WO2006005370A1 - Substrate for a device for electrophysiological measurements - Google Patents

Substrate for a device for electrophysiological measurements Download PDF

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Publication number
WO2006005370A1
WO2006005370A1 PCT/EP2004/007878 EP2004007878W WO2006005370A1 WO 2006005370 A1 WO2006005370 A1 WO 2006005370A1 EP 2004007878 W EP2004007878 W EP 2004007878W WO 2006005370 A1 WO2006005370 A1 WO 2006005370A1
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WO
WIPO (PCT)
Prior art keywords
substrate
layer
substrate according
measurements
electrophysiological
Prior art date
Application number
PCT/EP2004/007878
Other languages
French (fr)
Inventor
Uwe Sleytr
Dietmar Pum
Petra Christine Gufler
Original Assignee
Nano S Biotechnologie Gmbh
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Priority to PCT/EP2004/007878 priority Critical patent/WO2006005370A1/en
Publication of WO2006005370A1 publication Critical patent/WO2006005370A1/en

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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
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent

Definitions

  • This invention relates to a substrate for a device for elec ⁇ trophysiological measurements.
  • the planar chip replaces the pipette.
  • Patch-clamp-on-a-chip enables simultaneous patch-clamp recordings from many liposomes or cells.
  • the decisive factor in this system is the formation of a high-resistance gigaohmic seal between the chip and the lipid membrane. Lower resistances would lead to a low signal to noise ratio. In all systems difficulties with seal formation were ob ⁇ served, presumably because of non-sufficient contact area (nar ⁇ row sidewalls, sharp corners of the etched aperture) .
  • the present invention provides a substrate for a device for electrophysiological measurements comprising at least one perforation, wherein the substrate is covered at least in part with at least one protein and/or glyoprotein layer.
  • the at least one perfor ⁇ ation can exhibit different three dimensional shapes.
  • the perforation has the form of a frustum of a cone or a pyramid, of a cylinder , of a wedge, of a prism or of a cube.
  • substrate is defined as a structural component of a device for electrophysiological measurements whereon a membranous sample can adhere. Furthermore the substrate may function additionally as a barrier between two electrode compartments of a patch clamp device.
  • the "protein layer” refers to all kind of proteins capable to be immobilised on the surface of a substrate to be used in apparatus for electrophysiological measurements.
  • suitable proteins are S-layer pro ⁇ teins, antibodies, other proteins and glycoproteins and frag ⁇ ments thereof.
  • polypeptides and peptides may be immobilised on the surface of a substrate.
  • the substrate according to the present invention may be used in apparatus for electrophysiological measurements employing the path clamp technique.
  • apparatus are disclosed for example in the WO 96/13721 Al, WO 99/66329 Al and US 2002/0108869 Al.
  • the presence of more than one perforation enables the substrate to be used also in high throughput screenings.
  • the invention enables standardized and reproducible spatial binding properties of membranous samples for electrophysiological meas ⁇ urements.
  • the protein layer (glyosylated or non-glycosylated) can be bound covalently or non-covalently, e.g. by hydrophobic or hy- drophilic interactions, to the surface of the substrate.
  • Fur ⁇ thermore the substrate can be covered entirely or partially with a protein layer, whereby the protein layer may comprise more than one protein species.
  • the at least one protein layer provides anchoring/adhesion sites for membranous samples, e.g. cells, cell fragments, lipo ⁇ somes and other lipid membrane aggregates, on a substrate which can be used in patch-clamp devices known in the state of the art. Furthermore the protein layer plays an important role in the high-resistance gigaohmic seal formation between the lipid membrane and the chip surface. Moreover the proteins on the sur ⁇ face of the substrate can be used as a pre-blocking inert coat ⁇ ing, in order to prevent interfering effects and unspecific ad ⁇ sorption of cells, liposomes and other lipid aggregates.
