the doping of the forming polymer take place simultaneously. A base addition is not required.

Also on an electrode fiber 14 deposited polypyrrole is suitable for the detection of said hormonal and antioxidant-acting analytes. However, this is only possible with a polypyrrole obtained galvanostatically and subsequently overoxidized at a constant potential. In the galvanostatic generation addition of one of the said sterically hindered bases is also required.

Both polycarbazoles and polythiophenes as well as polypyrroles have conjugated n-electron pairs and conduct the electric current. All compounds consist of polymeric, aromatic ring systems and can interact with cyclic compounds in general and especially with those of said neuronal messengers and with ascorbic acid. This means that they hold them in the surface area of the working electrode 60 until an electrochemical event occurs. This is usually an oxidation of the neurotransmitters at the working electrode 60 leads to an electrical signal. But it can also take a reduction of the already oxidized hormonal messengers, if the working electrode 60 is controlled cathodically. However, oxidation and reduction often take place rapidly, since cyclic voltammetry (ZV) or fast cyclic voltammetry (SZV) is used as the measuring method in order to avoid depletion of the molecules or particle families to be determined in the medium.

In a cyclic voltammetric measurement, one attaches to the working electrode 60 a negative voltage and gradually increases it to a maximum positive value. From there one returns just as gradually back to the negative output value and detects for each applied voltage value one at the working electrode 60 flowing electricity. Whenever an oxidation or reduction event occurs, that current changes from a trace without such an event.

This fact is used to determine in a separate experiment that concentration range of an analyte in which the measured current changes are linear. For this purpose, determined peak current values (y-axis) of test samples of known concentration are used against amounts of neurotransmitter used, and a calibration range is obtained for an excellent range. Within this linear concentration range, unknown quantities of corresponding analyte can be mixed with the microsensor in a sample solution 1 reliably determined by measuring the peak currents. Thus, within this detection range, changes in the concentration of neurotransmitters in vivo can be reliably detected.

The electrode fibers 14 the different electrodes 40 . 50 . 60 and the one enveloping them as a carrier 11 serving quartz glass jacket give the microsensor 1 Stability. This is increased because the tetrode 15 According to the invention in a cannula open on both sides 16 glued, which are usually used adhesion promoter on epoxy or acrylic resin base. Said cannula 16 has the same length as the carrier 11 and has an outer diameter of 250 microns to 350 microns; if possible it is 305 μm. Its wall is between 1 .mu.m and 3 .mu.m, preferably 1.5 microns thick and consists of an electrically shielding acting glass jacket, which does not necessarily have to resort to high-melting silicate glasses or quartz. Should the cannula 16 act as a Faraday cage or electrically driven as a sheath electrode, it is made of stainless steel or another, oxidatively difficult to attack metal material of the same dimensions. Both cannula types 16 envelop the tetrode 15 in its longitudinal extent completely. Only at the contacting end 20 protrude exposed electrode fibers 14 out. This is the tetrode 15 before insertion into the cannula 16 Treated in a definable area with hydrofluoric acid and in this way their isolation coat 11 away.

The exposed electrode fibers 14 are on the microplate 30 attached, preferably soldered. Also contact wires 32 are held to it via solder joints and connect the microsensor 1 with an electrical source and / or an electronic measuring instrument. Uncovered ends of the electrode fibers 14 , Microplate 30 and ends of the contact wires 32 are embedded in an adhesive layer and thus form the contacting end 20 which is on the adjacent cannula 16 rests on it and enters into a firmly adhering adhesive bond. In another embodiment, the contacting end 20 not sticking to the cannula 16 put on, but so sunk into it and glued, that only the contact wires 32 protrude. For precise and stable fixation, one also uses the already mentioned synthetic resin adhesive or uses another, fast-curing polymer, which may also consist of two components.

