WO2005059534A1

WO2005059534A1 - Refillable microprobe

Info

Publication number: WO2005059534A1
Application number: PCT/DE2004/002722
Authority: WO
Inventors: Stefan Martin Hanstein
Original assignee: Justus-Liebig-Universität Giessen
Priority date: 2003-12-19
Filing date: 2004-12-13
Publication date: 2005-06-30
Also published as: DE10360714A1

Abstract

The invention relates to microprobes for enzymatically measuring gas concentrations, whose reservoir for the enzyme solution is configured as a flow chamber. The microprobes are provided with a seal that is highly permeable for the analyte and electrically insulating. The inventive microprobes are especially suitable for phytophysiological measurements and can for example be used for the high-resolution measurement of gases on individual stomata of plant leaves. Since the reservoir for the enzyme solution is configured as a flow chamber, the inventive microprobe can be used several times.

Description

Patent application

[Refillable micro probe] The present invention relates to a micro probe for the enzymatic measurement of gases in biological samples, which allows the enzyme solution to be removed, refilled and / or exchanged.

[Description and Introduction of the General Field of the Invention]

The qualitative and quantitative detection of gases as well as gases and ions dissolved in liquids play an important role in science and technology. The currently implemented probes and sensors allow the determination of gases, ions or ions formed from gaseous substances, for example in combustion plants, in the control of exhaust gases and in numerous biological systems. Various measurement methods are used for substance determination, the use of a specific analysis method depending on the character and expected concentration of the substance to be determined and the place of use or measurement (macro or micro scale).

Suitable for detection are those physical and / or chemical properties of the analyte which allow clear conclusions to be drawn about its nature and which change in proportion to its concentration. The measuring methods used include potentiometric and amperometric methods as well as measurements of conductivity, temperature, pressure or partial pressure, resonance frequency and magnetic susceptibility.

TM007 bility. Depending on the measuring arrangement and the nature of the analyte and the measuring device, the change in the properties of the analyte (primary substance) can be determined directly, or the analyte is converted into a secondary substance, which is then measured. In the latter case, the primary and secondary substance must have a defined mathematical relationship to each other.

In order to be able to determine analytes also in substance mixtures, measuring devices are often used, in which the actual measuring range is separated from the mixture to be examined by a semipermeable membrane. Ideally, only one or a few of the substances to be analyzed can pass through this membrane. This membrane can consist, for example, of glass, plastics / polymers or metallic compounds. Silicone membranes have long been used in measuring probes for carbon dioxide and oxygen. When used in conductive media, the high electrical resistance of silicones ensures that the electrical potential of the measurement solution does not affect the sensor circuit.

The present invention relates to a microprobe, with the aid of which substances in biological samples can be analyzed enzymatically on a microscale and which allows the enzyme solution to be removed, refilled and / or replaced. The microsensor according to the invention thus represents a further development of the microsensor described in S. Hanstein, D. de Beer and H. Felle (Sensors and Actuators 2001, B81, 107-114). The microsensor according to the invention also contains the silicone seal described in DE 102 39 264.1 , which is permeable and at the same time electrically insulating for the analyte to be measured. This micro-probe already described is very small, highly sensitive and selective, so that it does not impair or change the metabolism of biological samples. The micro-probe is suitable for cell biological measurements, eg for measuring the concentration of C0 ₂ and 0 ₂ as control variables of the cellular energy

TM007 alternate and the cellular mass absorption or for measuring the formation of C0 ₂ and NH ₃ in foci of infection on host cells or on microbial pathogens. By doping the electrolyte behind the silicone seal with a suitable enzyme, it is possible to selectively convert a certain primary analyte from a biological sample into a secondary analyte.

The present invention relates to a novel method for producing such a microprobe, which makes it possible to install a device for exchanging and / or refilling the enzyme electrolyte solution.

State of the art

Various electrodes and sensors are currently known with which gaseous substances or gases and ions dissolved in liquid can be determined. Various chemical and physical parameters are used to identify the analytes to be determined, if necessary also in mixtures of substances. Many measuring methods use different electrodes made of precious metals and their salts. Depending on the design of the electrode, the salt can be in dissolved or solid form. The analyte can be detected, for example, by amperometric or potentiometric measurement. Disadvantages for the measurement of very small or rapidly changing analyte concentrations are the mostly too high detection limit of the sensor, an insufficient selectivity for a certain analyte in a mixture and the time required until the measurement signal is received. All measuring arrangements have in common that there is a semipermeable membrane between the measuring medium and the detection system, which is only permeable to the analyte or the mixture of the substances to be analyzed, in order to increase the selectivity of the measurement.

TM007 DE 199 29 264 AI describes a transducer designed as a flow measuring cell for determining substance concentrations or substance activities in fluids. The transducer contains a cavity which serves for the flow of the analytes and which can contain fillings made of membrane and / or gels which recognize the substance. These membranes and / or gels can additionally contain biocomponents such as enzymes, antibodies or microorganisms. It is not possible to replace or replace these biocomponents. The transducer is not suitable for the determination of dry, gaseous analytes.

DE 694 02 705 T2 describes a sensor with which oxygen and carbon dioxide in body fluids can be determined. The sensor contains a PTFE membrane for the passage of the analytes, a silver / silver chloride electrode and aprotic solvents, to which no or only a little aqueous medium is added. The analytes are reduced electrochemically and measured amperometrically.

DE 196 02 861 C2 describes an oxygen sensor which consists of a dialysis membrane, a silver-silver chloride anode and a cathode made of silver or platinum. The membrane is made from a gel that contains both a salt and an enzyme. This sensor is designed as a flow-through device and can therefore only be used for analytes dissolved in carrier liquids, but not for dry gases. In addition, the enzyme contained in the membrane cannot be exchanged or refilled.