  • the in ⁇ vention enables a sensitive electrical acquisition of transmem- brane proteins and is a further step toward high-throughput screening for drug discovery efforts.
  • the at least one protein layer is modified with at least one functionality.
  • a modifica ⁇ tion of the protein layer with at least one functionality en ⁇ ables the substrate to interact with e.g. the membranous sample.
  • the interaction of the at least one protein layer with the mem ⁇ branous sample is helpful in stabilizing the membrane on the substrate and furthermore to create a gigaohmic seal.
  • the modi ⁇ fication of the protein layer can occur chemically (by covalent or by non-covalent binding) or by genetic engineering (by creat ⁇ ing e.g. fusion proteins).
  • a protein layer e.g. S-layer fusion proteins
  • S-layer fusion proteins enables specific interac ⁇ tions between the protein and specific groups (ligands, recept ⁇ ors) on the liposome, lipid bilayer or cell surface.
  • a protein layer in particular an S-layer, allows a tight and spe ⁇ cific binding of unlimited types of liposomes, lipid bilayer ag ⁇ gregates, whole cells or cell fragments in an extremely gentle way. Thereby gigaohmic seal formation (with or without a gentle suction) is greatly facilitated.
  • binding mechanisms and functionalities exhibited by the protein layer are the following (e.g. S-layer protein(s)):
  • Reactive groups/receptors on the S-layer recognize ligands in the lipid membrane; reactive groups/receptors in the lipid membrane recognize an S-layer-bound ligand; e.g. antigen-antibody, ligand-receptor, lectine-carbo- - A - hydrate-interactions, metal-affinity-tags (e.g. histidine) ; S-layer/streptavidin fusion protein lattice - specific binding via biotinylated molecules in cells/lipo- somes/lipid aggregates.
  • S-layer/streptavidin fusion protein lattice - specific binding via biotinylated molecules in cells/lipo- somes/lipid aggregates.
  • the at least one functionality is selected from the group consisting of proteins, polypeptides, peptides, ligands, chemical groups and mixtures thereof.
  • the functional groups of the at least one functionality may comprise all functional groups knwon in the state of the art, which can be used for protein modifications.
  • these groups comprise proteins (e.g. avidin, streptavidin, lect- ine, antibodies and fragments thereof), ligands (e.g. carbo ⁇ hydrates, biotin) and chemical groups (e.g. -OH, -COOH, -NH 2 ) .
  • proteins e.g. avidin, streptavidin, lect- ine, antibodies and fragments thereof
  • ligands e.g. carbo ⁇ hydrates, biotin
  • chemical groups e.g. -OH, -COOH, -NH 2
  • the at least one protein layer is composed of at least one S-layer.
  • S-layer proteins The chemical and structural features of S- layer proteins turned out to be advantageous for a substrate to be used in patch clamp applications.
  • S-layer proteins are routinely utilised in several technical applications.
  • the EP 0 306 473 Bl discloses the use of S-layer proteins as a carrier for haptens, immunogenic or im- munostimulant substances.
  • the immobilisation of molecules, par ⁇ ticularly of proteins, on S-layer proteins is described in the EP 0 362 339 Bl.
  • the WO 01/81425 Al discloses the use of a structure comprising carbohydrates (secondary cell wall polymer) as an anchoring means for S-layer proteins in order to bind them to a solid support.
  • the production of a layer of functional molecules on a substrate using S-layer pro ⁇ teins is disclosed.
  • the protein layer on the surface of a carrier is formed by creating an electrochemical potential difference between the solution and the surface.
  • the EP 0 189019 Bl discloses the making and the use of an ultra ⁇ filtration membrane employing S-layer proteins.
  • S-layer protein species from different organisms can be used.
  • a protein species is used as a native, a recombinant, or a recombinant protein optionally with distinct functional domain (s) , e.g. streptavidin, ligand, affinity tag.
  • the S-layer is composed of identical S-layer proteins, a mixture of at least two species, or different protein species with each comprising one or more specific functionalities. The ratio of the distinct proteins can be varied.