The microsensor 1 can be made even more robust by the cannula 16 including tetrode 15 and contacting end 20 in a capillary open at both ends 18 pushed in and fixed there with said adhesives shift resistant. In a compact design, only the contact wires protrude 32 from the capillary 18 out. Is this 18 for the microplate 30 However, too narrow-walled or should the board 30 can be connected with additional lines, the not yet enclosed in adhesive resin contacting end 20 also on the capillary 18 rest. It is only after completion of interconnection and soldering over the entire surface covered with adhesive and thereby end to the cannula 16 and on the capillary 18 established. The latter consists of glass, which does not necessarily have to resort to high-melting compounds. Also, flexurally elastic, inert plastics such as polytetrafluoroethylene (PTFE) are well suited as a protective capillary sheath.

The microplate 30 receives at the sensor head 12 resulting signals and transmitted loss-neutral to a meter. It also records electrical events from a source and also passes them to the sensor head without loss 12 , It consists of a copper-coated plastic sheet, which has a tapered shape. For example, it has a surface of 10 to 40 mm ² , if possible 25 mm ² , with edge lengths of 2 to 15 mm are common and those of 4 to 8 mm are preferred. Should the microplate 30 inside the cannula 16 or the capillary 18 their surface is much smaller and is only 0.5 mm ² or below. The on the board 30 attached, strip-shaped contact wires 32 have a diameter of 5 microns to 25 microns, ideally 15 microns and are made of a precious metal, preferably silver is used. They are isolated from each other by a surrounding silicone tube. This is broken when two or more of the usual four existing contact wires 32 be electrically interconnected by connection, as it happens, for example, to that of the counter electrode 40 to be swept to be swept reference surface.

To create the microsensor according to the invention 1 At least two metal wires of platinum or a platinum-metal alloy are placed in a quartz tube and this deformed under the Taylor method at elevated temperature under tension. The resulting multi-fiber microelectrode 10 is an insoluble composite of quartz carrier 11 and resulting electrode fibers 14 , He is polished smooth at its pointed end, so that the sensor head 12 arises. This one is for the tetrode 15 in 2 and 3 displayed. Its round or oval surface is perpendicular to the microsensor longitudinal axis and has the same diameter as the multifiber microelectrode 10 , The at least partially conical shape of the microsensor 1 comes in this embodiment either by a correspondingly deminsionierte cannula 16 or capillary 18 or it results from the combination of multifiber microelectrode 10 and microplate 30 ,

In a non-illustrated embodiment to polish the pointed end of the quartz carrier 11 such that the diameter of the resulting sensor head 12 smaller than the diameter of the multifiber microelectrode 10 is. Accordingly, this tapers conically to a blunt conical point. The punctiform regions of the electrode fibers 14 protrude at the sensor head 12 from the multifiber microelectrode 10 out. However, one can only polish the pointed end so far polishing that some electrode fibers 14 on the round surface of the sensor head 12 and further on a by the remaining truncated cone formed lateral surface of the multi-fiber electrode 10 emerge. This is the punctiform area of the centric electrode fiber 14 such a polished tetrode 15 on the flat surface of the sensor head 12 and those regions of the concentric electrode fibers 14 on the lateral surface of the tetrode 15 ,

In yet another in 1b drawn design polishes the multi-fiber microelectrode 10 at its Meßseitigen end to a uniform cone. Thus, a cone-shaped tip, the sensor head 12 forms and combines all punctiform areas on the conical surface.

The sensor head 12 opposite ends are treated to a length of 2 mm to 5 cm, but in most cases on a 1 cm long piece of hydrofluoric acid, around the four electrode fibers 14 from her carrier 11 to free. The exposed fibers 14 be on a microplate 30 Fixed at excellent locations with a resin-based adhesive or a two-component adhesive. Then you attach the contact wires 32 with said adhesives at other designated points on the microplate 30 and covers all adhesive dots with a solder layer of tin. This creates a permanently conductive and firm connection between electrode fibers 14 micro board 30 and contact wires 32 , To isolate the latter against each other, each contact wire 32 slipped over a silicone tube, so that in each case only a terminal piece of contact wire 32 remains uninsulated. Shall have multiple electrode fibers 14 the same electrical or electrochemical function Take over, connecting the corresponding, shaped as a strip of contact wires 32 with a solder point or twisted together with a pair of pliers.

