[go: up one dir, main page]

WO1996012957A1 - Bio-recognition-controlled, ion-flow modulating bio-sensor - Google Patents

Bio-recognition-controlled, ion-flow modulating bio-sensor Download PDF

Info

Publication number
WO1996012957A1
WO1996012957A1 PCT/AT1995/000197 AT9500197W WO9612957A1 WO 1996012957 A1 WO1996012957 A1 WO 1996012957A1 AT 9500197 W AT9500197 W AT 9500197W WO 9612957 A1 WO9612957 A1 WO 9612957A1
Authority
WO
WIPO (PCT)
Prior art keywords
biosensor according
channel
membrane
channels
sensor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/AT1995/000197
Other languages
German (de)
French (fr)
Inventor
Fritz Pittner
Thomas Schalkhammer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Priority to EP95933226A priority Critical patent/EP0734528A1/en
Publication of WO1996012957A1 publication Critical patent/WO1996012957A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/002—Electrode membranes

Definitions

  • the invention relates to a biosensor construction by on-off control of an ion channel via biorecognitive interactions
  • Enzyme sensors are known as detection systems for biological molecules, which use the catalytic effect of biomolecules to detect a specific biosignal e.g. the concentration of glucose in an unspecific chemical signal e.g. Convert concentration of hydrogen peroxide, which is then electrochemically converted into an electrical quantity (a current or a potential). It is known that these sensor types have a low sensitivity and have fundamental problems due to the unspecific electrochemistry on sensor surfaces which, in addition to hydrogen peroxide, can oxidize many other biological components.
  • a specific biosignal e.g. the concentration of glucose in an unspecific chemical signal e.g. Convert concentration of hydrogen peroxide, which is then electrochemically converted into an electrical quantity (a current or a potential).
  • Optical systems such as surface plasmon resonance sensors, which are able to convert the surface binding of biomolecules into an optical signal, are also known as detection systems for biological molecules.
  • detection systems for biological molecules.
  • the great expenditure on equipment and the sensitivity of these sensors which is better than that of the enzyme sensors but inadequate for many applications, makes this technique usable only in some areas.
  • the aim of the invention is to eliminate these fundamental restrictions by means of a novel sensor design.
  • the biocomponent should not be used in this sensor structure for the catalytic conversion of the analyte, but as a selective barrier to the surrounding solution.
  • the analyte then serves as the key to the on / off control of an ion channel. Through this ion channel, up to 10 7 ions per second and channel can flow into a narrow membrane electrode gap (10-300 ⁇ m) and be determined there directly electrochemically (about 3 pA per open channel). A disturbance tion of the measurement signal by other components of the solution is suppressed by a selective membrane with built-in channel proteins.
  • the present invention differs from WO 89/01159 in that the arrangement of a ligand at the channel entrance of the ion channel which is possible with "molecular modeling" is sub-nanometer accurate.
  • WO 89/01159 describes the binding of relatively large receptor molecules (antibodies, enzymes, lectins, ..) at the channel entrance.
  • relatively large receptor molecules antibodies, enzymes, lectins, ..
  • the present invention uses the highly precise positioning of a small ligand at the entrance to the channel, which is then recognized by a large molecule (analyte) and bound at the optimal location (channel entrance).
  • This procedure is not only much more precise, but also a reversal of the procedure of WO 89/01159 in which the receptor protein, immobilized on the channel, binds the analyte biorecognitively, but in the present bio-sensor a small ligand on the ion channel is recognized and bound by the analyte becomes.
  • the present invention differs from US Pat. No. 5,234,566 in that it uses individual membranes, not a large number of necessarily identical membranes.
  • the construction of the membrane is not only possible by "soap assembling" but can also be done by other techniques (e.g. LB films).
  • a dependency of the conductivity on the membrane potential is not desired; an optimal behavior of the channel should almost follow Ohm's law.
  • the present invention differs from EP 0 441 120 A2 in that the lipid membrane is not crosslinked to the electrode surface by bridging molecules. Continuous contact with two aqueous phases above and below membrane (sign of an unstable membrane) is not a necessary part of the present invention.
  • the use of a frame structure over which the lipid membrane extends further avoids apolar walls to which the lipid membrane borders, which would represent unstable and therefore leaky areas of the membrane.
  • the present invention does not include the direct crosslinking of the lipid membrane with the electrode surface required in WO 94/07593 - using bridging linker lipids.
  • the present invention also differs from EP 0 394 997 A1 in that the content of the invention does not use the structure of a biosensor using the binding of a molecule to an immobilized ligand for on / off control of an ion channel in a membrane the direct effect of organic molecules on the channel itself.
  • the multimeric structure of the porins is not compatible with claim 1 (stable molecule).
  • the new sensor principle is based on the following findings:
  • the very well investigated and easily accessible channel peptide gramicidin can serve as the ion channel as the basis of a new affinity sensor. Both the sequences and the exact molecular data of different Grandeidin derivatives have been determined in recent years. The sequence homologies are consistently high.
  • the ion channels are formed by 2 associated gramicidin molecules.
  • the spatial structure of the peptide is a 6.3 - helix that forms an open ion channel in the center and is dynamically anchored with tryptophan residues on the two membrane sides.
  • the second essential component of the new sensor concept is membrane technology.
  • the advantage of black lipid membranes i.e. free access to both sides of the membrane combined with the advantage of seif assembling membranes, i.e. the stable arrangement of a self-assembling lipid film.
  • the choice of a photogel polymer as a carrier matrix for the lipid film and the covalent coupling of the lipid monomer to this gel surface stabilizes the lipid film against any mechanical destruction or against the floating of the second lipid film of the double membrane.
  • the third innovation in the context of the sensor concept is to be the use of thin-film technological process steps in the context of the sensor construction.
  • the electrochemical sensor is to be manufactured using established thin-film technology processes.
  • the frame structure of the lipid film is to be built up with photoresists, for example the AZ series or with Proh mid lacquer.
  • the next step is then the filling of the hydrophobic frame structure with a hydrophilic gel photopolymer as a carrier system for the lipid membrane.
  • the on / off switch is then coupled to the gramicidin channel by selective chemical modification of the carboxy-terminal ethanolamine capping structure.
  • a small molecule recognized by biorecognition is intended to reversibly close the ion channel by binding a large molecule (see FIG. 1).
  • the invention thus aims to create a new type of highly sensitive sensor principle with the theoretical sensitivity of a single analyte molecule.
  • the sensitivity is limited only by the binding constant ligand / binding molecule and by the time required for the statistical meeting of the two molecules.
  • the invention aims at the construction of such sensors with methods compatible with thin-film technology such as thin-layer substrates, sputtering and vapor deposition of the metal electrodes, use of photoresists and thin-film technology lipid membrane supports produced with photosensitive polymers and also includes the novel structure of covalently bound Flat lipid membranes at the polymer-liquid interface.
  • the use of the invention can therefore e.g. as a blood HIV antibody sensor for HIV diagnosis and for monitoring the course of HIV infections.
  • the HIV tests currently on the market are too insensitive and therefore too unsafe in many cases.
  • a selective peptide from the V3 loop of the gp41 protein can therefore be bound to the channel entrance of the gramicidine as a recognition determinant.
  • the sequence LGLWGCSGKLIC (a partial sequence of the HIV protein gp41) now in turn binds to serum antibodies of almost 100% of all HIV-infected people.
  • the sequence LGMWGCSGKLIC must be used for HIV-infected people from West Africa.
  • a binding sequence for HIV-2 is LNSWGCAFRQVC.
  • the two cysteines form a disulfide bridge in both HIV 1 and HIV 2, which is essential for the full antigenicity of the peptide.
  • a basic amino acid is essential in the middle of the so-called V3 loop. For some rare sera, flanking sequences of about 5 amino acids can also improve the anti-genicity.
  • the binding of the serum antibody leads to a channel blockage within the scope of the invention. This effect can then be measured with high sensitivity and selectivity. After a test, the sensor is briefly washed with a chaotrope and the bound antibody is removed. The sensor can then be used for the next test.
  • the invention can also be used as a urine estrogen sensor for determining the fertile days.
  • the detection of the fertile days in the context of the female cycle is possible with good accuracy due to the clear urine-estrogen peak preceding 24-48 hours.
  • ELISA tests to determine this urine parameter (or LH) have been on the market for some time. However, these tests are only one-time tests and extremely expensive, so that continuous measurement by the patient is not possible.
  • the new sensor principle can enable a sensor head that has at least one cycle duration, i.e. can be used for about 28 days and allows much more precise information about the cycle status.
  • This test system can enable the exact measurement of the days ready for conception if the desire for children is not fulfilled, and can also be used as an extremely gentle method for regulating conception.
  • Monitoring the effect of anti-estrogens in the context of tumor therapy is another important area of application for the sensor. Even during pregnancy, the measurement of the level of estrogen is of diagnostic importance.
  • the sensor can detect estrogen and its 17-glucuronido and sulfoconjugates via specific antibody binding.
  • the amount of urinary oestrogens can then be determined as a time integration over open and closed times on one channel, or also as a mean current (or its time dependence) on some 10 to 100 ion channels.
  • the binding to the channel in a use assay see . Figure 1).
  • the sensor is covered with an analyte-permeable but antibody-impermeable membrane. In the intermembrane space there are antibodies that react selectively with estrogen and its urinary derivatives and compete for a similar ligand at the ion channel entrance. Thus, the ion flow through the membrane is increased from almost zero to a value proportional to the estrogen level due to an increasing amount of urea estrogen.
  • a commercially available type of dialysis membrane can be used as the outer membrane.
  • This sensor can then be easily changed, e.g. be used to measure the hormone progesterone.
  • the invention aims at a sensor with approximately the following
  • the chemical synthesis of an ion channel can be explained using the example of the ion channel peptide gramicidin.
  • the synthesis of the channel peptide gramicidin A and its covalent dimers of bisgramicidine is based on gramicidin D.
  • Pure gramicidin A can then be obtained with the aid of silica or reversed phase chromatography. This can be done with anhydrous Hydrochloric acid in an organic solvent such as methanol can be deformylated.
  • the product is then again purified using reversed phase chromatography.
  • the dimerization is then carried out by coupling with a malonic acid ester, tartaric acid ester or structurally related crosslinkers.
  • the diethyl ester of dicarboxylic acid and gramicidin A is dissolved in DMF and DPPA (diphenylphosphorylazide) is slowly added. After the reaction, the product is again purified using reversed phase chromatography.
  • DPPA diphenylphosphoryla
  • the affinity ligands are coupled to the carboxy-terminal ethanolamine (of gramicidin A) by activating the two free terminal OH groups of the molecule with e.g. Divinyl sulfone or chloroformic acid nitrophenyl ester.
  • the ligand can then be selectively terminally bound by its OH, NH2 or SH group.
  • Both terminal OH groups of the bisgramicidine can also be activated with vinyl sulfone or nitrophenyl chloroformate and reacted with a diamine (ethylenediamine) or a dicapto compound.
  • the bisgramicidine derivative formed by the reaction then carries two terminal reactive amino (or SH) groups.
  • the iodactamido-caproic acid N-hydroxysuccinimide esters can then be used to introduce the alkylating group required for coupling histidines into the molecule.
  • the estrogen is coupled at position 6 (estriol, estrone) or at position 17 (estradiol) of the conjugated ring systems.
  • 17 ⁇ -estradiol 17-hemisuccinate, 17ß-estradiol 3-glycidyl ether or 17ß-estradiol 6- (O-carboxymethyl) oxi serve as ligands for estradiol.
  • 17ß-estradiol 17-hemisuccinate and 17ß-estradiol 6- (O-carboxymethyl) oxime are to be covalently linked to the bisaminomodified bisgramicidin already described via an amide bond.
  • the N-terninal .amino group can be used for coupling on the one hand after protection of the amino group of the lysine (arginine) in the V3 loop.
  • some additional terminal histidines can be introduced into the peptide. These can then be linked to the carboxy terminus of gramicidine via alkylating crosslinkers.
  • the biosensor substrates are manufactured using thin-film technology.
  • the production follows the scheme, see examples.
  • coated sensor substrates produced by the processes mentioned are then modified by polymer thin-film technology processes for use in electrochemical ion channel biosensors.
  • the carrier of the lipid biomembrane is made up of two different photostructurable polymers: a frame structure made of hydrophobic, i.e. lipophilic photoresist and a hydrophilic photopolymer within the frame structure as a membrane carrier.
  • the frame structure made of hydrophobic, ie lipophilic, photoresist can be constructed using photostructurable polymers from the semiconductor industry.
  • established photoresists of type AZ Hoechst
  • a photostructurable polyimide varnish eg Probimide 408
  • hydrophilic polymer systems can also be constructed analogously. It can be biocompatible, hydrophilic and swellable Polymer precursors are cross-linked by bivalent photocrosslinkers.
  • a polymer network with controllable pore size is formed, which serves as a structurable carrier material. 1 for immobilizing the biocomponents, ie lipid membranes and ion channels.
  • polyvinylpyrrolidine polymers with hydrophilic side groups (-0H, ester amides,.) Can be applied to the sensor blanks as viscous solutions by spinning processes and by photocross linkers (mostly carbene and nitrene formers, for example 4,4'-biazidostilbene-2,2'-disulfonic acid) cross-linked with long-wave UV (350 - 390 n) to polymer films.
  • photocross linkers mostly carbene and nitrene formers, for example 4,4'-biazidostilbene-2,2'-disulfonic acid
  • long-wave UV 350 - 390 n
  • lipid films are not bound to each other or to the cell wall by covalent interactions, they can easily be extracted under disintegrating conditions. Hydrophobic agents, detergents and alkali are suitable for this.
  • To build a stable lipid membrane two basic problems have to be solved: stable binding of the lipid film to the base and preventing the upper lipid film from flowing off in the lipid double layer.
  • the lower lipid film is stably bound to the gel surface by hydrophobic, ionic or covalent coupling of e.g. Thiolipids on a chemically reactive surface
  • the second lipid film can be anchored to the first covalently bound lipid film by incorporating symmetrical bis-lipids. These also carry at least one thio group in order to be fixed on the gel matrix. A proportion of freely movable lipids enables the fluid structure of the lipid membrane.
  • the modified gramicidine channel is installed in the same step with the soap assembling of the lipid film.
  • the ligand-modified bisgramicidin still has a free reactive group on the second carboxy terminus of the bisgramicidin molecule. In an analogous manner to the lipids, this can couple covalently to the gel surface and thus anchor the channel firmly (i.e. covalently) in the membrane.
  • Electrochemical conductivity and current measurements similar to the patch clamp measurement on ion channels in intact cells or on membrane fragments in the tip of glass pipettes bound by "mega seal” are the fundamental measuring principle of the ion channel biosensor.
  • the sensors constructed in the manner mentioned deliver a current signal proportional to the ion flow as micro-conductivity biosensors.
  • Amplifiers equipped with OPA 128 (Burr Brown) input stages are suitable as measuring stations. All measuring stations for single ion channel measurements in the patch clamp technique in the range up to 0.1 pA are also optimally suitable for the novel sensors described in the context of the invention.
  • Mask aligner Karl Süss model MA 45, 360 n 350 W, 7.5 s or photoresist developer AZ-Developer / Hoechst 60 s
  • Gramicidin D is separated from the other gramicidines (B, C) on a Sl - 100 polyol column from Serva with a gradient of 0 - 80% methanol / water.
  • Example 3 Carboxy-Terminal Modification of Gramicidine 500 mg of gramicidin A are stirred with 10 ml of benzene, a 3 to 5-fold excess of the carboxy-modified ligand is added, and 10-fold molar excess of a carbodiimide (for example dicyciohexylcarbodiimide) is added. The reaction is carried out at room temperature in the dark. The course of the reaction is monitored on silica gel plates using thin layer chromatography (chloroform / methanol / water: 100/10/1).
  • reaction mixture is then purified on a preparative C-18 reversed phase column with methanol / water.
  • Example 4 Synthesis of bisgramicidine
  • the starting product is deformylated gramisidine A, which is synthetically obtained by cleavage of gramicidin A with anhydrous hydrochloric acid in methanol and subsequent purification using a preparative C-18 reversed phase column with methanol / water.
  • the dimerized gramicidin A is deformylated by adding a 5-fold molar excess of diphenylphosphorylazide to a solution of a molar part of deformylated gramicidin A and an excess (3-10) fold of the dicarboxylic acid (eg malonic acid) in dimethylformamide at below - 10 ° C.
  • the reaction time at 0 ° C is 24-48 hours. After the reaction has been stopped with ethanol, the reaction mixture is evaporated to dryness after a few organic / aqueous extraction steps and, after the residue has been taken up in a small amount of methanol, on a preparative C-18 reversed phase column with the solvent system methanol / water (90/10 to 95 / 5) cleaned.
  • Example 5 Structure of the hydrophobic frame structure * The polyimide photoresist Probimide 408 is applied to the sensor chip by spin coating (5000 rpm, 2 min). * Softbake at 110 ° C for 15 minutes * Exposure with photomask with UV (365 nm) 10 s
  • Example 6 Structure of the hydrophilic lipid carrier polymer
  • the hydrophilic photocrosslinked gel is built up according to the following instructions: 2.5% (w / v) PVP (polyvinylpyrrolidone MW: 360,000 or 1,000,000) in distilled water and 0.75% (w / v) 4.4 ' -Diazidostilben-2,2'-disulfonic acid in distilled water are mixed (3: 2 v / v).
  • the prepolymer solution is either pipetted into the photoresist frame structure or the sensor is coated with the prepolymer solution by an immersion or spin coating process.
  • Metal surfaces chemically or electrochemically blocked derivatized.
  • the electrode is cleaned electrochemically between -600 and +1200 mV (up to the range of oxygen evolution) versus Ag / AgCl.
  • the electro-chemical cleaning is carried out by applying a time-variable voltage to a sensor immersed in neutral buffer.
  • Example 9 Lipid coating of the sensor electrodes The lipids (2-10 ⁇ g / 100 cm 2 surface area) dissolved in trifluoroethanol are spread on an air / liquid phase boundary on an LB trough. In an automated LB system, the lipid film is compressed from a fluid to a semi-crystalline phase. The sensor is then drawn through the lipid film using a nano actuator from Physik-Instrumen ⁇ te / Germany (driven by a DC motor with a resolution of 60 nm).
  • Unsaturated lipid films are covered with a photomask and crosslinked for about 120 seconds by the light of a 50W medium pressure mercury vapor lamp.
  • the lipids are cross-linked both within the lipid layer and with a reactive carrier.
  • Modified channels are installed in an analogous manner with mixtures of lipid and channel peptides.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Detergent Compositions (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