  • the S-layer can be native or chemically modified. Furthermore S-layers can be recrystallized as mono-, bi- or multilayers.
  • inert surface character neutrally charged surfaces. These types of S-layers show no binding affinity or unspecific binding.
  • S-lay ⁇ ers Two-dimensional crystalline bacterial surface layers can be recrystallized in vitro into coherent lattices on a great variety of surfaces and interfaces, including silicon or glass (Sleytr et al., 2002, 2003; Pum et al., 2000) .
  • Lab-on-a- chip-technologies were facilitated with the patterning of S-lay ⁇ ers on solid supports by microlithography (Neubauer et al., 1997, 1998; US 6296700 Bl) or micromoulding in capillaries (Gy ⁇ rvary et al., 2003a) .
  • S-layers interact with the lipid head group region of phospholipids, leading to a stabilization of - S - lipid membranes. Stabilization was demonstrated on lipid mem ⁇ branes (Schuster et al., 1999) (Wetzer et al., 1997, 1998; Gu- fler et al., 2004) and on liposomes (K ⁇ pc ⁇ et al., 1995; Mader et al., 1999) . S-layers do not penetrate the hydrophobic region of mono- (e.g. tetraetherlipid) or bilayer membranes; therefore they have no impact on the integrity of the lipid aggregates, cells or cell membranes.
  • mono- e.g. tetraetherlipid
  • S-layers provide a natural environment for lipid membranes and biological materials: e.g. in many ar- chaeal organisms the S-layer is the only component external to the cytoplasmic membrane (Baumeister & Lembcke, 1992) .
  • a broad range of S-layer proteins with different physico-chemical sur ⁇ face properties and functionalities are available.
  • the imple ⁇ mentation of recombinant S-layer- (US 2002/0168728 Al) and S- layer-fusion proteins exhibiting functional domains enables the application of specific binding mechanisms (Moll et al. , 2002) . Due to the high regularity of the crystal lattice, functionalit ⁇ ies repetitively arranged in well defined positions and orienta ⁇ tions can be obtained. Furthermore a significant reduction in surface roughness of the substrate is observable after recrys- tallization (Gufler et al., 2004) .
  • the substrate comprises an electrically insulating material. It is a basic requirement of devices for electrophysiological meas ⁇ urements, e.g. patch clamp devices, that the current can only pass the susbstrate through the pores, transporters and channels part of a membranous structure present in the vicinity of a per ⁇ foration of the substrate. Therefore substrates used for patch clamp experiments may comprise electrically insulating material. The surface of the substrate or the entire substrate may com ⁇ prise an electrically insulating material.
  • the electrically insulating material material is selected from the group consisting of silicon, glass, solid state polymers and mixtures thereof.
  • Microfabricated chips made of silicon, glass, solid state polymers or other materials are preferably used.
  • Transparent chips, made e.g. of glass or syn ⁇ thetic polymers, can be used for simultaneous electrophysiolo ⁇ gical and optical or spectroscopical measurements (e.g. fluores ⁇ cence measurements, microscopical methods) .
  • part of the substrate may also comprise an electrode. In this case part of the substrate is electroconductive.
  • the perforation is preferably 100 ran to 10 ⁇ m, preferably 500 nm to 9 ⁇ m, more preferably 1 ⁇ m to 8 ⁇ m, particularly 2 ⁇ m to 7 ⁇ m wide.
  • the perforations of the chips are fabricated e.g. by nano- and micro-lithographic methods, laser or micromoulding techniques.
  • the dimension of the perforation is adjusted accord ⁇ ing to the size of the membranous sample, whereby different per ⁇ forations of the substrate can possess differing dimensions. This demonstrates once more the flexible use of the substrate according to this invention.
  • Wide refers to the physical dimensions of the perforations.