The connection of multifiber microelectrode 10 , Microplate 30 and different contact wires 32 represents a simple form of a microsensor 1 dar. It surrounds the micro-board 30 Full surface with one of the mentioned adhesives, which is also on adjacent parts of the contact wires 32 and the exposed electrode fibers 14 distributed. The contacting end thus formed 20 allowed to fully cure, coated the adjacent multi-fiber electrode 10 with glue and glue everything together on or in the cannula 16 firmly. The multifiber microelectrode 10 completely fills the cannula interior, while the contacting end 20 either end to the cannula 16 sticks or is stuck inside. Sometimes it is also appropriate, not yet cured Kontaktierungsende 20 complete with adhesive provided multifiber microelectrode 10 immediately on or in the cannula 16 to place where it is fixed in place after drying.

To the construction of the microsensor 1 To make it even more solid, one brushes the with micro-sensor glued 1 provided cannula 16 with one of the named adhesives and attaches them in the capillary 18 , This is or is pulled out at one end inversely conical. In the so spanned sufficiently large funnel-shaped cavity can be the micro-board 30 including contact wires 32 insert and fix for example by pouring epoxy resin.

In a not shown embodiment of the microsensor 1 becomes the multifiber electrode 10 including contacting end 20 only in the cannula 16 fixed against displacement and is in the serving as a guide capillary 18 mounted longitudinally displaceable. In yet another variant, also not shown, but very space-saving variant is the multi-fiber microelectrode 10 even inside the cannula 16 freely movable and is on the cannula side by the contacting end 20 and on the other side by an annular thickening from falling out of the cannula 16 prevented.

One of the electrode fibers 14 must as a reference electrode 50 be executed so that stable potentials are obtained in a measurement process. Therefore, at least one of the fibers 14 initially electrochemically coated with silver and the surface of the silver layer 70 then chemically converted to silver chloride.

The silver coating is dipped in the multifiber microelectrode 10 in an aqueous solution of 1 mmol / l to 100 mmol / l silver nitrate (AgNO ₃ ) containing a three-fold molar excess of ammonia (NH ₃ ). At the punctiform area of the fiber 14 now is deposited at a current density between 0.2 and 10 mA / cm ² galvanostatically reductively from silver. For this the fiber becomes 14 connected to a working electrode terminal of the potentiostat. Furthermore, the coating arrangement consists of an external reference electrode, namely a pure silver wire and an external counter electrode in the form of a platinum sheet. The galvanostatic coating process takes between 100 and 500 seconds, depending on the thickness of the applied silver layer, which is on average 3 microns thick.

The microsensor 1 is then removed from the working electrode port and placed in a 1 to 5 molar nitric acid solution. This contains between 0.1 and 4 mol / l potassium chloride (KCl). Depending on the selected concentration and number of punctiform areas to be coated, it is necessary to wait for 1 to 10 minutes until the oxidizing nitric acid (HNO ₃ ) forms an approximately 1.5 μm thick silver chloride layer 70 formed on each silvered dot-shaped area.

The Ag / AgCl electrode thus obtained has a time-stable potential of -38 mV to -42 mV compared to a saturated calomel electrode (SCE). This gives you an Ag / AgCl reference electrode 50 created in the multifiber microelectrode 10 is integrated. With measurements under constant chloride concentration, for example in the human organism or in cell cultures, one obtains with this electrode 50 a stable reference electrode of the second kind.