A micro-probe containing bacteria for the determination of nitrate is explained in WO 99/45376. However, the bacteria used for denitrification cannot be replaced or regenerated. The electrode described in DE 695 14 427 T2 for the enzymatic determination of glucose, acid

TM007 Substance and lactate in body fluids do not allow the enzymes used to be exchanged or replaced. DE 38 13 709 AI describes an electrode for the determination of glucose in body fluids and contains a polymer membrane containing catalysis. With this electrode, used enzyme can only be replaced by replacing the complete, enzyme-containing polymer membrane.

However, all the electrodes and sensors mentioned are not suitable for measuring physiologically relevant concentrations in the smallest volumes of biological samples.

The measuring principle of many sensors is based on the stoichiometric conversion of a primary analyte into a secondary analyte. For this purpose, biosensors often contain enzymes that convert a specific primary analyte specifically and quantitatively into a secondary analyte. In many cases, the measuring arrangement contains a circuit which reacts to the concentration or activity of the secondary analyte and delivers a measurement signal which is dependent on its concentration. In the case of potentiometric probes, which must detect measurement signals of the order of 50 μV due to the required sensitivity, the circuit of the sensor must be electrically isolated from the sample so that the electrical potential of the sample liquid does not falsify the measurement result. It is also advantageous to use suitable membranes to prevent dilution of the secondary analyte by diffusing out of the sensor. Silicones can be chemically designed to combine electrically insulating and (gas) permeable properties. The silicone seal for micro probes described in DE 102 39 264.1 fulfills the condition of combining electrically insulating and (gas) permeable properties in the smallest space, so that a micro probe which contains this seal enables the determination of very low gas concentrations in very small

Volumes allowed. The in S. Hanstein, D. de Beer and H.

Felle (Sensors and Actuators 2001, B81, 107-114) described TM007 tuators 2001, B81, 107-114) and the probe described in DE 102 39 264.1 with a silicone seal use an enzyme solution to convert the gaseous primary analyte quantitatively and stoichiometrically into the ionic secondary analyte to be measured. However, these probe designs do not allow the enzyme to be replaced and / or replenished, so that the usefulness of the probes for the determination of gases is limited by the shelf life of the enzyme.

task

It is an object of the present invention to provide a microsensor for the enzymatic measurement of gases, which allows low gas concentrations to be determined in the smallest volumes and the enzyme solution to be removed, refilled and / or exchanged.

Solution of the task

This object is achieved according to the invention by micro-probes which contain a miniaturized probe tip, an electrically insulating seal which is highly permeable to the gas to be measured and a reservoir for liquids.

“Miniaturized” is understood to mean a probe tip whose outer diameter is less than or equal to 5 μm, the probe tip containing a silicone seal which has a high permeability for the analyte to be measured and has electrically insulating properties. Under “Reservoir for liquids” is understood to be a device which allows the enzyme solution to be removed, refilled and / or replaced. The reservoir is preferably designed in the form of a flow-through chamber which, for example, consists of a container into which an inlet and an outlet device each protrude. Inlet and outlet are particularly preferably combined by

TM007 the probe is provided with only one access through which a filler capillary is introduced for filling and / or refilling the enzyme solution.

The present invention represents a further development of the microsensor described in S. Hanstein, D. de Beer and H. Felle (Sensors and Actuators 2001, B81, 107-114) and the microsensor with the silicone seal described in DE 102 39 264.1, by allowing the present invention to exchange and / or replenish the enzyme solution. Due to the possibility of being able to exchange and / or refill the enzyme solution, the microprobe according to the invention can be used multiple times and has a longer service life than previously described microprobes, thereby achieving a great time and cost advantage.

The microprobe according to the invention is used in the enzymatic determination of substances on a microscale. The invention permits the construction of very small, highly sensitive, selective and reusable sensors which do not impair or change the metabolism of biological samples. It is suitable for micro probes that are used in cell biology, e.g. to measure the concentration of C0 ₂ and 0 ₂ as control variables for cellular energy metabolism and cellular uptake or to measure the formation of C0 ₂ and NH ₃ in infection foci on host cells or on microbial pathogens. The microprobe according to the invention contains a silicone seal that is highly permeable to the analyte and is described in DE 102 39 264.1. By doping the electrolyte behind the silicone seal with a suitable enzyme (cf. DE 102 39 264.1) it is possible to selectively convert a certain primary analyte from a biological sample into a secondary analyte. In combination with a suitable measuring electrode it is possible to measure the secondary analyte amperometrically or potentiometrically. When using the inventive The microsensor is also designed as a flow chamber of the enzyme electrolyte solution and can therefore be used multiple times and is therefore more cost-effective than the probes of this type described hitherto. Due to its construction, the microsensor according to the invention is particularly suitable for probes which can be used repeatedly and which, for example, use carbon dioxide behind individual stomata (stomata) of plant leaves as Control variable of opening and closing movements of the stomata can be measured.

The microprobes according to the invention are provided by the production of two coordinated glass micropipettes with miniaturized tips made of commercially available glass capillaries, the production of an electrically insulating silicone seal that is permeable to the analyte in a first micropipette, and the attachment of an inlet tube (first capillary tube) the outside of a second micropipette, inserting the second into the first micropipette, then inserting a second capillary tube (outlet tube) and a reference electrode into the cavity between the first and second micropipette, fixing the parts pushed into one another and attaching a conventional electrode holder.