The proposal is for a novel, highly sensitive sensor principle, the bio-recognition-controlled, ion-flow modulating bio-sensor with the theoretical sensitivity of a single analyte molecule which makes use of the bond of effector molecules, in which effector molecules are any which bond to a ligand from the group consisting of hormones, peptides, enzyme inhibitors, environmental toxins, active pharmaceutical agents, thioles or chelate formers for the on/off control of an ion channel in a membrane.

Description

Biorekoσnitions-αesteuerter. lonenfluß modulierender BiosensorBiorekoσnitions-αsteuerter. Ion flow modulating biosensor

Die Erfindung bezieht sich auf eine Biosensorkonstruktion durch On-Off Steuerung eines Ionenkanals über biorekognitive WechselwirkungenThe invention relates to a biosensor construction by on-off control of an ion channel via biorecognitive interactions

Als NachweisSysteme für biologische Moleküle sind Enzymsenso¬ ren bekannt, die die katalytische Wirkung von Biomolekülen verwenden, um ein spezifisches Biosignal z.B. die Konzentra- tion von Glukose in ein unspezifisches chemisches Signal z.B. Konzentration von Wasserstoffperoxid umzuwandeln, welches sodann elektrochemisch in eine elektrische Größe (einen Strom oder ein Potential) umgewandelt wird. Es ist bekannt, daß diese Sensortypen eine geringe Sensitivität und grundlegende Probleme durch die unspezifische Elektrochemie an Sensorober¬ flächen, welche neben Wasserstoffperoxid viele andere biolo¬ gische Komponenten oxidieren kann, aufweisen.Enzyme sensors are known as detection systems for biological molecules, which use the catalytic effect of biomolecules to detect a specific biosignal e.g. the concentration of glucose in an unspecific chemical signal e.g. Convert concentration of hydrogen peroxide, which is then electrochemically converted into an electrical quantity (a current or a potential). It is known that these sensor types have a low sensitivity and have fundamental problems due to the unspecific electrochemistry on sensor surfaces which, in addition to hydrogen peroxide, can oxidize many other biological components.

Als Nachweissysteme für biologische Moleküle sind weiters optische Systeme wie Surface Plasmon Resonance - Sensoren bekannt, welche die Oberflächenbindung von Biomolekülen in ein optisches Signal umzuwandeln vermögen. Es ist jedoch weiters bekannt, daß der große apparative Aufwand und die zwar im Ver¬ gleich zu den Enzymsensoren bessere aber für viele Anwendungen trotzdem nicht ausreichende Empfindlichkeit dieser Sensoren diese Technik nur für manche Bereiche einsetzbar macht.Optical systems such as surface plasmon resonance sensors, which are able to convert the surface binding of biomolecules into an optical signal, are also known as detection systems for biological molecules. However, it is also known that the great expenditure on equipment and the sensitivity of these sensors, which is better than that of the enzyme sensors but inadequate for many applications, makes this technique usable only in some areas.

Die Erfindung zielt darauf ab durch einen neuartigen Sensor¬ aufbau diese grundlegenden Einschränkungen zu beseitigen. (siehe Fig. 1) Die Biokomponente soll in diesem Sensoraufbau nicht zur katalytischen Umsetzung des Analyten Verwendung finden, sondern als selektive Barriere zur umgebenden Lösung. Die Analyt dient sodann als Schlüssel zur On/Off Steuerung eines Ionenkanals. Durch diesen Ionenkanal können pro Sekunde und Kanal bis zu 107 Ionen in einen schmalen Membran - Elek¬ trodenspalt (10 - 300 um) fließen und dort direkt elektroche¬ misch (etwa 3 pA je offener Kanal) bestimmt werden. Eine Stö- rung des Meßsignals durch andere Komponenten der Lösung wird durch eine selektive Membran mit eingebauten Kanalproteinen unterdrückt.The aim of the invention is to eliminate these fundamental restrictions by means of a novel sensor design. (See Fig. 1) The biocomponent should not be used in this sensor structure for the catalytic conversion of the analyte, but as a selective barrier to the surrounding solution. The analyte then serves as the key to the on / off control of an ion channel. Through this ion channel, up to 10 7 ions per second and channel can flow into a narrow membrane electrode gap (10-300 μm) and be determined there directly electrochemically (about 3 pA per open channel). A disturbance tion of the measurement signal by other components of the solution is suppressed by a selective membrane with built-in channel proteins.