To a working electrode 60 at least one of the punctiform regions of the electrode fiber (s) must form 14 at the sensor head 12 be particle selectively coated. For an electrically conductive polycarbazole layer 80 becomes the multifiber microelectrode 10 with its reference electrode 50 transferred under argon into an electrochemical cell. This contains distilled and dried acetonitrile as solvent. Therein one of the abovementioned conductive salts is dissolved at a concentration of 0.1 mol / l. Furthermore, the solution contains as the monomer carbazole in a concentration of 1 mmol / l to 10 mmol / l and an equimolar addition of one of the abovementioned nitrogenous bases. All fibers to be coated 14 are connected to the high sensitivity potentiostat via the working electrode terminal. The reference electrode used in the multi-fiber microelectrode 10 integrated Ag / AgCl reference electrode 50 and as a counter electrode contacted a platinum wire with the potentiostat. With this three-electrode arrangement is now at the point-like area of each electrically connected working electrode 60 cyclic voltammetric a firmly adhering polycarbazole layer 80 generated. This is done in 4 to 20 cycles and preferably 6 to 10 cycles one opposite to the reference electrode 50 measured potential range between 0 V and 1.5 V with a constant potential change rate. This is in a range from 2 mV / s to 100 mV / s and preferably at 20 to 60 mV / s.

For quality control, the resulting polymer is at least cyclic voltammetric in a monomer-free cell - which contains only solvent and conductive salt - characterized by its charge and discharge curve with three cycles. At anodic potentials, a p-doping occurs and anions of the conducting salt migrate into the polymer layer 80 , At cathodic potentials, lead salt cations migrate into the n-doped specific layer 80 , By applying these different potentials, a maximum possible potential window is determined in which the electroactive specific layer 80 reversibly loading and unloading without being destroyed. The finished microsensor 1 is then removed from the cell, rinsed with solvent and dried in air. It takes the specific layer 80 their laden condition.

Should a working electrode 60 with a polythiophene layer 80 are coated, a sterically hindered base is not needed. Otherwise, as with the coating with polycarbazole to proceed, using as the monomer, a thiophene or one of its higher homologues with two to five thiophene units. Also provided with functional groups or substituents monomeric derivatives and their di- or trimers can be used. The doping is carried out according to the procedure described for polycarbazole.

The detection of aromatic neuronal messengers such as dopamine and serotonin can be performed on a microsensor 1 on at least one electrode fiber 14 instead of or together with at least one of said polymer layers 80 a polypyrrole layer 80 be deposited. To this end, pyrrole is dissolved instead of or together with carbazole and / or another monomer in an acetonitrile solution containing one of the abovementioned conductive salts and one of the sterically hindered nitrogen bases mentioned. The concentrations to be maintained are identical to those of the carbazole solution described above. microsensor 1 and potentiostat are connected to electrodes in the manner already described. However, a coating with polypyrrole takes place galvanostatically, with at least one anodically contacted electrode fiber 14 a constant current between 50 nA and 200 nA is applied for a time of 30 s to 2 min. The polymer layer thus obtained 80 must then be oxidized anodically again at a constant potential.

In addition to these electrochemical deposition process, a specific layer can be 80 an electroactive polymer on a point area of an electrode fiber 14 also deposited chemically or physicochemically. Suitable methods for this are evaporation and condensation or sublimation under reduced pressure or pulsed laser ablation.

With a microsensor 1 which, if possible, is cannulated or capillary sheathed for better shielding of interfering signals or for protection under mechanical stress, neurotransmitters can be qualitatively and quantitatively determined in solution and in vivo.

For a normal tetrode measurement operation, there is its counter electrode 40 from two connected, unoccupied electrode fibers 14 , its reference electrode 50 from a single fiber modified in the manner described 14 and its polymer aromatic occupied working electrode 60 is also single-lipped.

First, draw a calibration line for each neurotransmitter to be determined. To do this, immerse the microsensor connected to a potentiostat 1 in phosphate buffered saline (PBS), which has a pH of 7.4 and contains 0.9% w / v sodium chloride (NaCl). In addition, in this physiological solution a specifiable small amount of the neuronal messenger to be examined, for example dopamine or serotonin. A ZV cycle of about -300 mV to +700 mV is carried out with a constant scan rate of between 0.2 V / s and 10 V / s and then increases the dopamine concentration in definable steps. After each concentration change, a cyclic voltammogram is recorded and the measured curves obtained in this way are superimposed graphically. 4 and 5 show with a polythiophene microsensor 1 obtained results for dopamine at a scan rate of 1 V / s.