The commercially available glass capillaries are drawn into micropipettes on a device for pulling out glass capillaries known to the person skilled in the art. Glass capillaries with an outer diameter of 2.6 to 3.4 mm and an inner diameter of 1.3 to 1.7 mm are used for the outer glass micropipette of the probe, which can optionally have an inner filament.

It is known to the person skilled in the art that he usually has to carry out two pull-out operations with these devices in order to obtain capillaries with a diameter of up to 2 mm for the production of micro-pipettes. Since capillaries with an outer diameter of

TM007 If it is often not possible to pull out more than 2 mm into a micropipette in two pull-out operations, reducing the capillary diameter to about 2 mm by an upstream pull-in operation is preferred. Through two further pull-out operations, the narrowed capillary section is pulled out into a micropipette with an outer diameter of the tip that can be selected between 2 and 5 μm. For measurements in plant leaves, the maximum possible diameter is determined by the respective opening width of the leaf pores (stomata). The smaller the outside diameter, the less the probe body interferes with the gas exchange through the sheet pore. On the other hand, the measuring sensitivity of the probe increases with the outside diameter. In practice, the outer diameter of the probe tip is selected so that it is about 50% of the expected stomatal opening width.

The micropipette 3 is then silanized internally with a solution of 0.01-0.05% tributylchlorosilane in chloroform by a process known to the person skilled in the art.

A silicone seal is then produced as described in principle in DE 102 39 264.1:

The manufacturing process depends on the tip dimensions of the probe. The following procedure is used for probes with a diameter smaller than 4 μm:

A non-crosslinking silicone oil 10 with a low viscosity is filled into the container capillary 9 (see FIG. 2) and this is mounted horizontally under the objective 6 of a microscope. The freshly silanized micropipette 3 is filled with sterile distilled water until the tip tapers. For this purpose, the tip is immersed in distilled water and filled with water to a length of about 1 mm using the vacuum. The optional addition of a nonionic surfactant, for example 4% by volume n-butanol, facilitates the subsequent suction of the silicone. In order to

TM007 gens to minimize the risk of blocking the tip due to contamination of the water, further water is added from behind with a filling capillary 13 until the tip is filled with water until the tapering begins. Optionally, 50 ppm (w / v) chloramphenicol can be added to the water supplied from the rear.

The glass micropipette 3 to be sealed, which is to be sealed, is introduced into the container capillary 9. The adapter head 16 is placed on the other end 8 of the first glass micropipette 3 (see FIG. 4), at the end of which there is a 50 ml syringe 21. The rubber seal 15 is fastened with the help of the sealing screw 14 at the end 8 of the glass micropipette 3. The adapter device on the metal tube 17 is then clamped in a micromanipulator. The metal tube 17 is connected via a plastic hose 18 and a three-way valve 19 to a 50 ml syringe 21 with a Luer lock connector 20. By closing the three-way valve 19 and the piston stroke of the syringe 21, a negative pressure is generated and silicone 10, which does not crosslink under microscopic control, is sucked into the glass micropipette 3. Since the interfacial tension between the silicone phase and the aqueous phase in the tip 3 has to be overcome, this happens suddenly. The necessary sharp phase boundary without bulges is only achieved if the aqueous phase is protein-free. Excess silicone is pressed out of the tip in two steps: First, the syringe plunger is lowered until the stopper is a maximum of five times as long as it should be in the final seal. Then glass micropipette 3, adapter and syringe are removed from the container capillary 9. The plug is shortened to the final length of the seal by lowering the syringe plunger, excess silicone 10 which does not crosslink run off. The crosslinking silicone oil 12 is applied to the holder 11 and brought into contact with the glass micropipette 3

TM007 is filled with the non-crosslinking silicone oil 10 (see Fig. 3). The holder 11 with the crosslinking silicone 12 is fed onto the tip of the glass micropipette 3, and the crosslinking silicone 12 acts on the non-crosslinking silicone oil for at least 5 seconds, preferably 45 seconds. The glass micropipette 3 is then pulled out of the crosslinking silicone oil 12. After renewing the crosslinking silicone oil on the holder 11, the process consisting of supplying the holder 11 with crosslinking silicone 12 to the tip of the glass micropipette 3, the action of the crosslinking agent on the non-crosslinking silicone oil and pulling the glass micropipette 3 out of the crosslinker Silicone oil, repeated once. The interruption of the action and the limitation to two applications of the crosslinking silicone oil 12 prevent this. Adhesion to the outside of the glass micropipette 3 or:. pulling out the silicone oil mixture from the tip of the glass micropipette 3 when removing the glass micropipette 3 from the crosslinking silicone oil. The glass micropipette 3 must not protrude more than 10 μm into the crosslinking silicone oil 12, since otherwise the probe diameter is increased by crosslinking silicone oil adhering to the outside. Due to the two-stage filling of the silicone oils (first the non-cross-linking, then the cross-linking silicone oil) the cross-linking only occurs when the silicone oils are in the correct position within the probe tip. The low viscosity of the non-crosslinking silicone oil and the stated silanization of the micropipette 3 are prerequisites for the positionability of the mixture of both silicone oils within the tip of the glass micropipette 3 with a diameter of less than 4 μm. The mixing of the two silicone oils on a micro scale is accomplished by the diffusion movement of the silicone molecules.

In contrast to DE 102 39 264.1, no enzyme-containing solution, but rather sterile distilled water 7 is injected from behind

TM007 filled this first glass micropipette 3. “Rear” is understood to mean that surface of the silicone seal which faces away from the smaller opening of the micropipette. The silicone seal cures at room temperature in about 2-6 hours or alternatively can also be carried out at 40-80 ° C. in the presence of moisture, which accelerates the hardening process by a few hours. Particularly preferred are hardening times of 4 hours at room temperature or 1 hour at about 60 ° C. in the presence of moisture.