Die vorliegende Erfindung unterscheidet sich von WO 89/01159 durch die mit "Molecular Modelling" mögliche sub-Nanometer genaue Anordnung eines Liganden am Kanaleingang des Ionenka¬ nals WO 89/01159 beschreibt hingegen die Bindung von relative großen Rezeptormolekülen (Antikörper, Enzyme, Lektine,..) am Kanaleingang. Versuche zeigen jedoch, daß eine genaue Positio¬ nierung der Bindungsstelle des Rezeptormoleküls am Kanalein¬ gang technisch kaum möglich ist und daher eine Veränderung des Ionenstroms durch den Ionenkanal nur indirekt erfolgt, wobei der Effekt relativ gering ist. Die vorliegende Erfindung hin- gegen nutzt die hochgenaue Positionierung eines kleinen Ligan¬ den am Eingang des Kanals der sodann von einem großen Molekül (Analyt) erkannt und an der optimalen Stelle (Kanaleingang) gebunden wird. Diese Vorgangsweise ist nicht nur deutlich genauer sondern auch eine Umkehrung der Vorgangsweise von WO 89/01159 in dem das Rezeptorprotein, am Kanal immobili¬ siert, den Analyten biorekognitiv bindet, im vorliegenden Bio¬ sensor jedoch ein kleiner Ligand am Ionenkanal vom Analyten erkannt und gebunden wird.The present invention differs from WO 89/01159 in that the arrangement of a ligand at the channel entrance of the ion channel which is possible with "molecular modeling" is sub-nanometer accurate. WO 89/01159, on the other hand, describes the binding of relatively large receptor molecules (antibodies, enzymes, lectins, ..) at the channel entrance. However, experiments show that an exact positioning of the binding site of the receptor molecule at the channel entrance is hardly technically possible and therefore a change in the ion current through the ion channel takes place only indirectly, the effect being relatively small. The present invention, on the other hand, uses the highly precise positioning of a small ligand at the entrance to the channel, which is then recognized by a large molecule (analyte) and bound at the optimal location (channel entrance). This procedure is not only much more precise, but also a reversal of the procedure of WO 89/01159 in which the receptor protein, immobilized on the channel, binds the analyte biorecognitively, but in the present bio-sensor a small ligand on the ion channel is recognized and bound by the analyte becomes.

Die vorliegende Erfindung unterscheidet sich von US 5,234,566 durch die Verwendung einzelner Membranen, nicht einer Vielzahl zwangsweise identer Membranen. Auch ist der Aufbau der Membran nicht nur durch "seif assembling" möglich sondern kann auch durch andere Techniken erfolgen (z.B. LB-Filme) . Eine .Abhän- gigkeit der Leitfähigkeit vom Membranpotential ist nicht er¬ wünscht, ein optimales Verhalten des Kanals sollte nahezu dem Ohmschen Gesetz folgen.The present invention differs from US Pat. No. 5,234,566 in that it uses individual membranes, not a large number of necessarily identical membranes. The construction of the membrane is not only possible by "soap assembling" but can also be done by other techniques (e.g. LB films). A dependency of the conductivity on the membrane potential is not desired; an optimal behavior of the channel should almost follow Ohm's law.

Die vorliegende Erfindung unterscheidet sich von EP 0 441 120 A2 durch die nicht notwendige Vernetzung der Lipidmembran mit der Elektrodenoberfläche durch überbrückende Moleküle. Der dauernde Kontakt mit zwei wäßrigen Phasen ober- und unterhalb der Membran (Zeichen einer nicht stabilen Membran) , ist kein notwendiger Teil der vorliegenden Erfindung. Die Verwendung einer Rahmenstruktur über die sich die Lipidmembran weiterer¬ streckt vermeidet apolaren Wände, an die die Lipidmembran grenzt, welche instabile und daher undichte Stellen der Mem¬ bran darstellen würden.The present invention differs from EP 0 441 120 A2 in that the lipid membrane is not crosslinked to the electrode surface by bridging molecules. Continuous contact with two aqueous phases above and below membrane (sign of an unstable membrane) is not a necessary part of the present invention. The use of a frame structure over which the lipid membrane extends further avoids apolar walls to which the lipid membrane borders, which would represent unstable and therefore leaky areas of the membrane.

Die vorliegende Erfindung beinhaltet nicht die in WO 94/07593 geforderte direkte Vernetzung der Lipidmembran mit der Elek- trodenoberfläche -unter Verwendung überbrückender Linkerlipide. Ebenfalls unterscheidet sich die vorliegende Erfindung von EP 0 394 997 AI dadurch, daß der Inhalt der Erfindung den Auf¬ bau eines Biosensors unter Verwendung der Bindung eines Mole¬ küls an einen immobilisierten Liganden zur ein/aus Steuerung eines Ionenkanals in einer Membran nutzt und nicht den direk¬ ten Effekt organischer Moleküle auf den Kanal selbst. Weiters ist die multimere Strukur der Porine mit dem Anspruch 1 (sta¬ biles Molekül) nicht vereinbar. Das neuartige Sensorprinzip beruht auf folgenden Erkenntnissen:The present invention does not include the direct crosslinking of the lipid membrane with the electrode surface required in WO 94/07593 - using bridging linker lipids. The present invention also differs from EP 0 394 997 A1 in that the content of the invention does not use the structure of a biosensor using the binding of a molecule to an immobilized ligand for on / off control of an ion channel in a membrane the direct effect of organic molecules on the channel itself. Furthermore, the multimeric structure of the porins is not compatible with claim 1 (stable molecule). The new sensor principle is based on the following findings:

Als Ionenkanal kann das sehr gut untersuchte und leicht zu¬ gängliche Kanalpeptid Gramicidin als Basis eines neuen Affini¬ tätssensors dienen. Sowohl die Sequenzen als auch genaue Mole¬ küldaten unterschiedlicher Grandeidinderivate konnten in den letzten Jahren bestimmt werden. Die Sequenzhomologien sind durchwegs hoch. Die Ionenkanäle werden dabei durch 2 assozi¬ ierte Moleküle Gramicidin gebildet. Die Raumstruktur des Pep- tids ist dabei eine 6.3 - Helix die zentral einen offenen Ionenkanal bildet und mit Tryptophanresten an den beiden Membranseiten dynamisch verankert ist.The very well investigated and easily accessible channel peptide gramicidin can serve as the ion channel as the basis of a new affinity sensor. Both the sequences and the exact molecular data of different Grandeidin derivatives have been determined in recent years. The sequence homologies are consistently high. The ion channels are formed by 2 associated gramicidin molecules. The spatial structure of the peptide is a 6.3 - helix that forms an open ion channel in the center and is dynamically anchored with tryptophan residues on the two membrane sides.

Es konnte durch patch cla p Techniken gezeigt werden, daß an beiden Seiten der Membran Gramicidin - Monomere eingeschränkt frei floaten. Kommt es zu einer Assoziation von 2 Molekülen Gramicidin unterschiedlicher Membranseiten so bildet sich ein durchgängiger Ionenkanal, (siehe Fig. 1) Chemische Modifikationen der Gramicidinpeptide am N - Terminus können nach Deformylierung (= Abspaltung der Cappingstruktur des Peptids) 2 Moleküle Gramicidin zu einem kovalenten Dimer (z.B. mit Weinsäure oder Malonsäure als Bisamide) verknüpfen. (siehe Abbildung) Diese Gramicidin-Dimer bilden die im Sinne der Patentanmeldung notwendigen stabilen Ionenkanäle in natür¬ lichen und artifiziellen Membranen. Untersuchungen des Ionen¬ kanals zeigten eine Inhibierbarkeit des Ionentransfer durch eine starke Bindung von divalenten Kationen, öffnet oder schließt sich ein einzelner Ionenkanal in der Membran so kann dies direkt durch einen Ionenstrom von 107 Ionen/s durch die Membrankanal gemessen werden. Bisgramicidinmoleküle als Ionen¬ kanäle im Sinne der Patentanmeldung sind durch ihre hohe Stabilität, die gut chemische Charakterisierung, die Möglich- keit einer Vielzahl chemischer Modifikationen und insbesondere durch die hohe Leitfähigkeit der Kanäle besonders gut geeig¬ net.It was shown by patch cla p techniques that gramicidin monomers float freely on both sides of the membrane. If there is an association of 2 molecules of gramicidin from different membrane sides, a continuous ion channel is formed (see FIG. 1) Chemical modifications of the gramicidin peptides at the N - terminus can link 2 molecules of gramicidin to a covalent dimer (eg with tartaric acid or malonic acid as bisamides) after deformylation (= cleavage of the capping structure of the peptide). (See figure) These gramicidin dimers form the stable ion channels in natural and artificial membranes necessary for the purposes of the patent application. Investigations of the ion channel showed that the ion transfer could be inhibited by a strong binding of divalent cations, if a single ion channel opened or closed in the membrane, this could be measured directly by an ion current of 10 7 ions / s through the membrane channel. Bisgramicidin molecules as ion channels in the sense of the patent application are particularly suitable due to their high stability, the good chemical characterization, the possibility of a large number of chemical modifications and in particular the high conductivity of the channels.

Die zweite essentielle Komponente des neuen Sensorkonzepts ist die Membrantechnologie. Es wird dabei der Vorteil der black lipid membranes d.h. der freie Zugang zu beiden Membranseiten mit dem Vorteil der seif assembling membranes d.h der stabilen Anordnung eines selbst aufbauenden Lipidfilms kombiniert. Die Wahl eines Photogelpolymers als Trägermatrix für den Lipidfilm und die kovalente Kopplung der Lipidmonomer an diese Gelober¬ fläche stabilisiert den Lipidfilm gegen jede mechanische Zer¬ störung, bzw. gegen das Floaten des zweiten Lipidfilms der Doppelmembran.The second essential component of the new sensor concept is membrane technology. The advantage of black lipid membranes i.e. free access to both sides of the membrane combined with the advantage of seif assembling membranes, i.e. the stable arrangement of a self-assembling lipid film. The choice of a photogel polymer as a carrier matrix for the lipid film and the covalent coupling of the lipid monomer to this gel surface stabilizes the lipid film against any mechanical destruction or against the floating of the second lipid film of the double membrane.

Die dritte Neuerung im Rahmen des Sensorkonzepts soll die Ver¬ wendung von dünnschichttechnologischen Verfahrensschritten im Rahmen des Sensoraufbaus sein. Dabei soll zuerst der elektro¬ chemische Sensor nach etablierten dünnschichttechnologischen Verfahren gefertigt werden. Sodann soll mit Photolacken z.B der AZ-Serie oder mit Proh mid-Lack die Rahmenstruktur des Lipidfilms aufgebaut werden. Der nächste Schritt ist sodann die Füllung der hydrophoben Rahmenstruktur mit einem hydrophi¬ len Gel Photopolymer als Trägersystem für die Lipidmembran.The third innovation in the context of the sensor concept is to be the use of thin-film technological process steps in the context of the sensor construction. First, the electrochemical sensor is to be manufactured using established thin-film technology processes. Then the frame structure of the lipid film is to be built up with photoresists, for example the AZ series or with Proh mid lacquer. The next step is then the filling of the hydrophobic frame structure with a hydrophilic gel photopolymer as a carrier system for the lipid membrane.