  • the term “wide” for a circular perforation refers to its diameter and "wide” for a quadratic perforation is referred to the distance between the nooks of such a perforation.
  • the substrate is sub ⁇ stantially planar.
  • the geometry of a substrate can be manifold.
  • the substrate can be planar like chips or exhibit a spherical form.
  • the substrate is chemically or physically modified. Not only the protein layer on the surface of the substrate can be modified, but also the sur ⁇ face of the substrate itself. Such modifications allow creating zones in which e.g. no protein layer can adhere and other zones where an interaction between surface of the substrate and the membranous sample is prevented. Other modifications may prevent an unspecific binding of the membranous sample on the surface of the substrate, where no protein layer is present.
  • Such a modification is preferably selected from the group consisting of ionizing radiation, atomic radicals, corona treat ⁇ ment, silane groups, chemical groups and mixtures thereof.
  • Chem ⁇ ical groups may comprise carbohydrates (e.g. secondary cell wall polymer), biotin and reactive groups.
  • Other chemical groups and modification methods are disclosed in several publications and textbooks (Schalkhammer, 2002; Wong, 1991) .
  • the substrate can be used for electrophysiological measurements, preferably for patch clamp measurements.
  • apparatus for patch clamp experiments can employ a substrate ' as disclosed herein.
  • the substrate according to the present invention can be used for a device for simultaneous electrophysiological measurements, preferably for patch clamp measurements, and op ⁇ tical or spectroscopical measurements, if the substrate com ⁇ prises a transparent electrically insulating material.
  • Another aspect of the present invention relates to a method for measuring an electrical property of a membranous sample us ⁇ ing a substrate as disclosed.
  • the membranous sample is selected from the group consisting of cells, cell fragments, cell membranes, liposomes, artificial lipid membranes, lipid layers and mixtures thereof.
  • the membranous sample comprises next to lipids and other cell membrane components particularly transporters and ion channels.
  • the substrate of the present invention may be preferably used in a method for the simultaneous electrophysiological and optical or spectroscopical measurements of a membranous sample.
  • Fig.l shows two examples of substrate - and perforation (a)- structures (sections: IA and IB; top view: 1C) .
  • the substrate is provided with a plurality of perforations.
  • Fig.2 shows S-layer coatings.
  • the S-layer adhesion site (a) for the positioning of cells/liposomes/artificial membranes is recrystallized around the aperture (section: 2A, top view: 2B) .
  • An inert coating (b) of the residual chip surface can be .per ⁇ formed by using another S-layer protein species (section: 2C, top view: 2D) .
  • the S-layer adhesion sites (Fig. 2, marked with a) can be structured or non-structured.
  • the S-layer patterns can be performed simple, economical, reproducible and versatile. Structuring can be performed by (i) nano- and micromoulding techniques (Gy ⁇ rvary et al.
  • the rings, squares/rectangles and bands can be thin (a,f) or broad (b,g) .
  • the apertures can be surrounded by a single S-layer zone (a,b,f,g) or by multiple zones (c,d e, h , i , j) .
  • the distance between two zones (d,i) as well as the thickness of the different zones (e, j) can be varied.
  • the rings, rectangles or bands can be continuous (a-e, f-j ) or non- continuous (k-n) .
  • Different zones may be covered with different types of S-layers.
  • a further structuring embodiment is obtained by time-depend ⁇ ent control of the recrystallization process.
  • 2-dimensional growth of S-layer domains takes place in all directions, until neigh ⁇ bouring areas meet (Gy ⁇ rvary et al., 2003a) .
  • Fig.4 shows the composition of the S-layer lattice (protein type, lattice symmetry, functionality) within the adhesion site (A, B, C, D) and within one zone (E, F, G) .
  • Variable architec ⁇ tures (as shown in Fig.3) exhibiting zones of S-layer lattices composed of proteins from either the same (Fig. 4A) or different (Fig. 4B) organisms (native or recombiant; single or variable functionalities) can be used.