The following method is used for probes with a probe tip diameter greater than or equal to 4 μm: the use of non-crosslinking silicone oil 10 can be dispensed with by dipping the dry, freshly silanized outer glass micropipette 3 into crosslinking silicone oil 12 as described above , The silicone seal then hardens as described above. After the silicone seal has hardened, sterile distilled water, which preferably contains 4% by volume of n-butanol, is filled into the glass micropipette 3 from behind. The glass micropipette is immediately 3, such as Parafilm ^® closed with extensible closure film and stored at 4-8 ° C, until the aqueous phase to 12-36 reaches the seal h.

A second glass capillary with an inner filament is drawn out into a micropipette in a one- or two-stage process, the tip dimensions of which allow it to be placed within the tip of the first micropipette (see below). A two-stage pull-out of the second glass capillary is preferred. The tip of the second pipette must be within the first micropipette up to the distances indicated below. The second micropipette is silanized by the process known to the person skilled in the art S. Hanstein, D. de Beer and H. Felle (Sensors and Actuators 2001, B81, 107-114). The filament fills the tip with a proton-sensitive hydrophobic cock known to those skilled in the art.

TM007 tail for the production of the pH-sensitive microelectrode described in S. Hanstein, D. de Beer and H. Felle (Sensors and Actuators 2001, B81, 107-114), which is understood to mean the end of the glass micropipette, which has the larger diameter. (Cocktail: see exemplary embodiment point 3, “Process for the production of the pH-sensitive microelectrode”). The silanization in the assembled probe prevents the penetration of aqueous solution and reference buffer (see below) into the glass tip. The glass micropipette 25 is then Filled with a solution of a proton-sensitive cocktail and PVC in THF as described in DE 102 39 264.1 Evaporation of the solvent THF forms a solid PVC gel. The solid PVC gel is first made with undiluted proton-sensitive cocktail and then overlaid with a reference buffer 24.

In the present invention, an inlet tube 2 for liquids is then attached to the outside of this second glass micropipette 25 and fixed with a fastening means 22. A commercially available cannula is suitable as an inlet tube. It is bent using tweezers as shown in Fig. 5. The second glass micropipette 25 with the fixed inlet tube 2 is inserted into the first glass micropipette 3, but not yet advanced to the silicone seal 23. Then a reference electrode and possibly an outlet tube 5 for liquids (see above) are pushed between the two glass micropipettes. Then the second, inner glass micropipette 25 is inserted so far into the first glass micropipette 3 that the distance from the tip of the second pipette to the tip of the first pipette is between 5 and 100 microns, about 5-25 microns with an outer diameter outer pipette 3 greater than or equal to 4 μm and 25-100 μm with an outer diameter below 4 μm. The inner micropipette 25 projects as in DE

TM007 102 39 264.1 describes at its end facing away from the silicone seal about 2.5 cm beyond the first, outer micropipette 3. The two micropipettes 3 and 25, the outlet tube 5 and the reference electrode 26 are fastened to one another with a fastening means 22. The cavity formed in this way between the outer (3) and inner (25) micropipette, into which inlet (2) and possibly outlet tubes (5) and the reference electrode 26 project, forms the probe chamber 28 for filling in solutions. It is advantageous to use special commercially available glass adhesives as fasteners, which harden within 12 hours using UV light.

The protruding end of the second glass micropipette 25 is connected to a conventional electrode holder 27 after the fastener has hardened. The second glass micropipette 25, the commercially available proton-sensitive cocktail and the reference buffer (electrolyte) 24, together with a working electrode to be installed, form the pH-sensitive microelectrode 1. A conventional electrode holder, in which an electrode is integrated, is advantageously used as the working electrode and which allows the pH-sensitive microelectrode to be connected to another electrode.

Before the microsensor is used, the probe chamber 28 is rinsed with fresh enzyme electrolyte 4. The access to the inlet tube and the entrances to the inlet and outlet tubes are protected against evaporation with a water-impermeable film known to the person skilled in the art (for example parafilm, see below). After an equilibration time of about 1 hour for wide peaks to 3 hours for narrow peaks at room temperature, the microsensor can be used.

TM007 Because of its lower melting point, borosilicate glass is more suitable than aluminum silicate gas as the starting material for the shaping of the glass micropipettes. Aluminum silicate and quartz glass are more chemically resistant, and their shape adaptation, due to their higher melting points, requires the particularly powerful glass drawing devices known to the person skilled in the art. The two glass micropipettes can consist of the same or different types of glass. A non-crosslinking silicone oil with trimethyl siloxy end groups with a viscosity between 0.02 and 0.2 Stokes, for example, can be used as the non-crosslinking silicone oil, preferably Dow Corning product (R) 200 fluid with a viscosity of 0.1 Stokes (25 ° C) and an activity of 100%.

The crosslinking silicone is preferably a mixture of dimethylsiloxane with terminal hydroxyl groups and trimethylsiloxane, for example Dow Corning Product (R) 1340 RTV Coating or alternatively mixtures of a silanol with a viscosity of 50-120 cSt, e.g. Dow Corning product DC 2-1273, or a silanol with a viscosity of 2,000 cSt, e.g. Dow Corning product DC 3-0133, each mixed with 5-10% by weight methyltrimethoxysiloxane. For example, a commercially available cannula with a length of 25-45 mm and a diameter of 0.3-0.6 mm can be used as the inlet tube.