Die Kopplung des On/Off Schalters auf den Gramicidin Kanal erfolgt sodann durch selektive chemische Modifikation der carboxyterminalen - Ethanolamin Cappingstruktur. Dabei soll ein kleines durch Biorekognition erkanntes Molekül durch die Bindung eines großes Moleküls den Ionenkanal reversibel ver¬ schließen, (siehe Fig. 1)The on / off switch is then coupled to the gramicidin channel by selective chemical modification of the carboxy-terminal ethanolamine capping structure. A small molecule recognized by biorecognition is intended to reversibly close the ion channel by binding a large molecule (see FIG. 1).

Die Erfindung zielt somit darauf ab eine neuartiges hochsensi¬ tives Sensorprinzip mit der theoretischen Empfindlichkeit eines einzigen Analytmoleküls zu schaffen. Die Empfindlichkeit wird nur durch die Bindungskonstante Ligand/Bindungsmolekül und durch den Zeitbedarf des statistischen Aufeinandertref fens der beiden Moleküle begrenzt.The invention thus aims to create a new type of highly sensitive sensor principle with the theoretical sensitivity of a single analyte molecule. The sensitivity is limited only by the binding constant ligand / binding molecule and by the time required for the statistical meeting of the two molecules.

Weiters zielt die Erfindung auf den Aufbau solcher Sensoren mit Dünnschichttechnolgie-kompatiblen Methoden wie Dünn- Schicht Substraten, Sputtern und Aufdampfen der Metallelektro¬ den, Einsatz von Photolacken und mit photosenstiven Polymeren hergestellten dünnschichttechnolgischen Lipidmembranträgem ab und beinhaltet darüber hinaus auch den neuartigen Aufbau von kovalent gebunden Lipidmembranen an Polymer-Flüssigkeits- Grenz f lachen .Furthermore, the invention aims at the construction of such sensors with methods compatible with thin-film technology such as thin-layer substrates, sputtering and vapor deposition of the metal electrodes, use of photoresists and thin-film technology lipid membrane supports produced with photosensitive polymers and also includes the novel structure of covalently bound Flat lipid membranes at the polymer-liquid interface.

Die moderne medizinisch-biochemische Analytik stellt immer größere Anforderungen an die Selektivität und Spezifität von Meßsystemen.Modern medical-biochemical analysis places increasing demands on the selectivity and specificity of measuring systems.

Schnellere und insbesondere einfachere Testverfahren in der Diagnostik ermöglichen vielfach erst die routinemäßige Bestim¬ mung wichtiger körpereigener und körperfremder Substanzen in der Arztpraxis oder zumindest abseits etablierter Universi- tätskrankenanstalten. Darüber hinaus kann auch die direkte Messung von Blut- oder Urininhaltsstoffen durch den Patienten neuartige Möglichkeiten eröffnen. Der Einsatz des neuen Sensortyps ermöglicht die Nutzung aller biochemischen und chemischen Affinitätreaktionen zwischen kleinen diagnostisch oder umweitanalytisch relevanten Ligan- denmolekülen und einem großen den Ionenkanal sperrenden Mole¬ kül.Faster and, in particular, simpler test methods in diagnostics often enable the routine determination of important endogenous and foreign substances in the doctor's office or at least outside of established university hospitals. In addition, the direct measurement of blood or urine substances by the patient can open up new possibilities. The use of the new sensor type enables the use of all biochemical and chemical affinity reactions between small ligand molecules which are relevant for diagnosis or for further analysis and a large molecule which blocks the ion channel.

Der Einsatz der Erfindung kann daher z.B. als Blut HIV-Anti¬ körper Sensor zur HIV-Diagnostik und zur Verlaufskontrolle von HIV-Infektionen erfolgen. Die derzeit im Handel befindlichen HIV-Tests sind trotz hoher Standards im diagnostischen Fenster von etwa 3 - 6 Wochen zu unsensitiv und damit in vielen Fällen zu unsicher. Dies führt einerseits zu quälender Unsicherheit für Betroffene bei unklaren Testergebnissen führen anderer- seits können dadurch in Blutbanken immer noch trotz sorgfälti¬ ger Überprüfungen positive Seren in den Verkehr gelangen.The use of the invention can therefore e.g. as a blood HIV antibody sensor for HIV diagnosis and for monitoring the course of HIV infections. Despite the high standards in the diagnostic window of around 3 to 6 weeks, the HIV tests currently on the market are too insensitive and therefore too unsafe in many cases. On the one hand, this leads to excruciating uncertainty for those concerned with unclear test results, and on the other hand, despite careful checks, positive sera can still get into circulation in blood banks.

Insbesondere in Ländern der dritten Welt sind die bestehenden Einmaltests zu teuer aber auch in vielen Fällen zu kompliziert in der Handhabung.In third world countries in particular, the existing one-time tests are too expensive, but in many cases too complicated to use.

Es kann daher ein selektives Peptid aus dem V3 - loop des gp41 Proteins als Erkennungsdeterminante an den Kanaleingang des Gramicidins gebunden werden.A selective peptide from the V3 loop of the gp41 protein can therefore be bound to the channel entrance of the gramicidine as a recognition determinant.

Die Sequenz LGLWGCSGKLIC (eine Teilsequenz des HIV-Proteins gp41) bindet nun ihrerseits an Serumantikörper von nahezu 100 % aller HlV-infizierten Personen. Zusätzlich muß die Sequenz LGMWGCSGKLIC für HIV-infizierte aus Westafrika verwen- det werden. Eine bindende Sequenz für HIV-2 lautet LNSWGCAFRQVC. Dabei bilden jeweils die beiden Cysteine sowohl bei HIV 1 als auch HIV 2 eine Disulfidbrücke die für die volle Antigenität des Peptids essentiell ist. In der Mitte der dadurch gebildeten sogenannten V3 - loop ist eine basische Aminosäure essentiell. Für manche seltene Seren können auch noch flankierende Sequenzen von etwa 5 Aminosäure die Anti¬ genität verbessern. Die Bindung des Serumantikörpers führt im Rahmen der Erfindung zu einer Kanalblockade. Dieser Effekt kann dann mit hoher Sensitivität und auch Selektivität gemessen werden. Nach einem Test wird der Sensor kurz mit einem Chaotrop gewaschen und der gebundene Antikörper entfernt. Sodann kann der Sensor für den nächsten Test verwendet werden.The sequence LGLWGCSGKLIC (a partial sequence of the HIV protein gp41) now in turn binds to serum antibodies of almost 100% of all HIV-infected people. In addition, the sequence LGMWGCSGKLIC must be used for HIV-infected people from West Africa. A binding sequence for HIV-2 is LNSWGCAFRQVC. The two cysteines form a disulfide bridge in both HIV 1 and HIV 2, which is essential for the full antigenicity of the peptide. A basic amino acid is essential in the middle of the so-called V3 loop. For some rare sera, flanking sequences of about 5 amino acids can also improve the anti-genicity. The binding of the serum antibody leads to a channel blockage within the scope of the invention. This effect can then be measured with high sensitivity and selectivity. After a test, the sensor is briefly washed with a chaotrope and the bound antibody is removed. The sensor can then be used for the next test.

Der Einsatz der Erfindung kann weiters auch als Harn Östrogen- sensor zur Bestimmung der fruchtbaren Tage erfolgen. Die Erkennung der fruchtbaren Tage im Rahmen des weiblichen Zyklu- ses ist durch die 24-48 Stunden vorangehende deutliche Harn- Östrogenspitze mit guter Genauigkeit möglich. ELISA- Tests zur Bestimmung dieses Harn-Parameters (oder von LH) sind seit einiger Zeit im Handel. Jedoch sind diese Tests nur Einmaltest und überaus teuer, so daß eine Dauermessung durch den Patien¬ ten nicht möglich ist. Das neue Sensorprinzip kann einen Sensorkopf ermöglichen der zumindest eine Zyklusdauer d.h. etwa 28 Tage verwendet werden kann und deutlich genauere Aus- sagen über den Zykluszustand ermöglicht. Dieses Testsystem kann sowohl bei unerfülltem Kinderwunsch die genaue Messung der empfängnisbereiten Tagen ermöglichen als auch als eine äußerst schonende Methode zur Empfängnisregelung verwendet werden. Die Überwachung der Wirkung von Antiöstrogenen im Rah- men der Tumortherapie stellt einen weiteren wichtigen Einsatz¬ bereich des Sensors dar. Auch im Verlauf einer Schwangerschaft ist die Messung des Östrogenspiegels von diagnostischer Bedeu¬ tung.The invention can also be used as a urine estrogen sensor for determining the fertile days. The detection of the fertile days in the context of the female cycle is possible with good accuracy due to the clear urine-estrogen peak preceding 24-48 hours. ELISA tests to determine this urine parameter (or LH) have been on the market for some time. However, these tests are only one-time tests and extremely expensive, so that continuous measurement by the patient is not possible. The new sensor principle can enable a sensor head that has at least one cycle duration, i.e. can be used for about 28 days and allows much more precise information about the cycle status. This test system can enable the exact measurement of the days ready for conception if the desire for children is not fulfilled, and can also be used as an extremely gentle method for regulating conception. Monitoring the effect of anti-estrogens in the context of tumor therapy is another important area of application for the sensor. Even during pregnancy, the measurement of the level of estrogen is of diagnostic importance.

Der Sensor kann über spezifische Antikörperbindung Östrogen und dessen 17-Glucuronido- und Sulfokonjugate erkennen. Die Menge der Harnöstrogene kann sodann als Zeitintegration über offene und geschlossene Zeiten an einem Kanal, oder auch als Mittelwertsstrom (bzw. dessen Zeitabhängigkeit ) an einigen 10 bis 100 Ionenkanälen bestimmt werden. Als großer Unterschied zu den vorangegangenen HIV Sensortyp soll hier jedoch in reversibler Weise die Bindung an den Kanal in einem Verdrän- gungsassay genutzt werden (siehe .Abbildung 1) . Als zusätzlich Komponente wird hier der Sensor mit einer analytpermeablen aber antikörperimpermeablen Membran überzogen. Im Intermem- branraum befinden sich Antikörper die selektiv mit Östrogen und seinen Harnderivaten reagieren und um einen gleichartigen Liganden am Ionenkanaleingang kompetitieren. So wird durch eine steigende Menge Harnöstrogen der Ionenfluß durch die Mem¬ bran von beinahe Null auf einen dem östrogenspiegel proportio¬ nalen Wert erhöht.The sensor can detect estrogen and its 17-glucuronido and sulfoconjugates via specific antibody binding. The amount of urinary oestrogens can then be determined as a time integration over open and closed times on one channel, or also as a mean current (or its time dependence) on some 10 to 100 ion channels. As a big difference to the previous types of HIV sensors, the binding to the channel in a use assay (see .Figure 1). As an additional component, the sensor is covered with an analyte-permeable but antibody-impermeable membrane. In the intermembrane space there are antibodies that react selectively with estrogen and its urinary derivatives and compete for a similar ligand at the ion channel entrance. Thus, the ion flow through the membrane is increased from almost zero to a value proportional to the estrogen level due to an increasing amount of urea estrogen.

Als äußere Membran kann dabei ein kommerziell erhältlicher Typ einer Dialysemembran verwendet werden.A commercially available type of dialysis membrane can be used as the outer membrane.