Dopamine and serotonin are oxidized or reduced at specific potentials or potential levels specific to the particular neurotransmitter. So take out 5 in that signals for the reduction of dopamine at -20 mV are particularly pronounced. Applying the currents measured at this point to the amount of dopamine used gives the in 6 calibration line shown. This can be used in a concentration range of 20 .mu.mol / l to 700 .mu.mol / l for the quantitative determination of an unknown amount of neurotransmitters in a liquid medium.

As it turned out 7 the majority of serotonin is oxidized at +450 mV, but with for this compound in the presence of dopamine, a cathodic reduction signal is not observed. With the microsensor according to the invention 1 Accordingly, at least the two neurotransmitters dopamine and serotonin can be quantified side by side, since their specific signals of electrochemical activity, ie their potential layers, are spaced apart from one another and can therefore be evaluated independently of one another. If dopamine and serotonin coexist in a sample to be examined, the first-mentioned neurotransmitter can be detected down to a concentration of 20 .mu.mol / l and for the second-mentioned compound, the detection limit is 10 .mu.mol / l.

There are dopamine, serotonin and norepinephrine (their structural formulas in 8th are shown) in equimolar amounts of 71 .mu.mol / l in a measurement solution, so only the typical for dopamine and serotonin zyklovoltammetrischen curves can be obtained. Thus, norepinephrine does not influence the redox behavior of the other two neurotransmitters under these concentration and measurement conditions.

With the microsensor 1 one also determines kinetics of neurotransmitters. For this one pursues at a constant potential, in which selectively only a certain active substance is converted at the sensor surface, the temporally changing current. This gives a picture of the temporal change in the concentration of a neurotransmitter.

With the microsensor according to the invention 1 is an in vivo determination of neuronal messengers in extracorporeal cell and tissue cultures but also in the brain possible. In particular, serotonin and dopamine can be detected qualitatively and quantitatively in real time. It is also possible to detect their changes in quantity due to neurophysiological processes or medications in a certain intercellular space region, which is very important for a deeper understanding of neuropathological diseases, such as senile dementia and Parkinson's disease.

With the microsensor 1 Moreover, it is possible to study neurochemical processes in the cell interior and in cell interstices, for example on synaptically or electro-synaptically connecting nerve fibers outside and within the organism, and to influence neuronal events electrically.

In examinations of the brain, a hole approximately 1 cm in diameter is drilled into a lifeless or locally anesthetized skull and positioned in a stereotactic frame. This frame has a semi-arcing ring with fixed instruments for interventions inside the brain, such as a 64-channel multi-electrode delivery system. Through the opened skullcap and the meninges (dura) is a stylet (= serving as a guide lining with inwardly guided mandrel) inserted into the brain interior, by gently displacing cell tissue and blood vessels until a target region, such as the thalamus is reached. Now the mandrel is pulled out of the guide of the stylet and the microsensor according to the invention 1 introduced.

In order to determine the nature and amount of at least one neuronal messenger in cultured cell cultures as well as living or inanimate organisms, one moves the microsensor connected to a high sensitivity potentiostat 1 up to about two micrometers or less of a cell tissue to be examined. There it covers an area of about 7 μm ² to 520 μm ² . Between one and five of the already described current / voltage cycles (ZV cycles) are run through and the microsensor 1 then gradually shifted. For each surface area, current and voltage values are taken from the associated cyclic voltammogram (ZV) in the manner already described and compared with the recorded calibration lines in order to qualitatively and quantitatively determine at least one neurotransmitter.

The The invention is not limited to one of the above-described embodiments limited, but in more diverse Way changeable.