Capillary tubes are used as outlet tubes, for example commercially available untreated or deactivated capillary columns for gas chromatography (“fused silica, Si0 ₂ ) with an inner diameter greater than or equal to 100 μm and 200-400 μm outer diameter. In the case of the probe type without a separate outlet tube (see above), a disposable glass capillary made of borosilicate glass is inserted through the cannula protruding into the probe to fill the probe chamber. The fine glass capillary with a corresponding outer diameter is

TM007 produced by a process known to the person skilled in the art on a single-stage drawing device.

Cyanoacrylate or special UV-curing glass adhesives are preferably used as fasteners. For fixing the inlet tube, a commercial Cyanacrylatklefoer or Scotch ^® Super Glue (Beiersdorf AG, Hamburg) is preferably used, for example UHU- superglue with a Vi skosität of 70 MPa (Buehl / Baden UHU GmbH & Co. KG,). To attach the two glass micropipettes to one another, for example, the UV glass adhesive (Greven adhesive, Heddesheim, Germany) or alternatively the methacrylate adhesive Acrifix 1 92 ^® (Röhm GmbH, Darmstadt) can be used.

In the ready-to-use micro probe, the enzyme electrolyte solution 4 serves to convert a primary analyte quantitatively and stoichiometrically into a secondary analyte, which is then measured. A particularly suitable enzyme is, for example, carbonic anhydrase.

The enzyme can be stabilized with suitable antioxidants. Suitable antioxidants are, for example, ascorbic acid, glutathione, rosemic acid, benzoic acid and catechins.

Potentiometric (pH, NH ₄ ⁺ ) or amperometric micro probes are used as transducers to measure the concentration of primary or secondary analyte behind the silicone seal (see S. Hanstein, D. de Beer and H. Felle, Sensors and Actuators 2001 , B81, 107-114). They are pushed into the glass micropipette 3 from the end that is not provided with the seal.

An electrode that is non-toxic and contains a metal and its salt or consists of a metal and its salt is used as the reference electrode. Advantageous

TM007 a silver-silver chloride electrode is used as the reference electrode. A conventional electrode holder is advantageously used as the working electrode, in which an electrode is integrated i which contains a metal and its salt, preferably a noble metal and its salt.

Ausführungsbeispie1

1. Process for three-stage extraction of the outer glass micropipette A large-diameter outer glass micropipette 3 made of borosilicate glass with an inner diameter of 1.6 mm and an outer diameter of 3 mm is used (for example from Hilgenberg GmbH, Malsdorf, Germany) . The three-stage extraction takes place with the vertical pulling device LM-3 / PA (List Medical, Göttingen, Germany) with 1 mm thick standard heating coil made of Pt / Ir (80/20, respectively wt.%), Inside diameter of the coil 4 , 5 mm. Inner glass micropipette 25: borosilicate, 1 mm AD, 0.5 mm ID, Clark Electromedical Instruments, now Harvard Apparatus, Holliston, Ma, USA). 1st train from 16 mm height (upper stop) to 5 mm height with a current of 21 A; Then fix the capillary 7 mm higher in the upper holder. 2. Train of 12 mm in height to the lower stop (2 mm) at a current of 13 + 0.5 A depending on the spiral condition and the desired tip diameter. Outer glass micropipette 3: borosilicate, 3 mm AD, 1.6 mm ID, Clark Electromedical Instruments, now Harvard Apparatus, Holliston, Ma, USA). 1st train from 16 mm high to 12 mm high at 21 A; Then fix the capillary 1.5 mm higher in the upper holder. 2nd train from 13.5 mm to 7 mm at 21 A; Fasten the capillary (afterwards?) 5 mm higher in the upper holder. 3. Draw from 12 mm to the lower stop at 12.6 ± 0.5 A depending on the spiral condition and the desired tip diameter.

TM007 Before the seal is made, the micropipettes 3 and 25 are silanized on the inside as described.

2. Process for making the seal The freshly silanized microcapillary is filled with sterile distilled water as described and the silicone seal is made as described. Dow Corning Product 200 (R) Fluid with a viscosity of 0.1 Stokes (25 ° C) and an activity of 100% is used as the non-crosslinking silicone oil. The container capillary 9 used has an inner diameter of 2 mm. Dow Corning Product (R) 1340 RTV Coating serves as the cross-linking silicone oil.

3. Process for the production of the pH-sensitive microelectrode

Another glass micropipette 25 (outer diameter 1 mm, inner diameter 0.5 mm) made of borosilicate glass or chemically more resistant aluminum silicate glass, both with inner filament, is silanized as described above. A person skilled in the known proton-sensitive hydrophobic cocktail, preferably Fluka Product # 95297, hydrogen ionophore II Cocktail A, Selectophore ^® is dissolved in a mixture of 40 mg PVC / ml THF in a ratio of 30:70 (V / V) , This (hydrophobic) solution is filled into the second glass micropipette from behind using a filling capillary. The evaporation of the THF forms a firm PVC gel. The solid PVC gel is first overlaid with undiluted proton-sensitive cocktail, then with reference buffer. Reference buffer: 100 M 2- [N-morpholino-] ethanesulfonic acid is mixed with a solution of 100 mM tris (hydroxymethyl) aminomethane and adjusted to pH 8.3, then 100 mM KC1 are added. A conventional electrode holder is used as the working electrode, in which an electrode is integrated and which allows the pH-sensitive microelectrode to be connected to a further electrode.