In einfacher Weise kann dieser Sensor sodann leicht verändert auch z.B. zur Messung des Hormons Progesteron verwendet werden.This sensor can then be easily changed, e.g. be used to measure the hormone progesterone.

Die Erfindung zielt darauf ab einen Sensor mit etwa folgendenThe invention aims at a sensor with approximately the following

Arbeitsschritten aufzubauen:To build up work steps:

1.) Chemische Synthese eines lonenkanals z.B. Bisgramicidin A und kovalente Kopplung der Liganden an den Kanaleingang1.) Chemical synthesis of an ion channel e.g. Bisgramicidin A and covalent coupling of the ligands to the channel entrance

2. ) Dünnschichttechnologischer Sensoraufbau2.) Thin-film technological sensor construction

3. ) Polymermembranaufbau des Sensors 4.) Lipidcoating des Sensors,3.) polymer membrane structure of the sensor 4.) lipid coating of the sensor,

5.) Einbau des ligandenmodifizierten Ionenkanals5.) Installation of the ligand-modified ion channel

6.) Einbau in ein Kunststoffgehäuse6.) Installation in a plastic housing

7. ) Für reversible Messungen Aufbringen des reversiblen7.) For reversible measurements apply the reversible

Bindungsmoleküls und Einbau einer semiper eablen Membran 8.) Messung mit dem IonenkanalsensorBinding molecule and incorporation of a semiper eable membrane 8.) Measurement with the ion channel sensor

Die chemische Synthese eine Ionenkanals kann am Beispiel des Ionenkanalpeptids Gramicidin erläutert werden. Die Synthese des Kanalpeptids Gramicidin A und dessen kovalenten Dimeren des Bisgramicidins geht von Gramicidin D aus. Mit Hilfe von silica oder reversed phase Chromatographie kann sodann reines Gramicidin A gewonnen werden. Dieses kann mit wasserfreier Salzsäure in einem organischen Lösungsmittel z.B. Methanol deformyliert werden. Das Produkt wird sodann wiederum mit reversed phase Chromatographie gereinigt. Die Dimerisierung erfolgt dann durch Kopplung mit einem Malonsäureester, Wein- säureester oder strukturell verwandten Crosslinkern. Dabei wird der Diethylester der Dicarbonsäure und Gramicidin A in DMF gelöst und langsam DPPA (Diphenylphosphorylazid) zuge¬ geben. Nach der Reaktion wird das Produkt wiederum mit reversed phase Chromatographie gereinigt.The chemical synthesis of an ion channel can be explained using the example of the ion channel peptide gramicidin. The synthesis of the channel peptide gramicidin A and its covalent dimers of bisgramicidine is based on gramicidin D. Pure gramicidin A can then be obtained with the aid of silica or reversed phase chromatography. This can be done with anhydrous Hydrochloric acid in an organic solvent such as methanol can be deformylated. The product is then again purified using reversed phase chromatography. The dimerization is then carried out by coupling with a malonic acid ester, tartaric acid ester or structurally related crosslinkers. The diethyl ester of dicarboxylic acid and gramicidin A is dissolved in DMF and DPPA (diphenylphosphorylazide) is slowly added. After the reaction, the product is again purified using reversed phase chromatography.

Die Kopplung der Affinitätsliganden an das carboxyterminale Ethanolamin (des Gramicidin A) erfolgt durch Aktivierung der beiden freien terminalen OH-Gruppen des Moleküls mit z.B. Divinylsulfon oder Chlora eisensäure-nitrophenylester. Sodann kann der Ligand durch seine OH, NH2, oder SH Gruppe selektiv terminal gebunden werden.The affinity ligands are coupled to the carboxy-terminal ethanolamine (of gramicidin A) by activating the two free terminal OH groups of the molecule with e.g. Divinyl sulfone or chloroformic acid nitrophenyl ester. The ligand can then be selectively terminally bound by its OH, NH2 or SH group.

Es können auch beide terminalen OH-Gruppen des Bisgramicidins mit Vinylsulfon oder Chlorameisensäure-nitrophenylester akti- viert und mit einem Diamin (Ethylendiamin) oder einer Di er- captoverbindung umgesetzt werden. Das sich durch die Umsetzung bildenden Bisgramicidinderivat trägt sodann zwei terminale reaktive Amino (oder SH) -Gruppen. Sodann kann mit Iodactamido-capronsäure-N-hydroxysuccinimid-estern die zur Kopplung von Histidinen nötige alkylierende Gruppe in das Molekül eingeführt werden.Both terminal OH groups of the bisgramicidine can also be activated with vinyl sulfone or nitrophenyl chloroformate and reacted with a diamine (ethylenediamine) or a dicapto compound. The bisgramicidine derivative formed by the reaction then carries two terminal reactive amino (or SH) groups. The iodactamido-caproic acid N-hydroxysuccinimide esters can then be used to introduce the alkylating group required for coupling histidines into the molecule.

Die Kopplung von östrogenen erfolgt an der Position 6 (Östriol, Östron) oder an Position 17 (Östradiol) der konju- gierten Ringsysteme. Als Liganden für östradiol dienen dabei 17ß-Östradiol 17-hemisuccinat, 17ß-Östradiol 3-gylcidylether oder 17ß-Östradiol 6- (O-carboxymethyl)oxi . 17ß-Östradiol 17-hemisuccinat und 17ß-Östradiol 6- (O-carboxymethyl) oxim sollen an das bereits beschriebene bisaminomodifizierten Bis- gramicidin über eine Amidbindung kovalent verknüpft werden. 17ß-Östradiol 3-gylcidylether kopplet direkt an das bereits beschriebene bisaminomodifizierten Bisgramicidin. Zur Kopplung des HlV-Peptid kann einerseits nach Schutz der Aminogruppe des Lysins (Arginins) im V3-loop die N-terninale .Aminogruppe zur Kopplung verwendet werden. Als zweite Strate- gie können einige zusätzliche terminale Histidine in das Peptid eingeführt werden. Diese können dann über alkylierende Crosslinker an den Carboxyterminus des Gramicidins gebunden werden.The estrogen is coupled at position 6 (estriol, estrone) or at position 17 (estradiol) of the conjugated ring systems. 17β-estradiol 17-hemisuccinate, 17ß-estradiol 3-glycidyl ether or 17ß-estradiol 6- (O-carboxymethyl) oxi serve as ligands for estradiol. 17ß-estradiol 17-hemisuccinate and 17ß-estradiol 6- (O-carboxymethyl) oxime are to be covalently linked to the bisaminomodified bisgramicidin already described via an amide bond. 17ß-estradiol 3-gylcidyl ether coupled directly to the bisaminomodified bisgramicidin already described. For coupling the HIV peptide, the N-terninal .amino group can be used for coupling on the one hand after protection of the amino group of the lysine (arginine) in the V3 loop. As a second strategy, some additional terminal histidines can be introduced into the peptide. These can then be linked to the carboxy terminus of gramicidine via alkylating crosslinkers.

Die Fertigung der Biosensorsubstrate erfolgt durch dünn- schichttechnologische Verfahren. Die Fertigung folgt dabei dem Schema siehe Beispiele.The biosensor substrates are manufactured using thin-film technology. The production follows the scheme, see examples.

Die nach den genannten Verfahren hergestellten beschichteten Sensorsubstrate werden sodann durch Polymer- dünnschichttech- nologische Verfahren für den Einsatz in elektrochemischen Ionenkanal-Biosensoren modifiziert.The coated sensor substrates produced by the processes mentioned are then modified by polymer thin-film technology processes for use in electrochemical ion channel biosensors.

Der Träger der Lipidbiomembran baut sich aus zwei unterschied- liehen photostrukturierbaren Polymeren auf: Einer Rahmenstruk¬ tur aus hydrophobem d.h. lipophilem Photolack und einem hydro¬ philes Photopolymer innerhalb der Rahmenstruktur als Membran¬ träger.The carrier of the lipid biomembrane is made up of two different photostructurable polymers: a frame structure made of hydrophobic, i.e. lipophilic photoresist and a hydrophilic photopolymer within the frame structure as a membrane carrier.

Die Rahmenstruktur aus hydrophobem d.h. lipophilem Photolack kann durch photostrukturierbaren Polymere aus der Halbleiter¬ industrie aufgebaut werden. Es eignen sich dafür einerseits etablierte Photolack vom Typ AZ (Hoechst) die durch nachfol¬ gendes Einbacken bei Temperaturen über 150 Grad völlig ver- netzten und daher völlig unlöslich werden. Ein photostruk- turierbarer Polyimidlack (z.B. Probimide 408) weist ebenfalls eine außergewöhnlich gute chemische Resistenz auf muß jedoch bei Temperaturen zwischen 350 und 400 Grad eingebrannt werden. Ausgehend von den in den letzten Jahren entwickelten hydropho- ben photostrukturierbaren Polymeren aus der Halbleiterindu¬ strie können analog auch hydrophile Polymersysteme aufgebaut werden. Dabei können biokompatible, hydrophile und quellfähige Polymervorstufen durch bivalente Photovernetzer vernetzt werden. Es bildet sich ein Polymernetzwerk mit steuerbarer Porengröße, welches als strukturierbares Trägermateric-.l zur Immobilisierung der Biokomponenten d.h. Lipidmembranen und Ionenkanäle dient.The frame structure made of hydrophobic, ie lipophilic, photoresist can be constructed using photostructurable polymers from the semiconductor industry. On the one hand, established photoresists of type AZ (Hoechst) are suitable for this, which through subsequent baking completely crosslink at temperatures above 150 degrees and therefore become completely insoluble. A photostructurable polyimide varnish (eg Probimide 408) also has exceptionally good chemical resistance, but must be baked at temperatures between 350 and 400 degrees. Starting from the hydrophobic photostructurable polymers from the semiconductor industry developed in recent years, hydrophilic polymer systems can also be constructed analogously. It can be biocompatible, hydrophilic and swellable Polymer precursors are cross-linked by bivalent photocrosslinkers. A polymer network with controllable pore size is formed, which serves as a structurable carrier material. 1 for immobilizing the biocomponents, ie lipid membranes and ion channels.

Insbesondere Polyvinylpyrrolidinpolymere mit hydrophilen Seitengruppen (-0H, Ester amide, . ) können als viskose Lösungen durch Spinnprozesse auf die Sensorrohlinge aufgebracht werden und durch Photocrosslinker (meist Carben und Nitren - Bildner z.B. 4,4'-Biazidostilben-2,2'-disulfonsäure) mit langwelligem UV (350 - 390 n ) zu Polymerfilmen vernetzt werden. Die reak¬ tiven funktioneilen Gruppen des Polymers können dann aktiviert und zur Kopplung von Proteinen, Peptiden und Lipiden verwendet werden.In particular, polyvinylpyrrolidine polymers with hydrophilic side groups (-0H, ester amides,.) Can be applied to the sensor blanks as viscous solutions by spinning processes and by photocross linkers (mostly carbene and nitrene formers, for example 4,4'-biazidostilbene-2,2'-disulfonic acid) cross-linked with long-wave UV (350 - 390 n) to polymer films. The reactive functional groups of the polymer can then be activated and used to couple proteins, peptides and lipids.