Electrochemical coatings of the electrode fibers 14 With conductive polymers, in addition to acetonitrile, it is also possible to carry out other oxidation-resistant, aprotic solvents, such as dimethylsulfoxide (DMSO), dimethyletheramide (DMF), dioxane, tetrahydrofuran (THF), alicyclic or aliphatic ethers and very oxidation-resistant, cyclic ketones. In this case, polymer aromatic conductors can be prepared not only by anodic oxidation of aromatic starting compounds but also by their cathodic reduction. By starting compound are meant all heterocyclic aromatic monomers.

In addition to aromatic, the oxidation accessible neurotransmitters and ascorbic acid (vitamin C) can also be nitric oxide (NO) with the microsensor 1 determine.

It can be seen that a microsensor ( 1 ) has an at least partially tapered shape. It consists at least of a 20 μm to 200 μm thick carrier ( 11 ), in the two or more electrode fibers ( 14 ) are embedded in a form-fitting manner. They are made of a metal or a metal alloy, have diameters of 4 microns to 18 microns and span the carrier ( 11 ) in its longitudinal direction stretching completely. At the measuring side they protrude out of it and form punctiform areas. Some of them are with a layer ( 70 ) and / or with a specific layer ( 80 ), which consists of an electroactive polymer, for example polycarbazole, polythiophene or polypyrrole. Point-shaped areas and the measuring end of the support ( 11 ) form the sensor head ( 12 ), which is planar or tapered flat. Against destruction, the microsensor ( 1 ) through a cannula ( 16 ) or a capillary ( 18 ) protect. The layers) ( 70 ) and the specific layers () 80 ) of at least one multifiber microelectrode ( 10 ) existing microsensor ( 1 ) are produced electrochemically, inter alia by means of cyclic voltammetry from electrically conductive solutions of monomers or salts and give the microsensor ( 1 ) particle-selective properties. These are used to target substances, such as neurotransmitters and their concentrations in solution or in tissue compartments.

All from the claims, Description and drawing of the features and advantages, including constructive details, spatial Arrangements and method steps, both individually also be essential to the invention in the most diverse combinations.

1

microsensor

10

Multi-fiber microelectrode

11

carrier

12

sensor head

14

Electrode filament (s)

15

tetrode

16

cannula

18

capillary

20

contacting end

30

micro board

32

contact wires

40

counter electrode

50

reference electrode

60

working electrode

70

layer

80

specific layer

Claims (40)