TM007 4. Production of the reference electrode

A silver-silver chloride electrode is used as the reference electrode 26 (installation in the first glass micropipette). Production: approx. 1 mm of the Teflon coating of a Teflon-coated silver wire (diameter of the silver core 100 μm) is removed by heat and the bare silver tip is chlorinated in a 3M KCl solution at 100 μA for 3 minutes.

5. Manufacture of the micro probe

The assembly is carried out as described above. An inlet tube 2 is attached to the pH-sensitive microelectrode 1 using cyanoacrylate adhesive (e.g. Tesa ^{® superglue} (Beiersdorf AG, Hamburg, Germany); UHU superglue (viscosity 70 mPa; UHU GmbH & Co. KG, Bühl / Baden, Germany) , for example, are in the form of a curved cannula (Ex .: 1 Sterican ^®, 0.4 x 40 mm (B. Braun Melsungen AG fixed, Melsungen) Examples of outlet tubes. 5: fused silica capillary tube untreated,, ID 0.15 mm, OD 0.22 mm (order no. 0642472) or fused silica capillary tube deactivated (non-polar) ID 0.15 mm, OD 0.22 mm (order no. 06424460), SGE (Deutschland) GmbH, Darmstadt; Fused-silica capillaries untreated, ID 0.1 mm, OD 0.363 mm (Art. 23735) or fused-silica capillaries deactivated (non-polar), ID 0.1 mm, OD 0.363 mm (Art. 25740-U), Su - pelco, Taufkirchen, Germany Before the two glass micropipettes are attached, the outlet tube can be attached to the inner glass micropipette with glue, or it is possible to use an inlet instead of an inlet ss and outlet to attach only a single access, eg a cannula, to the inner glass micropipette. For later filling, this access can be used to insert an inlet tube. With the help of the adapter (Fig. 4), liquid can be brought through the inlet tube into the finished micro probe. Pressure and fluid equalization are carried out via the simple access.

TM007 The two glass micropipettes are attached to each other with adhesive, preferably UV glass adhesive (Greven adhesive, Heddesheim, Germany). The adhesive cures in 12 hours by lighting with a UV lamp. The free end of the second glass micropipette 25 facing away from the tip is then inserted into a conventional electrode holder. This electrode holder contains an Ag-AgCl plate encased in plastic, which serves as a working electrode (see above).

The area of the outer glass micropipette in which the chlorinated tip of the silver electrode is located is provided with a 5 mm wide ring made of acrylic paint, since the electrical potential at the Ag / AgCl electrode is light-sensitive. The probe is now ready for transport and storage.

In order to use the microprobe for C0 ₂ measurement, the probe chamber 28 is rinsed with a carbonic anhydrase solution. For this purpose, a 1% stock solution of chloramphenicol in ethanol, a buffer solution of 1 mM NaHC0 ₃ and 100 mM NaCl (pH 8.3) and a 0.1 M neutral Na ascorbate solution (antioxidant) are first prepared. The enzyme solution is then made from 0.4 ml of the NaHC0 ₃ buffer solution described, 3 mg of lyophilized carbonic anhydrase (Serva, Heidelberg, Germany), 2 μl of chloramphenicol stock solution and 4 μl of a 0.1 M Na ascorbate solution was preset to pH 8.3 with KOH. This solution is placed in a sterile 1 ml disposable syringe and used to rinse and fill the probe chamber 28. After removing the syringe, the inlet and outlet of the probe are closed with Parafilm® (American National Can, Neenah, USA). After three hours of equilibration at room temperature, the probe is ready for use. Storage takes place in the refrigerator at 4-8 ° C.

TM007 LIST OF REFERENCE NUMBERS

pH-sensitive microelectrode inlet tube (first capillary tube) outer glass micropipette (first pipette) Enzy (electrolyte) solution

Outlet tube (second capillary tube) Microscope objective Sterile distilled water Place for attaching the adapter Container-capillary non-cross-linking silicone Holder cross-linking silicone

Filling capillary with sterile distilled water sealing screw rubber seal adapter head

Metal tube for clamping the adapter in the micromanipulator

Plastic tubing (only the beginning and end drawn) Three-way valve Luer lock connection Syringe (50 ml) Fastener, silicone seal

Electrolyte of the pH-sensitive microelectrode (reference buffer) inner glass micropipette (second pipette) reference electrode

Place for attaching the electrode holder Probe chamber for filling in enzyme (electrolyte) solution

7 Legends

Fig. 1 :

Schematic representation of the refillable microprobe according to the invention: Between an outer glass micropipette 3 and an inner glass micropipette 25 designed as a pH-sensitive microelectrode 1, an inlet tube 2 and an outlet tube 5 are attached, which fill, refill and / or exchange the enzyme electrolyte solution allow in the probe chamber 28. The drawn 3mm bar is to be understood as a yardstick.

FIG.

Schematic representation of the introduction of the non-crosslinking silicone oil 10 into the glass micropipette 3 under microscopic control 6. The tip of the glass micropipette 3 is filled with distilled water 7. The adapter is placed on the rear end 8 of the capillary. The water-filled glass micropipette is introduced into the container capillary 9 with the silicone oil 10. A vacuum is generated by the piston stroke of the adapter syringe (see FIG. 4) and silicone oil 10 is sucked into the glass micropipette 3.

Fi_, Schematic representation of the introduction of the crosslinking silicone oil 12. The illustration summarizes two processes. First (shown on the right), the sterile distilled water already sucked in through the tip of 3 is filled up from behind with the help of a filling capillary until the beginning of the rejuvenation of 3, the opening of the

Glass micropipette is understood, which has the larger diameter. Then (shown on the left) silicone oil applied to the holder 11 is brought into contact with the glass micropipette 3.