  • Different zones of an S-layer ad ⁇ hesion site can be composed of one functionality (Fig. 4C) or variable functionalities (Fig. 4D) .
  • One zone can be composed of an S-layer lattice with one single functionality (Fig.
  • One zone can be composed of S-layer proteins from different organisms exhibiting varying lattice symmetry (pA, pB, pC) and/or functionalities (Fig. 4G) .
  • Fig.5 shows the fabrication of an S-layer adhesion site by using the micromoulding technique (e.g. PDMS mould) .
  • the micromoulding technique e.g. PDMS mould
  • Fig.6 shows additional modification of the substrate surface devoid of S-layer lattice within the adhesion site.
  • Fig.7 shows S-layer proteins as means for smoothing the sharp corners of the chip perforations.
  • Fig.8 shows the closing- and opening-behaviour of single gramicidin pores reconstituted in anchored and clamped giant liposomes.
  • EXAMPLE The application of 2-dimensional protein crystals for gramicidin ion channel recordings from giant liposomes placed on a planar chip-device
  • S-layer proteins Two-dimensional bacterial surface layer (S-layer) proteins were applied for coating of planar silicon chips and subsequent binding of giant unilamellar vesicles (GUVs) that were function- alized with gramicidin.
  • the GUVs contained a low amount of biot- inylated lipids.
  • a genetically engineered S-layer protein that exhibited a fused heterotetrameric streptavidin domain was re- crystallized around the aperture of the microstructured silicon chip forming an S-layer adhesion site.
  • Successful binding via biotin-streptavidin interactions between GUV and the S-layer- streptavidin fusion protein layer (one GUV per aperture) was ob ⁇ served. Gigaseal formation was monitored by resistance measure ⁇ ments.
  • the phospholipids EggPC and Biotin-cap-DPPE (Avanti Polar Lipids, Alabaster, AL) were dissolved and stored at -20 0 C in chloroform (Merck, Darmstadt, Germany) at a concentration of 0.1M.
  • chloroform Merck, Darmstadt, Germany
  • IM KCl aqueous solution of IM KCl was used.
  • the buffer was prepared in MilliQ-water (Millipore, minim ⁇ um resistance >18M0hms cm) and thoroughly degassed and filtered through a 0.2- ⁇ m filter (Sartorius AG, G ⁇ ttingen, Germany) be ⁇ fore use. All chemicals were purchased from Sigma Aldrich (Vi ⁇ enna, Austria) .
  • a silicon wafer (NMRC, Cork, Ireland, 1x1 cm 2 , contact angle 64°) was structured by standard lithographic techniques and an ⁇ isotropic ion etching, resulting in an aperture with a diameter of ⁇ 1-7 ⁇ m.
  • the aperture was placed in the centre of the wafer (one pore/chip) .
  • the chips were cleaned by ultrasonic treatment twice for 20 min in 2% Hellmanex (Hellma, M ⁇ llheim, Germany) and twice for 20 min in absolute ethanol. After each sonication step, the slides were rinsed thoroughly in MilliQ-water and fi ⁇ nally dried in a stream of dry nitrogen. Cleaned chips were stored in clean air until usage.
  • S- layer protein SbpA from the gram-positive strain B.
  • sphaericus CCM 2177 (Czech Collection of Microorganisms) were performed as described elsewhere (Sleytr et al., 1986) .
  • the wildtype SbpA stock solution was diluted with to a final concentration of 0.1 mg/ml.
  • Molecular cloning, gene expression, isolation, refolding and purification procedures of S-layer streptavidin fusion pro ⁇ teins (rSbpA/strp) were described elsewhere (Moll et al., 2002) (Huber et al. , submitted) .
  • rSbpA/strp proteins recrystallized on hydrophobic silicon as a monolayer with the streptavidin domain exposed to the aqueous solution.
  • An appropriate amount of rSbpA/strp was desintegrated in 4M GHCl for 5 min.
