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WO1996012957A1 - Biodetecteur de modulation de flux d'ions, regule par bioreconnaissance - Google Patents

Biodetecteur de modulation de flux d'ions, regule par bioreconnaissance Download PDF

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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
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WIPO (PCT)
Prior art keywords
biosensor according
channel
membrane
channels
sensor
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PCT/AT1995/000197
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German (de)
English (en)
Inventor
Fritz Pittner
Thomas Schalkhammer
Original Assignee
Fritz Pittner
Thomas Schalkhammer
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 Fritz Pittner, Thomas Schalkhammer filed Critical Fritz Pittner
Priority to EP95933226A priority Critical patent/EP0734528A1/fr
Publication of WO1996012957A1 publication Critical patent/WO1996012957A1/fr

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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.

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Abstract

L'invention concerne un nouveau principe de détection à grande sensibilité, un biodétecteur de modulation de flux d'ions, régulé par bioreconnaissance, ayant la sensibilité théorique d'une molécule d'analyte unique, qui fait appel à la liaison de molécules effectrices, lesdites molécules effectrices étant toutes molécules pouvant se lier à un ligand du groupe des hormones, des peptides, des inhibiteurs d'enzyme, des toxines de l'environnement, des principes actifs pharmaceutiques, des thiophènes ou des chélateurs, pour moduler on/off un canal d'ions dans une membrane.
PCT/AT1995/000197 1994-10-19 1995-10-11 Biodetecteur de modulation de flux d'ions, regule par bioreconnaissance WO1996012957A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP95933226A EP0734528A1 (fr) 1994-10-19 1995-10-11 Biodetecteur de modulation de flux d'ions, regule par bioreconnaissance

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ATA1970/94 1994-10-19
AT0197094A AT402935B (de) 1994-10-19 1994-10-19 Biorekognitions-gesteuerter, ionenfluss-modulirender biosensor

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WO1996012957A1 true WO1996012957A1 (fr) 1996-05-02

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Cited By (7)

* Cited by examiner, † Cited by third party
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WO1997025616A1 (fr) * 1996-01-11 1997-07-17 Australian Membrane And Biotechnology Research Institute Groupage sanguin par capteur a canal ionique
WO1997029366A1 (fr) * 1996-02-08 1997-08-14 Australian Membrane And Biotechnology Research Institute Biocapteurs de detection des enzymes
EP0791176A1 (fr) * 1994-11-16 1997-08-27 Australian Membrane And Biotechnology Research Institute Dispositif et procede de detection
WO1997043274A1 (fr) * 1996-05-13 1997-11-20 Australian Membrane And Biotechnology Research Institute Composants ameliores d'un reservoir
EP1019526A1 (fr) * 1996-09-27 2000-07-19 The Australian National University Technique d'evaluation de l'activite canal ionique d'une substance
WO2003095669A1 (fr) * 2002-05-10 2003-11-20 The Texas A & M University System Detection stochastique par interactions covalentes
AU768862B2 (en) * 1998-06-12 2004-01-08 Xention Limited High throughput screen

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0791176A4 (fr) * 1994-11-16 1999-11-10 Au Membrane & Biotech Res Inst Dispositif et procede de detection
EP0791176A1 (fr) * 1994-11-16 1997-08-27 Australian Membrane And Biotechnology Research Institute Dispositif et procede de detection
WO1997025616A1 (fr) * 1996-01-11 1997-07-17 Australian Membrane And Biotechnology Research Institute Groupage sanguin par capteur a canal ionique
WO1997029366A1 (fr) * 1996-02-08 1997-08-14 Australian Membrane And Biotechnology Research Institute Biocapteurs de detection des enzymes
US6417009B1 (en) 1996-05-13 2002-07-09 Australian Membrane And Biotechnology Institute Reservoir components
WO1997043274A1 (fr) * 1996-05-13 1997-11-20 Australian Membrane And Biotechnology Research Institute Composants ameliores d'un reservoir
EP1019526A1 (fr) * 1996-09-27 2000-07-19 The Australian National University Technique d'evaluation de l'activite canal ionique d'une substance
EP1019526A4 (fr) * 1996-09-27 2004-08-25 Univ Australian Technique d'evaluation de l'activite canal ionique d'une 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 (fr) * 2002-05-10 2003-11-20 The Texas A & M University System Detection stochastique par interactions covalentes
US8404449B2 (en) 2002-05-10 2013-03-26 The Texas A&M University System Stochastic sensing through covalent interactions

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EP0734528A1 (fr) 1996-10-02
ATA197094A (de) 1997-02-15
AT402935B (de) 1997-09-25

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