Da Lipidfilme sowohl untereinander als auch an der Zellwand nicht durch kovalente Wechselwirkungen gebunden sind, können sie leicht unter desintegrierenden Bedingungen extrahiert werden. Dazu eignen sich hydrophobe Agenzien, Detergentien und Alkali . Zum Aufbau einer stabilen Lipidmembran müssen zwei grundsätzliche Probleme gelöst werden: Stabile Bindung des Lipidfilms an die Unterlage und das Verhindern des Abfloatens des oberen Lipidfilms im Lipiddoppellayer .Since lipid films are not bound to each other or to the cell wall by covalent interactions, they can easily be extracted under disintegrating conditions. Hydrophobic agents, detergents and alkali are suitable for this. To build a stable lipid membrane, two basic problems have to be solved: stable binding of the lipid film to the base and preventing the upper lipid film from flowing off in the lipid double layer.

Die stabile Bindung des unteren Lipidfilms an die Gelober¬ fläche erfolgt durch hydrophobe, ionische oder kovalente Kopp¬ lung von z.B. Thiolipiden an eine chemisch reaktive OberflächeThe lower lipid film is stably bound to the gel surface by hydrophobic, ionic or covalent coupling of e.g. Thiolipids on a chemically reactive surface

Dabei wird vor der Aufbringung des z.B. seif assembling oder LB Thio-Lipidfilms auf die Oberfläche dieselbe in folgender Weise modifiziert: Einführung reaktiver OH-Gruppen (z.B. durch Sauerstoffplasma) und Kopplung der OH-Gruppen mit bivalenten OH und SH reaktiven Crosslinkern wie z.B. Divinylsulfon. Sodann wird der in der genannten Weise aktivierte Sensor in ein Bad mit in organischem LM gelösten Thio-Lipiden getaucht, es erfolgt das seif assembling.Before applying the eg seif assembling or LB thio-lipid film to the surface, the same is modified in the following way: introduction of reactive OH groups (eg by means of oxygen plasma) and coupling of the OH groups with bivalent OH and SH reactive crosslinkers such as divinyl sulfone. The sensor activated in the manner mentioned is then immersed in a bath with thiolipids dissolved in organic LM, and the soap is assembled.

Der zweite Lipidfilm kann am ersten kovalent gebunden Lipid¬ film durch den Einbau von symetrischen Bis-Lipiden verankert werden. Diese tragen ebenfalls mindestens eine Thiogruppe um an der Gelmatrix fixiert zu sein. Ein Anteil an frei beweg¬ lichen Lipiden ermöglicht die fluide Struktur der Lipidmem- bran.The second lipid film can be anchored to the first covalently bound lipid film by incorporating symmetrical bis-lipids. These also carry at least one thio group in order to be fixed on the gel matrix. A proportion of freely movable lipids enables the fluid structure of the lipid membrane.

Der Einbau des modifizierten Gramicidinkanals erfolgt im gleichen Arbeitsschritt mit dem seif assembling des Lipid¬ films. Das Liganden - modifizierte Bisgramicidin besitzt noch eine freie reaktive Gruppe am zweiten Carboxyterminus des Bis- gramicidinmoleküls. Diese kann in analoger Weise wie die Lipide kovalent mit der Geloberfläche koppeln und so den Kanal fix (d.h. kovalent) in der Membran verankern.The modified gramicidine channel is installed in the same step with the soap assembling of the lipid film. The ligand-modified bisgramicidin still has a free reactive group on the second carboxy terminus of the bisgramicidin molecule. In an analogous manner to the lipids, this can couple covalently to the gel surface and thus anchor the channel firmly (i.e. covalently) in the membrane.

Durch Wechselstrom-Leitfähigkeit, Phasenwinkel und Impedanz kann die Ausbildung von Inhomogenitäten und die Dichtheit der Lipidmembranschichten auf Metalloberflächen direkt beobachtet und quantifiziert werden. Insbesondere die Bildung seif assembling von Filmen äußert sich direkt in einer Änderung der Sensorkapazität.The formation of inhomogeneities and the tightness of the lipid membrane layers on metal surfaces can be directly observed and quantified by alternating current conductivity, phase angle and impedance. In particular, the formation of self-assembling films manifests itself directly in a change in the sensor capacity.

Elektrochemische Leitfähigkeit- und Strommessungen ähnlich der Patch clamp Messung an Ionenkanälen in intakten Zellen oder an Membranfragmenten in der Spitze von Glaspipetten durch "mega seal" gebunden sind das fundamentale Meßprinzip des Ionen- kanalbiosensors.Electrochemical conductivity and current measurements similar to the patch clamp measurement on ion channels in intact cells or on membrane fragments in the tip of glass pipettes bound by "mega seal" are the fundamental measuring principle of the ion channel biosensor.

Die in der genannten Weise aufgebauten Sensoren liefern als Mikroleitfähigkeitsbiosensoren ein dem Ionenfluß proportiona- les Stromsignal. Als Meßplätze sind z.B. mit OPA 128 (Burr Brown) Eingangsstu¬ fen ausgestattet Verstärker geeignet. Auch alle Meßplätze für Einzelionenkanalmessungen in der patch clamp Technik im Bereich bis 0.1 pA sind für den neuartigen im Rahmen der Er- findung beschriebenen Sensoren optimal geeignet.The sensors constructed in the manner mentioned deliver a current signal proportional to the ion flow as micro-conductivity biosensors. Amplifiers equipped with OPA 128 (Burr Brown) input stages are suitable as measuring stations. All measuring stations for single ion channel measurements in the patch clamp technique in the range up to 0.1 pA are also optimally suitable for the novel sensors described in the context of the invention.

Die Erfindung wird nachfolgend an Hand von Ausführungsbeispie¬ len näher erläutertThe invention is explained in more detail below on the basis of exemplary embodiments

Beispiel 1: Aufbau der SensorsubstrateExample 1: Structure of the sensor substrates

• Verwendung von Sensorsubstraten aus Aluminiumoxid, Glas oder• Use of sensor substrates made of aluminum oxide, glass or

Kunststoff (Polycarbonat)Plastic (polycarbonate)

• Herstellung von Photomasken über CAD, PS-File, Photoplotter, Eisenoxidmaske, bzw. Kunststofffilmmaske• Production of photomasks via CAD, PS file, photoplotter, iron oxide mask or plastic film mask

• Photolack AZ5218 E, positiv 5000 rpm, 30 s• Photoresist AZ5218 E, positive 5000 rpm, 30 s

• Mask-aligner: Karl Süss Modell MA 45, 360 n 350 W, 7.5 s oder Photolack-Entwickler AZ-Developer/Hoechst 60 s• Mask aligner: Karl Süss model MA 45, 360 n 350 W, 7.5 s or photoresist developer AZ-Developer / Hoechst 60 s

• Einbacken 120°C, 30 min • Hochvakuumbedampfung bzw. Sputtern der Elektroden z.B. 50 nm Titan, 100 nm Platin, oder Gold bzw. Palladium, Anlage: Balzers, ESQ 110, BB800 059BD mit 270° Elektronen- strahlumienkkanone bei 2.10-5 Torr, 150°C 1 nm/s bzw. Labor¬ anlage der Fa. Balzers • Floaten: 30 min, 80°C, Ultraschall mit AZ-Remover• Einbacken 120 ° C, 30 min • Hochvakuumbedampfung or sputtering of the electrodes, for example, 50 nm titanium, 100 nm platinum, or gold or palladium complex: Balzers, ESQ 110, BB800 059BD 270 ° electron strahlumienkkanone at 2.10- 5 Torr , 150 ° C 1 nm / s or laboratory system from Balzers • Float: 30 min, 80 ° C, ultrasound with AZ remover

• Isopropanol Waschschritt• Isopropanol wash step

• Sägen der Wafer mit 100 um Diamantsäge mit Computersteuerung• Sawing the wafers with 100 µm diamond saw with computer control

• Aufbringen einer Ag/AgCl-Referenzelektrode durch Ag-Poly- er-Lack und Curing bei 110°C, 30 min• Application of an Ag / AgCl reference electrode through Ag polymer lacquer and curing at 110 ° C, 30 min

Beispiel 2: Reinigung des Gramicidin AExample 2: Purification of Gramicidin A

Gramicidin D wird auf einer Sl - 100 Polyol Säule der Firma Serva mit einem Gradient von 0 - 80 % Methanol/Wasser von den anderen Gramicidinen (B,C) abgetrennt. Beispiel 3: Carboxyterminale Modifikation des Gramicidins 500 mg Gramicidin A werden mit 10 ml Benzol gerührt, ein 3 - 5 facher Überschuß des Carboxygruppen modifizierten Liσanden zugegeben und mit 10 fächern molaren Überschuß eines Carbodiimids (z.B. Dicyciohexylcarbodiimid) versetzt. Die Reaktion wird bei Raumtemperatur im Dunkeln vorgenommen. Der Verlauf der Reaktion wird mit auf Siligagel-Platten mit Hilfe der Dünnschichtchromatographie (Chloroform/Methanol/Wasser: 100/10/1) verfolgt.Gramicidin D is separated from the other gramicidines (B, C) on a Sl - 100 polyol column from Serva with a gradient of 0 - 80% methanol / water. Example 3: Carboxy-Terminal Modification of Gramicidine 500 mg of gramicidin A are stirred with 10 ml of benzene, a 3 to 5-fold excess of the carboxy-modified ligand is added, and 10-fold molar excess of a carbodiimide (for example dicyciohexylcarbodiimide) is added. The reaction is carried out at room temperature in the dark. The course of the reaction is monitored on silica gel plates using thin layer chromatography (chloroform / methanol / water: 100/10/1).

Das Reaktionsgemisch wird sodann auf einer präparativen C-18 reversed phase Säule mit Methanol/Wasser gereinigt.The reaction mixture is then purified on a preparative C-18 reversed phase column with methanol / water.

Beispiel 4: Synthese des Bisgramicidins Das Ausgangsprodukt ist desformyliertes Gramisidin A, welches durch Spaltung von Gramicidin A mit wasserfreier Salzsäure in Methanol und nachfolgende Reingung mit präparativen C-18 reversed phase Säule mit Methanol/Wasser synthetisch gewonnen wird.Example 4: Synthesis of bisgramicidine The starting product is deformylated gramisidine A, which is synthetically obtained by cleavage of gramicidin A with anhydrous hydrochloric acid in methanol and subsequent purification using a preparative C-18 reversed phase column with methanol / water.

Die Dimerisierung des ist desformyliertes Gramicidin A erfolgt durch Zusatz eines 5 fachen molaren Überschußes von Diphenyl- phosphorylazid zu einer Lösung von einem molaren Teil desfor- myliertem Gramicidin A und einem Überschuß (3 - 10) fach der Dicarbonsäure (z.B. Malonsäure) in Dimethylformamid bei unter - 10 °C. Die Reaktionsdauer bei 0 °C beträgt 24 - 48 Stunden. Nach Stoppen der Reaktion mit Ethanol wird das Reaktionsge¬ misch nach einigen organisch/wässrigen Extraktionsschritten zur Trockene eingeengt und nach Aufnehmen des Rückstands in einer kleien Menge Methanol auf einer präparativen C-18 reversed phase Säule mit dem Laufmittelsystem Methanol Wasser (90/10 bis 95/5) gereinigt.The dimerized gramicidin A is deformylated by adding a 5-fold molar excess of diphenylphosphorylazide to a solution of a molar part of deformylated gramicidin A and an excess (3-10) fold of the dicarboxylic acid (eg malonic acid) in dimethylformamide at below - 10 ° C. The reaction time at 0 ° C is 24-48 hours. After the reaction has been stopped with ethanol, the reaction mixture is evaporated to dryness after a few organic / aqueous extraction steps and, after the residue has been taken up in a small amount of methanol, on a preparative C-18 reversed phase column with the solvent system methanol / water (90/10 to 95 / 5) cleaned.