Microsensor for the qualitative and quantitative determination of at least one ingredient in a liquid medium consisting of a multifiber microelectrode ( 10 ) with at least two electrode fibers ( 14 ), of which at least one as a counter electrode ( 40 ) and / or as a reference electrode ( 50 ) and with a support ( 11 ), which contains all the electrode fibers ( 14 ) at the same time enclosing one-piece, compared to the liquid medium indifferenter electrical insulator, in which the electrode fibers ( 14 ) are isolated from each other, each of them connected to the carrier ( 11 ) is materially directly connected, characterized in that the microsensor ( 1 ) has an at least partially conical shape and at least one of the fibers ( 14 ) a working electrode ( 60 ) which on the measurement side has a specific layer ( 80 ) is provided from a biocompatible, electroactive polymer. Microsensor according to claim 1, characterized in that the multifiber microelectrode ( 10 ) with a microplate ( 30 ) is connected to the contact wires ( 32 ) are soldered from a precious metal. Microsensor according to one of Claims 1 or 2, characterized in that the support ( 11 ) is a round or oval quartz glass jacket. Microsensor according to one of Claims 1 to 3, characterized in that the electrode fibers ( 14 ) are made of a noble metal, such as platinum and / or a platinum / tungsten alloy and have a diameter of 4 to 18 microns and in a preferred embodiment of 10 to 12 microns. Microsensor according to one of Claims 1 to 4, characterized in that at least one electrode fiber ( 14 ) with a layer ( 70 ) and that different electrode fibers ( 14 ) with different layers ( 70 ) and / or with different specific layers ( 80 ) are provided. Microsensor according to claim 5, characterized in that the layer ( 70 ) and / or the specific layer ( 80 ) only on a part of the electrode fiber ( 14 ), which is not attached to the carrier ( 11 ) adjoins. Microsensor according to one of Claims 2 to 6, characterized in that the electrode fibers ( 14 ) on one side, which in the direction of a contacting end ( 20 ), from the surrounding carrier ( 11 ) and to the microplate ( 30 ) are soldered. Microsensor according to claim 7, characterized in that the exposed side of the electrode fibers ( 14 ), the microplate ( 30 ) and ends of the contact wires ( 32 ) are embedded in an adhesive matrix and together the contacting end ( 20 ) form. Microsensor according to one of Claims 1 to 8, characterized in that it comprises n electrode fibers ( 14 ), where n is an integer value of 2 to 50, preferably 3 to 13 and be preferably from 4 to 6 and the individual fibers ( 14 ) are arranged, for example, equidistant from each other. Microsensor according to one of Claims 1 to 9, characterized in that a centrally arranged electrode fiber ( 14 ) of n electrode fibers ( 14 ) is preferably concentrically surrounded, wherein n assumes an integer value of 1 to 49, preferably from 2 to 12 and most preferably from 3 to 6. Microsensor according to one of Claims 1 to 10, characterized in that the electrode fibers ( 14 ) are connected so that they form independently electrically responsive micro-disc electrodes with electrical shielding. Microsensor according to one of Claims 1 to 11, characterized in that the multifiber microelectrode ( 10 ) has a diameter of less than or equal to 250 microns. Microsensor according to one of Claims 7 to 12, characterized in that the multifiber microelectrode ( 10 ) at one end, the end of contacting ( 20 ), a sensor head ( 12 ), which has a circular or oval, or at least partially conically tapered, smooth polished surface. Microsensor according to one of Claims 1 to 13, characterized in that the multifiber microelectrode ( 10 ) a tetrode ( 15 ), in which three electrode fibers ( 14 ) a fourth, centrally in the carrier ( 11 ) arranged electrode fiber ( 14 ) surrounded concentrically. Microsensor according to Claim 14, characterized in that the tetrode ( 15 ) is a circular or oval fiber with a diameter of 50 to 110 microns, in a preferred embodiment of 70 to 90 microns and thereby has a length of 5 to 15 cm, and preferably from 10 to 12 cm. Microsensor according to one of Claims 1 to 15, characterized in that the multifiber microelectrode ( 10 ) into a cannula ( 16 ) is glued, for example by means of an epoxy or acrylic resin-based adhesive. Microsensor according to claim 16, characterized in that the cannula ( 16 ) consists of a glass material or of stainless steel and has an outer diameter of 250 .mu.m to 350 .mu.m and preferably from 280 .mu.m to 305 .mu.m. Microsensor according to one of Claims 16 or 17, characterized in that the multifiber microelectrode ( 10 ) and the cannula ( 16 ) at the contacting end ( 20 ) are fixed, for example by an adhesive bond. Microsensor according to one of Claims 16 to 18, characterized in that the cannula ( 16 ) in a capillary ( 18 ), for example, with acrylic or epoxy resin is glued in her. Microsensor according to claim 19, characterized in that the capillary ( 18 ) consists of glass and / or of a flexible plastic. Microsensor according to one of Claims 2 to 20, characterized in that two of the electrode fibers ( 14 ) via a contact wire ( 32 ) as