  • the protein was dialysed against MilliQ-water for 2 h.
  • the protein solution was adjusted to a concentration of lmg/ml with MilliQ-water, centrifuged in an Eppendorf Centrifuge and stored at 4°.
  • the dried film was hydrated to a final lipid concen ⁇ tration of 1% (w/v) by adding a standard buffer containing 2% CHAPS (Sigma, Vienna, Austria) .
  • the detergent was either removed by dialysis or by addition of Bio Beads SM-2 (Sigma, Vienna, Austria) .
  • Glass microscopic slides (ELKA, Germany) were cleaned in Hellmanex and absolute ethanol (ultrasonic treatment) , thor ⁇ oughly rinsed with MilliQ-water and dried in a stream of nitro ⁇ gen. Small drops ( ⁇ 20 ⁇ l) or wide stripes (for larger amounts) of the vesicle suspension were deposited onto the glass slides.
  • the suspension was partially dehydrated for 3 h/4°C in a desic ⁇ cator.
  • Giant liposomes exhibiting a size distribution from 20 ⁇ m to 80 ⁇ m were obtained by rehydration: an appropriate amount of standard buffer solution was deposited on the top of the dehyd ⁇ rated drop. Size and size distribution of GUVs were examined by transmission electron microscopy (Philips, Eindhoven, The Neth ⁇ erlands) of negatively stained samples.
  • Formvar/carbon coated EM copper grids (Athene old/300 mesh) were deposited on a drop of the sample (5min) . Afterwards the sample was fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.0 and stained in 2% uranyl acetate.
  • Poly(dimethylsiloxane) PDMS (Sylgard 184; DOW corning), is used to fabricate the molds from these masters using the standard procedure (Kumar et al., 1994) (Kim et al., 1995) .
  • the mold was carefully placed onto the manufactured silicon chip (microscopical control) such that the aperture was circularily surrounded by the two horseshoe-like capillaries. Starting from inlets the microchannels (20 ⁇ m channel width) were filled with the rSbpA/strp recrystallization solution.
  • the voltage-clamp setup was mounted on a steel core bread ⁇ board (CS-11-4, Newport, Darmstadt, Germany) .
  • the headstage of the patch clamp amplifier (EPC 9, HEKA Electronic, Lambrecht/Pfalz, Germany) was fixed on a translation stage (M- 426, Newport, Darmstadt, Germany) which could be moved vertic ⁇ ally (Motorizer 860 A, Newport, Darmstadt, Germany; regulated DC power supply: Kenwood PR 18-1.2, Tokyo, Japan) .
  • the headstage of the amplifier, together with measurement chamber were placed within a grounded Faraday cage.
  • the S-layer covered chip was placed horizontally into a home-made teflon-chamber.
  • the chip was sealed into the chamber by a silicone paste (Bayer AG, Leverkusen, Germany) und thus divided the chamber into two com ⁇ partments.
  • Ag/AgCl redox electrodes were used for voltage-clamp recordings.
  • the reference electrode was placed in the lower, the working electrode in the upper compartment.
  • the lower compart ⁇ ment comprised a vacuum device in order to apply a suction to the clamped liposome.
  • the S-layer covered surface was facing the upper compartment. During this procedure, desiccation of the S- layer was carefully prevented.
  • the cham ⁇ ber was filled with the standard buffer solution.
  • a small amount of the freshly prepared biotinylated gramicidin-GUV suspension was pipetted onto the S-layer.
  • a single liposome was bound to the rSbpA/strp adhesion zone. After application of a gentle suc ⁇ tion, seal formation was monitored by resistance measurements. 2.7 Signal recording and data acquisition
  • the current response of a given voltage function was meas ⁇ ured at 25 0 C to calculate the electrophysiological parameter of the clamped liposome patch.