TM007 Fig. 4:

Cross-sectional representation: adapter for sucking in non-crosslinking silicone oil 10 into the tip of a glass micropipette 3. The adapter consists of a sealing screw 14, rubber seal 15, adapter head 16, a metal tube 17 for clamping the adapter in the micromanipulator and a plastic hose 18. The Plastic hose 18 is connected to a three-way valve 19 for ventilation. the three-way valve 19 has a Luer lock connection 20 to a 50 ml syringe 21.

Fig. 5:

Schematic representation of the finished micro probe: The micro probe consists of two concentric, glass micropipettes pushed into each other (3 and 25). The inner glass micropipette 25 contains a proton-sensitive cocktail covered with reference buffer 24. Inner glass micropipette 25, proton-sensitive cocktail and working electrode covered with reference buffer together form the pH-sensitive microelectrode 1. In this case, the electrode used is preferably one that is integrated in a conventional electrode holder (see FIG. 27). The pH-sensitive microelectrode 1 is positioned in the tip of the outer glass micropipette 3. The tip of the pH-sensitive microelectrode (1) is located about 20 μm behind the tip of the outer glass micropipette 3, which is sealed with a silicone seal 23. The space between the outer glass micropipette 3 and the pH-sensitive microelectrode 1 is filled with an enzyme solution 4. A reference electrode 26 connects the enzyme solution 4 to the ground. Before assembling the outer 3 and inner 25 glass micropipette, an inlet tube 2 is attached to the outside of the inner glass micropipette, which allows the filling, refilling or replacement of enzyme solution 4 in the finished microprobe.

TM007 The two glass micropipettes are then pushed into one another, the inner micropipette 25 not yet being pushed as far as the silicone seal 23. Then, if necessary, an outlet tube 5 can be pushed between the two micropipettes before the inner glass micropipette 25 is pushed as far as the silicone seal 23. Both glass micropipettes (3 and 25), inlet 2 and possibly outlet tubes 5 are fastened to one another with the aid of a fastening means 22. The rear end 27 of the pH-sensitive microelectrode is connected to a conventional electrode holder.

Fig. 6:

Schematic representation of the tip of the finished micro probe:

A silicone seal 23 is located in the tip of the outer, first glass micropipette 3. The space behind this silicone seal 23 is filled with enzyme electrolyte 4. A proton-sensitive cocktail 24 is located in the tip of the second glass micropipette 25. Inlet 2 and outlet tubes 5 for the enzyme electrolyte 4 are located in the space between the two glass capillaries 3 and 25.

The first, outer glass micropipette tapers in two stages. Inlet and outlet tubes end before the first taper begins. 5 shows the first taper. The second taper can only be seen in FIG. 6.

TM007

Claims

Expectations

1 . Micro probe for measuring gas concentrations, consisting of - an outer glass micropipette (3) containing an electrically insulating seal (23) that is highly permeable to the gas to be measured in the tip of the outer glass micropipette (3), an inner (25 ) Glass micropipette which are pushed into the outer glass micropipette (3) such that a cavity is formed in the form of a probe chamber (28), the tip of the inner glass micropipette (25) not touching the silicone seal (23), an enzyme solution (4), which is located in the probe chamber (28) between the first (3) and second (23) glass micropipette, a pH-sensitive microelectrode (1) consisting of the inner glass micropipette (25), one hydrophobic proton-selective cocktail, a reference buffer (24) and a working electrode, a reference electrode (26) which protrudes into the rear end of the probe chamber (28) and is immersed in the enzyme solution (4), a fastening means (22) which the two Glass M ikropipetten (3 and 25) and the reference electrode (26) at the rear end of the probe chamber, and an electrode holder which is connected to the rear end of the inner glass micropipette (25), characterized in that the probe tip of the microprobe is miniaturized The cavity (28), which is designed as a probe chamber, of the glass micropipettes (3 and 25) pushed into one another is a flow chamber.

TM007 the probe chamber (28) has inlet (2) and outlet devices (5), and outer (3) and inner (25) glass micropipette, reference electrode, inlet and outlet device (2 and 5) at the rear end of the microsensor with a fastening means (22) are attached to each other. 2. Micro probe according to claim 1, characterized in that the miniaturized probe tip has an outer diameter less than or equal to 5 microns. 3. A method for producing a microsensor, characterized in that the following steps are carried out in succession: I. pulling out a first glass capillary to the micropipette,

2. Fill the tip of this extended first glass micropipette with a liquid,

3. Suck a non-crosslinking silicone oil into the liquid-filled first glass micropipette, 4. Push out excess non-crosslinking silicone oil, 5. Immerse the tip of the first glass micropipette in a drop of crosslinking silicone oil, 6. Tip of the first glass micropipette for Leave in the cross-linking silicone oil for 5 to 60 seconds, 7. Pull the first glass micropipette out of the cross-linking silicone oil, 8. Repeat steps 5 to 7 once, 9. Harden the silicone seal, 10. Pull out a second glass capillary that has an inner filament has, for micropipette, II. filling the second glass micropipette with a solution of a hydrophobic proton-sensitive cocktail and a liquid polymer, the loading

TM007 filling from the side of the second glass micropipette facing away from the tip, 12. curing of the mixture of hydrophobic proton-sensitive cocktail and polymer, 13. covering of the cured mixture with a hydrophobic proton-sensitive cocktail and a reference buffer, 14. attaching and attaching an inlet tube for liquids on the outside of the second glass micropipette, 15. connecting a working electrode to the second glass micropipette, 16. tip of the second glass micropipette while maintaining a distance between the tip of the second glass micropipette and the silicone seal in the first glass - Insert the micropipette, 17. Insert an outlet tube for liquids and a reference electrode between the two glass micropipettes, 18. Insert the second glass micropipette into the first glass micropipette until the distance between the tip of the second pipette and the tip of the first pipette is between 5 and 100 μm, 19th at de Attach the glass micropipettes, the outlet tube and the reference electrode to each other using a fastener, 20. Connect the rear end of the second glass micropipette to an electrode holder, 21. Cure the fastener.