counterelectrode ( 40 ) are interconnected. Microsensor according to one of Claims 1 to 21, characterized in that a centric and a concentrically arranged electrode fiber ( 14 ) interconnected the counter electrode ( 40 ) form. Microsensor according to one of Claims 5 to 22, characterized in that the layer ( 70 ) on the reference electrode ( 50 ) is present and consists of deposited silver, which is chemically converted to silver chloride on its surface. Microsensor according to one of Claims 1 to 23, characterized in that the specific layer ( 80 ) is selective for at least one ingredient and consists of an electrically conductive polymer. Microsensor according to Claim 24, characterized that this conductive Polymer conjugated to each other, heterocyclic Aromatics exists. Microsensor according to claim 24 or 25, characterized that this conductive Polymer is a polycarbazole and / or a polythiophene. Microsensor according to claim 24 or 25, characterized that this conductive Polymer is a polypyrrole. Microsensor according to one of Claims 1 to 27, characterized in that the layer ( 80 ) is dopable or doped, for example, with compounds which have a steric and / or charge-altering influence on them. Microsensor according to one of Claims 1 to 28, characterized in that the layer ( 80 ) is doped with special ions, for example with metal or halide ions or with ions of quaternary ammonium salts, boron halides, phosphorohalo or perchlorates. Microsensor according to one of Claims 1 to 29, characterized in that the working electrode ( 60 ) in uncoated form has a roughened, for example by means of sulfuric acid etched surface. Method for producing a microsensor ( 1 ) for the determination of at least one ingredient in a liquid medium, in particular according to one of claims 1 to 30, characterized in that a. that at least two noble metal wires placed in a quartz tube and this is deformed in the heat under tension according to Taylor, b. that the deformed quartz tube is removed on one side, so that the metal wires are used as electrode fibers ( 14 ), c. that one of the exposed electrode fibers ( 14 ) opposite measuring area of the multi-fiber microelectrode ( 10 ) to a sensor head ( 12 ) smooth surface is polished, d. and that at least one of the electrode fibers ( 14 ) on the sensor head ( 12 ) with an electroactive layer ( 70 ) and / or with an electroactive specific layer ( 80 ). Method according to claim 31 or 32, characterized in that the sensor head ( 12 ) is polished so that it has a conical shape or forms a partial cone without apex. Process according to claim 31, characterized in that as electroactive layer ( 80 ) a conductive polymer is deposited electrochemically or chemically. Method according to one of Claims 31 to 33, characterized in that the multifiber microelectrode ( 10 ) on the exposed electrode fibers ( 14 ) with a microplate ( 30 ) is connected. Method for determining at least one ingredient in a liquid medium with a microsensor ( 1 ) with sensor head ( 12 ), in particular according to one of claims 1 to 34, characterized in that a. that the microsensor ( 1 ) is connected to a high sensitive potentiostat, b. that he uses the sensor head ( 12 ) is introduced into the liquid medium, for example within a tissue compartment, and c. that current-voltage curves are recorded by means of cyclic voltammetry. Process according to claim 35, characterized in that that he from at least one of the current-voltage curves for at least a measuring peak an associated one Read voltage value and this one basis by comparison values Ingredient assigns. Method according to one of claims 35 or 36, characterized that he from at least one of the current-voltage curves for a measuring peak one associated Read current value and this on the basis of an ingredient characteristic Current / quantity calibration line allocates a corresponding amount. Method for the analytical determination of at least one ingredient present in an organism, for example within the brain, using a microsensor ( 1 ), in particular according to one of claims 1 to 37, characterized in that a. that a circular hole is drilled in a skull, through which a mandrin consisting of food and thorn is introduced into a target region of the brain, b. that the mandrel is pulled out and the microsensor according to the invention ( 1 ) and brought to the target region, c. that current-voltage curves are recorded by means of cyclic voltammetry, d. that one superimposes the obtained curves on a screen or on a measuring device, reads voltage values and identifies an ingredient by assignment to calibration values e. and reading current values which are subtracted from a calibration line specific to the ingredient thereby quantitatively determining the ingredient. Use of the microsensor ( 1 ) in particular according to one of claims 1 to 38 for the qualitative and quantitative detection of neurotransmitters and / or antioxidants in solutions and on or in biological material. Use of the microsensor ( 1 ), in particular according to one of claims 1 to 39, for shifting equilibria between oxidized and reduced forms of a neurotransmitter or a hormone within cell tissue assemblies and intercellular spaces.