Beispiel 5: Aufbau der hydrophoben Rahmenstruktur * Der Polyimidphotolack Probimide 408 wird durch Spincoaten auf den Sensorchip aufgebracht (5000 rpm, 2 min) . * Softbake bei 110 °C für 15 Minuten * Belichtung mit Photomaske mit UV (365 nm) 10 sExample 5: Structure of the hydrophobic frame structure * The polyimide photoresist Probimide 408 is applied to the sensor chip by spin coating (5000 rpm, 2 min). * Softbake at 110 ° C for 15 minutes * Exposure with photomask with UV (365 nm) 10 s

* Postbake bei 100 °C für 5 Minuten* Postbake at 100 ° C for 5 minutes

* Entwicklen mit Tauch- oder Sprayentwickler 60 s (z.B. QZ 3301) * Curing: Linear in 1 h auf 350 °C, dann 350 °C für 30 Minuten* Develop with immersion or spray developer for 60 s (e.g. QZ 3301) * Curing: Linear in 1 h to 350 ° C, then 350 ° C for 30 minutes

Beispiel 6: Aufbau des hydrophilen Lipidträgerpolymers Das hydrophile photovernetzte Gel wird nach folgender Vor¬ schrift aufgebaut: 2.5 % (w/v) PVP (Polyvinylpyrrolidon MW: 360.000 oder 1.000.000) in destilliertem Wasser und 0.75 % (w/v) 4.4'-Diazidostilben-2,2 '-disulfonsäure in destilliertem Wasser werden gemischt (3:2 v/v). Die Präpolymerlösung wird entweder in die Photolackrahmenstruktur pipettiert oder der Sensor wird durch ein Eintauch- oder Spinbeschichtungsverfahren mit der Präpolymerlösung überzogen.Example 6: Structure of the hydrophilic lipid carrier polymer The hydrophilic photocrosslinked gel is built up according to the following instructions: 2.5% (w / v) PVP (polyvinylpyrrolidone MW: 360,000 or 1,000,000) in distilled water and 0.75% (w / v) 4.4 ' -Diazidostilben-2,2'-disulfonic acid in distilled water are mixed (3: 2 v / v). The prepolymer solution is either pipetted into the photoresist frame structure or the sensor is coated with the prepolymer solution by an immersion or spin coating process.

Das Photopolymer wird durch Belichten mit einer 50 W Quecksil¬ berdampflampe für 5 s durch Einwirken von UV-A und UV-B gehär- tet und überschüssiger Vernetzer durch einen Waschschritt in Phosphatpuffer pH = 7.0 0.1 M entfernt.The photopolymer is cured by exposure to a 50 W mercury vapor lamp for 5 s by exposure to UV-A and UV-B, and excess crosslinker is removed by a washing step in phosphate buffer pH = 7.0 0.1 M.

Beispiel 7: Aufbau des BiosensorsExample 7: Structure of the biosensor

Der Ablauf der Beschichtungsschritte und des Meßaufbaus ist in Figur 2 zusammengefaßt.The sequence of the coating steps and the measurement setup is summarized in Figure 2.

Beispiel 8: Reinigung von PlatinsensorelektrodenExample 8: Cleaning platinum sensor electrodes

Nach der Verwendung von löslichen Thio-Verbindungen sindAfter using soluble thio compounds

Metalloberflächen chemisch bzw. elektrochemisch blockiert = derivatisiert. Um die kovalent Bindung der Thiole mit der Elektrodenoberfläche zu brechen wird die Elektrode elektroche¬ misch zwischen -600 und +1200 mV (bis in den Bereich der Sauerstoffentwicklung) versus Ag/AgCl gereinigt. Die elektro¬ chemische Reinigung erfolgt durch Anlegen einer zeitlich variablen Spannung an einen in neutralen Puffer eingetauchten Sensor. Beispiel 9: Lipidbeschichtung der Sensorelektroden Die in Trifluorethanol gelösten Lipide (2-10 μg/100 cm2 Ober¬ fläche) werden an einer Luft/Flüssigkeit Phasengrenze auf einem LB-Trog gespreitet. In einem automatisierten LB-System wird der Lipidfilm von einer fluiden in eine semikristalline Phase komprimiert. Der Sensor wird sodann durch den Lipidfilm gezogen wobei ein Nanoaktuator der Firma Physik-Instrumen¬ te/Deutschland (von einem DC-Motor mit der Auflösung von 60 nm angetrieben) verwendet wird.Metal surfaces chemically or electrochemically blocked = derivatized. In order to break the covalent bond between the thiols and the electrode surface, the electrode is cleaned electrochemically between -600 and +1200 mV (up to the range of oxygen evolution) versus Ag / AgCl. The electro-chemical cleaning is carried out by applying a time-variable voltage to a sensor immersed in neutral buffer. Example 9: Lipid coating of the sensor electrodes The lipids (2-10 μg / 100 cm 2 surface area) dissolved in trifluoroethanol are spread on an air / liquid phase boundary on an LB trough. In an automated LB system, the lipid film is compressed from a fluid to a semi-crystalline phase. The sensor is then drawn through the lipid film using a nano actuator from Physik-Instrumen¬ te / Germany (driven by a DC motor with a resolution of 60 nm).

Ungesättigte Lipidfilme werden mit einer Photomaske bedeckt und durch das Licht einer 50W Mitteldruck Quecksilberdampf¬ lampe etwa 120 Sekunden vernetzt. Dabei werden die Lipide sowohl innerhalb der Lipidschicht als auch mit einem reaktiven Träger vernetzt.Unsaturated lipid films are covered with a photomask and crosslinked for about 120 seconds by the light of a 50W medium pressure mercury vapor lamp. The lipids are cross-linked both within the lipid layer and with a reactive carrier.

Der Einbau modifizierter Kanäle erfolgt in analoger Weise mit Mischungen aus Lipid und Kanalpeptiden. Modified channels are installed in an analogous manner with mixtures of lipid and channel peptides.

Claims

Patentansprüche: Claims: 1. Biosensor, dadurch gekennzeichnet, daß als Meßprinzip ein Ionenstrom durch die Kanäle von in Lipidmembranen eingebauten kanalbildenden Molekülen verwendet wird, wobei durch die Bin¬ dung von Effektormolekülen, welche beliebige Moleküle dar¬ stellen die an einen Liganden aus der Gruppe von Hormonen, Peptiden, Enzyminhibitoren, Umwelttoxinen, Pharmawirkstoffen, Thiolen, Chelatbildnern und toxischen Substanzen mit einem Molekulargewicht unter 2000 Dalton und Kombinationen davon binden, der Ionenstrom verringert wird, und dabei die Ver¬ ringerung durch die Bindung von Effektormolekülen ein am Kanal kovalent immobilisierten Ligandenmolekülen bewirkt wird, und jeder Kanal aus einem stabilen Molekül oder mehreren stark gebundenen, nicht in Untereinheiten dissozierenden Molekülen besteht.1. Biosensor, characterized in that an ion current through the channels of channel-forming molecules built into lipid membranes is used as the measuring principle, with the binding of effector molecules, which represent any molecules, to a ligand from the group of hormones, peptides , Enzyme inhibitors, environmental toxins, active pharmaceutical ingredients, thiols, chelating agents and toxic substances with a molecular weight below 2000 daltons and combinations thereof bind, the ion current is reduced, and the reduction is caused by the binding of effector molecules, a ligand molecule covalently immobilized on the channel, and each channel consists of a stable molecule or several strongly bound molecules that do not dissociate into subunits. 2. Biosensor nach Anspruch 1, dadurch gekennzeichnet, daß als Meßsignal der Ionenstrom durch die Kanäle gemessen wird, wobei die Messung elektrochemisch über die Ionenkonzentrationen, als Leitfähigkeit quer durch Membran, als Kapazität der Membran oder als Konzentration fluoreszierender Ionen wie Europium im inneren Membrankompartiment erfolgen kann.2. Biosensor according to claim 1, characterized in that the ion current through the channels is measured as a measurement signal, the measurement being carried out electrochemically via the ion concentrations, as conductivity across the membrane, as the capacity of the membrane or as a concentration of fluorescent ions such as europium in the inner membrane compartment can. 3. Biosensor nach Anspruch 1 und 2, dadurch gekennzeichnet, daß die Lipidmembran über Metall- oder Halbleiterelektroden direkt oder mit einem Submembranvolumen von weniger als 3 cm3 angebracht ist, wobei die auf dem Sensorsubstrat aufgebrachten Elektroden durch Dünnschichttechnologie, Dickschichttechnolo- gie oder aus dünnen gewalzten Metallfolien gefertigt sind.3. Biosensor according to claim 1 and 2, characterized in that the lipid membrane is attached directly via metal or semiconductor electrodes or with a sub-membrane volume of less than 3 cm 3 , the electrodes applied to the sensor substrate by thin-film technology, thick-film technology or from thin rolled metal foils are made. 4. Biosensor nach Anspruch 1 und 2, dadurch gekennzeichnet, daß die Lipiάmembran durch wasserhaltige und/oder leitfähige kovalent oder ionisch vernetzte Polymere, Biopolymere, Gele, Dendrimere oder kristalline Festkörper getragen wird. 4. Biosensor according to claim 1 and 2, characterized in that the lipid membrane is carried by water-containing and / or conductive covalently or ionically crosslinked polymers, biopolymers, gels, dendrimers or crystalline solids. 5. Biosensor nach Anspruch 1, 2 und 4, dadurch gekennzeichnet, daß die Lipidmembran auf den wasserhaltigen und oder leitfähi¬ gen kovalent oder ionisch vernetzten Polymeren, Biopolymeren, Gelen, Dendrimeren oder kristallinen Festkörpern kovalent gebunden ist.5. Biosensor according to claim 1, 2 and 4, characterized in that the lipid membrane is covalently bound to the water-containing and / or conductive covalently or ionically crosslinked polymers, biopolymers, gels, dendrimers or crystalline solids. 6. Biosensor nach Anspruch 1 und 2, dadurch gekennzeichnet, daß der Rahmen für die Lipidmembran durch Photostrukturieren eines Dünnfilms von Polymeren, Gläsern, Keramiken, Kohlen- stoff, Metallen oder Halbleiter erzeugt sind.6. Biosensor according to claim 1 and 2, characterized in that the frame for the lipid membrane is produced by photostructuring a thin film of polymers, glasses, ceramics, carbon, metals or semiconductors. 7. Biosensor nach Anspruch 1 und 2, dadurch gekennzeichnet, daß als Kanalmoleküle Peptide oder Proteine verwendet werden.7. Biosensor according to claim 1 and 2, characterized in that peptides or proteins are used as channel molecules. 8. Biosensor nach Anspruch 1, 2 und 7, dadurch gekennzeichnet, daß als Kanal ein Peptidkanal mit einer 6.3 Helix verwendet wird.8. Biosensor according to claim 1, 2 and 7, characterized in that a peptide channel with a 6.3 helix is used as the channel. 9. Biosensor nach Anspruch 1, 2, 7 und 8, dadurch gekennzeich- net, daß als Kanäle Peptidkanäle aus Gramicidinen und deren kovalent verknüpften Dimeren insbesondere N- terminal ver¬ knüpften Dimeren eingesetzt werden.9. Biosensor according to claim 1, 2, 7 and 8, characterized in that peptide channels made of gramicidines and their covalently linked dimers, in particular N-terminally linked dimers, are used as channels. 10. Biosensor nach Anspruch 1, 2 und 7, dadurch gekennzeich- net, daß als Kanäle kovalent vernetzte Alantethicin Kanäle ver¬ wendet werden.10. Biosensor according to claim 1, 2 and 7, characterized in that covalently crosslinked alantethicin channels are used as channels. 11. Biosensor nach Anspruch 1, 2 und 7, dadurch gekennzeich¬ net, daß als Kanäle Bakterientoxine verwendet werden.11. Biosensor according to claim 1, 2 and 7, characterized gekennzeich¬ net that bacterial toxins are used as channels. 12. Biosensor nach Anspruch 1, 2 und 7, dadurch gekennzeich¬ net, daß die Bindung des Liganden am oder nahe dem Carboxyter- minus des Kanalpeptids erfolgt.12. Biosensor according to claim 1, 2 and 7, characterized gekennzeich¬ net that the binding of the ligand takes place on or near the carboxyter minus the channel peptide. 13. Biosensor nach Anspruch 1 und 2 dadurch gekennzeichnet, daß als Kanäle synthetische zyklische Peptide oder zyklische aus Zuckereinheiten aufgebaute Moleküle wie hydrophob modifi¬ zierte Cyclodextrine verwendet werden.13. Biosensor according to claim 1 and 2, characterized in that synthetic cyclic peptides or cyclic as channels Molecules built up from sugar units such as hydrophobically modified cyclodextrins can be used. 14. Verwendung des Biosensors nach Anspruch 1 und 2 zur Mes- j sung von Hormonen, viralen Proteinen und Peptiden, bakteriel¬ len Proteinen und Peptiden, Umweltgiften, humanen Proteinen und artifiziellen Toxinen. 14. Use of the biosensor according to claim 1 and 2 for measuring hormones, viral proteins and peptides, bacterial proteins and peptides, environmental toxins, human proteins and artificial toxins.
PCT/AT1995/000197 1994-10-19 1995-10-11 Bio-recognition-controlled, ion-flow modulating bio-sensor Ceased WO1996012957A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP95933226A EP0734528A1 (en) 1994-10-19 1995-10-11 Bio-recognition-controlled, ion-flow modulating bio-sensor