4. The method for producing a microprobe according to claim 3, characterized in that the first glass capillary is pulled out to the micropipette according to step 1 in three stages.

TM007

5. The method for producing a microprobe according to claim 3, characterized in that the pulling out of the first glass capillary to the micropipette according to step 1 takes place in two stages.

6. The method for producing a microprobe according to claim 3, characterized in that the z: wide glass capillary is pulled out to form the micropipette according to step 10 in two stages.

7. The method for producing a microprobe according to claim 3, characterized in that the pulling out of the second glass capillary to the micropipette is carried out in one step according to step 10.

8. A method for producing a microprobe according to one of claims 3 to 7, characterized in that the reference buffer, which is used according to step 13 to overlay the cured mixture of hydrophobic proton-sensitive cocktail and polymer, by mixing 100 mM 2- [ N-Morpholino] ethanesulfonic acid with a solution of 100 mM tris (hydroxymethyl) aminomethane, adjusting the pH of this mixture to 8.3 and adding 100 mM KC1 is obtained.

9. A method for producing a microprobe according to one of claims 3 to 8, characterized in that the liquid with which the tip of the first glass micropipette is filled according to step 2 is water, preferably sterile distilled water.

TM007

10. The method for producing a microprobe according to one of claims 3 to 9, characterized in that the tip of the first glass micropipette is left in step 3 in claim 3 for 40-50 seconds in the crosslinking silicone oil, preferably for 45 seconds.

11. A method for producing a microprobe according to one of claims 3 to 10, characterized in that glass micropipettes made of borosilicate glass, aluminum silicate glass or quartz glass are used.

12. A method for producing a microprobe according to one of claims 3 to 11, characterized in that the first glass micropipette has an inner filament.

13. A method for producing a microprobe according to one of claims 3 to 11, characterized in that the first glass micropipette has no inner filament.

14. A method for producing a microprobe according to one of claims 3 to 13, characterized in that the first glass micropipette is silanized before filling with a liquid according to step 2.

15. A method for producing a microprobe according to one of claims 3 to 14, characterized in that the second glass micropipette is silanized according to step 11 before filling with a hydrophobic proton-sensitive cocktail and a liquid polymer.

16. A method for producing a microprobe according to one of claims 3 to 15, characterized in that the liquid with which the first glass micropipette according to

TM007 Step 2 is filled, water to which chloramphenicol has been added, preferably sterile distilled water to which 50 ppm chloramphenicol (w / v) are added.

17. A method for producing a microprobe according to one of claims 3 to 16, characterized in that the silicones used are permeable to gases and electrically insulating.

18. A method for producing a microprobe according to one of claims 3 to 17, characterized in that the inlet tube is a cannula with a length of 25-45 mm and a diameter of 0.3-0.6 mm, preferably around a cannula 40 mm long and 0.4 mm in diameter.

19. A method for producing a microprobe according to one of claims 3 to 18, characterized in that the outlet tube is a capillary tube with an inner diameter of 100-350 μm and an outer diameter of 200-400 μm.

20. A method for producing a microprobe according to claim 19, characterized in that the outlet tube is a capillary tube made of quartz glass, preferably a capillary tube made of untreated or deactivated quartz glass.

21. A method for producing a microprobe according to one of claims 3 to 20, characterized in that the working electrode according to method step 15 is dispensed with and instead after the fastening of the

TM007 two glass micropipettes, the outlet tube and the reference electrode are connected by a fastening means according to method step 19, the rear end of the second glass micropipette with a conventional electrode holder, the electrode holder containing an electrode made of a metal and a salt of this metal.

22. A method for producing a microprobe according to claim 21, characterized in that the working electrode consists of a silver-silver chloride plate which is encased in plastic.

23. A method for producing a microprobe according to one of claims 3 to 21, characterized in that the electrodes are non-toxic electrodes, preferably silver-silver chloride electrodes.

24. Use of a microsensor according to one of claims 1 and 2 for the enzymatic measurement of gas concentrations, characterized in that the following steps are carried out in succession before the start of the measurement: rinsing the probe chamber of the microsensor with enzyme solution, inlet and outlet tubes of the microsensor with a Close evaporation protection, allow microsensor to equilibrate for 3 h at room temperature.

25. Use of a micro probe according to claim 24 for the enzymatic measurement of gas concentrations, characterized in that the micro probe is used for measuring gases from the group of carbon dioxide, ammonia and oxygen, preferably for the measurement of carbon dioxide. TM007

26. Use of a microprobe according to claim 25 for the enzymatic measurement of gas concentrations, characterized in that the enzyme is carbonic anhydrase.

27. Use of a micro probe according to one of claims 25 and 26, characterized in that the micro probe is used for investigations of biological systems.

28. Use of a micro probe according to one of claims 25 to 27, characterized in that the micro probe is used for investigations of gases in phytophysiological systems, preferably for the measurement of carbon dioxide and / or ammonia in plant leaves, very particularly preferably for high-resolution carbon dioxide and / or ammonia measurements on individual stomata of plant leaves.

TM007