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AT0197094A AT402935B (en) 1994-10-19 1994-10-19 BIORECOGNITION-CONTROLLED, ION FLOW-MODULATING BIOSENSOR
ATA1970/94 1994-10-19

Publications (1)

Publication Number Publication Date
WO1996012957A1 true WO1996012957A1 (en) 1996-05-02

Family

ID=3525002

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AT1995/000197 Ceased WO1996012957A1 (en) 1994-10-19 1995-10-11 Bio-recognition-controlled, ion-flow modulating bio-sensor

Country Status (3)

Country Link
EP (1) EP0734528A1 (en)
AT (1) AT402935B (en)
WO (1) WO1996012957A1 (en)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997025616A1 (en) * 1996-01-11 1997-07-17 Australian Membrane And Biotechnology Research Institute Ion channel sensor typing
WO1997029366A1 (en) * 1996-02-08 1997-08-14 Australian Membrane And Biotechnology Research Institute Enzyme detection biosensors
WO1997043274A1 (en) * 1996-05-13 1997-11-20 Australian Membrane And Biotechnology Research Institute Improved reservoir components
EP0791176A4 (en) * 1994-11-16 1999-11-10 Au Membrane & Biotech Res Inst Detection device and method
WO2003095669A1 (en) * 2002-05-10 2003-11-20 The Texas A & M University System Stochastic sensing through covalent interactions
AU768862B2 (en) * 1998-06-12 2004-01-08 Xention Limited High throughput screen
EP1019526A4 (en) * 1996-09-27 2004-08-25 Univ Australian METHOD FOR DETERMINING THE ION CHANNEL ACTIVITY OF A SUBSTANCE

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989001159A1 (en) * 1987-07-27 1989-02-09 Commonwealth Scientific And Industrial Research Or Receptor membranes
WO1990002327A1 (en) * 1988-08-18 1990-03-08 AUSTRALIAN MEMBRANE AND BIOTECHNOLOGY RESEARCH INSTITUTE LTD., Commonwealth Scientific and Industrial Research Organization Improvements in sensitivity and selectivity of ion channel membrane biosensors
EP0394997A1 (en) * 1989-04-26 1990-10-31 Europäisches Laboratorium Für Molekularbiologie (Embl) Biospecific membranes incorporating porins and their use
EP0441120A2 (en) * 1990-01-09 1991-08-14 Yeda Research And Development Co. Ltd. Biosensors
WO1993010212A1 (en) * 1991-11-18 1993-05-27 Case George D Thin membrane sensor with biochemical switch
WO1994012875A1 (en) * 1992-12-03 1994-06-09 Australian Membrane And Biotechnology Research Institute Analyte detection by competitive inhibition of ion channel gating
WO1995016206A1 (en) * 1993-12-06 1995-06-15 Biosystems Technology Corporation Composition and method for detection of analytes

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1104883A3 (en) * 1992-10-01 2001-07-18 Australian Membrane And Biotechnology Research Institute Improved sensor membranes

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989001159A1 (en) * 1987-07-27 1989-02-09 Commonwealth Scientific And Industrial Research Or Receptor membranes
WO1990002327A1 (en) * 1988-08-18 1990-03-08 AUSTRALIAN MEMBRANE AND BIOTECHNOLOGY RESEARCH INSTITUTE LTD., Commonwealth Scientific and Industrial Research Organization Improvements in sensitivity and selectivity of ion channel membrane biosensors
EP0394997A1 (en) * 1989-04-26 1990-10-31 Europäisches Laboratorium Für Molekularbiologie (Embl) Biospecific membranes incorporating porins and their use
EP0441120A2 (en) * 1990-01-09 1991-08-14 Yeda Research And Development Co. Ltd. Biosensors
WO1993010212A1 (en) * 1991-11-18 1993-05-27 Case George D Thin membrane sensor with biochemical switch
WO1994012875A1 (en) * 1992-12-03 1994-06-09 Australian Membrane And Biotechnology Research Institute Analyte detection by competitive inhibition of ion channel gating
WO1995016206A1 (en) * 1993-12-06 1995-06-15 Biosystems Technology Corporation Composition and method for detection of analytes

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
M. ERAY ET AL: "Highly stable bilayer lipid membranes (BLMs) formed on microfabricated polyimide apertures.", BIOSENSORS & BIOELECTRONICS, vol. 9, pages 343 - 351, XP000566057 *
Y. UMEZAWA ET AL: "Development of biological and synthetic receptor-embedded lipid membrane assemblies with molecular recognition and signal transduction/amplification functions.", NEW FUNCTIONALITY MATERIALS, VOLUME B SYNTHESIS AND FUNCTION CONTROL OF BIOFUNCTIONALITY MATERIALS., AMSTERDAM, NL, pages 111 - 116, XP000566146 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0791176A4 (en) * 1994-11-16 1999-11-10 Au Membrane & Biotech Res Inst Detection device and method
WO1997025616A1 (en) * 1996-01-11 1997-07-17 Australian Membrane And Biotechnology Research Institute Ion channel sensor typing
WO1997029366A1 (en) * 1996-02-08 1997-08-14 Australian Membrane And Biotechnology Research Institute Enzyme detection biosensors
WO1997043274A1 (en) * 1996-05-13 1997-11-20 Australian Membrane And Biotechnology Research Institute Improved reservoir components
US6417009B1 (en) 1996-05-13 2002-07-09 Australian Membrane And Biotechnology Institute Reservoir components
EP1019526A4 (en) * 1996-09-27 2004-08-25 Univ Australian METHOD FOR DETERMINING THE ION CHANNEL ACTIVITY OF A SUBSTANCE
AU768862B2 (en) * 1998-06-12 2004-01-08 Xention Limited High throughput screen
US6936462B1 (en) 1998-06-12 2005-08-30 Xention Discovery Limited High throughput screen
US10006902B2 (en) 1998-06-12 2018-06-26 Sophion Bioscience A/S High throughput screen
WO2003095669A1 (en) * 2002-05-10 2003-11-20 The Texas A & M University System Stochastic sensing through covalent interactions
US8404449B2 (en) 2002-05-10 2013-03-26 The Texas A&M University System Stochastic sensing through covalent interactions

Also Published As

Publication number Publication date
EP0734528A1 (en) 1996-10-02
AT402935B (en) 1997-09-25
ATA197094A (en) 1997-02-15

Similar Documents

Publication Publication Date Title
AT402934B (en) USE OF A SPECIAL AQUEOUS COMPOSITION FOR CLEANING AND DISINFECTING OBJECTS IN THE BREWING INDUSTRY
JP3293793B2 (en) Gating of receptor membranes and ionophores
DE69114870T3 (en) Biosensors.
DE69305160T2 (en) LIPID MEMBRANES FOR BIOSENSOR APPLICATIONS
DE69719817T2 (en) BIOSENSOR DEVICE AND METHOD
US20170227486A1 (en) Sensors for the detection of analytes
US7625951B2 (en) Stimuli-responsive hydrogel microdomes integrated with genetically engineered proteins for high-throughput screening of pharmaceuticals
US20020068295A1 (en) Multimeric biopolymers as structural elements and sensors and actuators in microsystems
Mihailescu et al. Platform with biomimetic electrochemical sensors for adiponectin and leptin detection in human serum
DE10036907A1 (en) Coating for various types of substrates and processes for their production
EP0734528A1 (en) Bio-recognition-controlled, ion-flow modulating bio-sensor
KR20190049223A (en) Nano-biosensor with electrode for enhanced sensing TNF-alpha by deposition of gold nanoparticle and covalent TNF-alpha antibody binding
Brzózka et al. Enhanced performance of potassium CHEMFETs by optimization of a polysiloxane membrane
EP1032826A1 (en) Method for producing laterally organized structures on supporting surfaces
WO1997020203A1 (en) Novel membranes and membrane dna/rna sensors
EP1018011B1 (en) Metal ion specific capacity affinity sensor
US12383490B2 (en) Generic high-capacity protein capture and tunable electrochemical release
Liang et al. Interdigitated conductometric immunosensor for determination of interleukin‐6 in humans based on dendrimer G4 and colloidal gold modified composite film
Moreira et al. Chitosan-based molecularly imprinted polymers for point-of-care C-reactive protein detection
EP1623224B1 (en) Biocompatible sensor electrode assembly and method for the production thereof
Weiss-Wichert et al. A new analytical device based on gated ion channels: a peptide-channel biosensor
AT403215B (en) Ion-flow-modulating DNA and RNA sensor
WO1995008770A1 (en) Process for coating surfaces with biological molecules and other receptor molecules
CN115753922A (en) A flexible portable molecularly imprinted electrochemical sensor and its preparation method and detection method
BR102020010979A2 (en) Flexible platinum electrodes used as electrochemical biosensors for the determination of biomarkers related to parkinson's disease and manufacturing method

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

ENP Entry into the national phase

Ref country code: US

Ref document number: 1996 666278

Date of ref document: 19960619

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 1995933226

Country of ref document: EP

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWP Wipo information: published in national office

Ref document number: 1995933226

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1995933226

Country of ref document: EP