  • Conductance was measured via rectan ⁇ gular voltage pulses of up to 20OmV across the membrane. All the data handling (voltage stimulation, data acquisition, data ' stor ⁇ age, data analysis) was performed on a Power Macintosh 7600/120 computer (Apple Computer GmbH, Ismaning, Germany) .
  • the setting of the two built-in Bessel filters of the EPC 9 amplifier for the current-monitor signal was 10 and 1.5 kHz, respectively.
  • GUVs functionalized with biotinylated lipids and gramicidin were chosen as an appropriate system for single chan ⁇ nel recordings.
  • the size of GUVs varied from ⁇ 20-80 ⁇ m, the size distribution was fairly balanced. In some cases, multilamellar vesicles were obtained, which were not used.for the experiments.
  • the GUVs were used immediately after preparation or for later experiments devided into aliquots and stored at -80 0 C until us ⁇ age.
  • the residual chip surface around the rSbpA/strp adhesion site was coated with wildtype SbpA.
  • the out ⁇ er surface of SbpA is charge-neutral.
  • adhesion and/or fusion of GUVs to larger lipid aggregates or bilayered structures were observed.
  • a significant decrease in unspecific adsorption and/or fusion processes of GUVs was achieved. Since liposomes only adsorbed slightly and at a very low amount, they could be washed away easily by gentle rinsing of the upper compartment of the measurement cell.
  • a single GUV was bound per S-layer adhe ⁇ sion site. In some experiments either the GUV was not centered and thus no gigaseal was obtained or more smaller GUVs per adhe ⁇ sion site were bound.
  • the challenge of preparing lipid membranes on silicon chips is the recording of single ion carriers and channels.
  • the gener ⁇ al functionality of this concept was demonstrated with gramicid ⁇ in, a peptide known to self-integrate into one leaflet of a lip ⁇ id bilayer and to form single channels if dimerization occurs.
  • the closing- and opening-behaviour of single gramicidin pores reconstituted in the anchored and clamped giant liposomes was observed (Fig. 8) .
  • a voltage typically around 200 mV was applied across the bilayer.
  • Gram ⁇ icidin is selective for monovalent cations and as KCl solution was used, K+ currents were recorded.
  • the present example shows that stable gigaohm seals over mi ⁇ crometer-sized holes can be obtained.
  • the self-positioning of biotinylated giant liposomes can easily be achieved by the high binding affinity of biotin to the streptavidin hetrotetramers which are presented in a well-defined and oriented way by the S- layer lattice.
  • no suction is necessary for the positioning of the target molecules, which is always problem as this leads often to clogging of the aperture. If the target molecule is on the right position, suction guarantees the giga-seal formation between the liposome and the aperture.

Abstract

A substrate for a device for electrophysiological measurements comprising at least one perforation, wherein the substrate is covered at least in part with at least one protein and/or glycoprotein layer.

Description

Substrate for a device for electrophysiological measurements
This invention relates to a substrate for a device for elec¬ trophysiological measurements.
In recent years the patch-clamp technique became state of the art for electrophysiological measurements of ion channels and transporters located in cells, cell fragments or artificial lipid membranes. The classic patch-clamp technique uses a glass pipette, which is gently pressed against the lipid membrane. A very small area is isolated, which is investigated (Sakmann & Neher, 1995) . The electric currents through reconstituted ion channels and pores are recorded in a highly specific and sensit¬ ive way. However, classic patch-clamping is time-consuming and not convenient for practical devices and automation. Recently, automated patch-clamp devices ("patch clamp on a chip" (Sigworth & Klemic, 2002) ) were introduced, using planar microstructured chips made of PDMS (Klemic et al., 2002), silicon (Pantoja et al., 2001; Schmidt et al., 2000) or glass (Fertig et al., 2002; Brϋggemann et al., 2003) for recording from whole cells (Fertig et al., 2OO2V Klemic et al., 2002), liposomes (Schmidt et al. , 2000) and painted artificial membranes (Pantoja et al., 2001) .
The planar chip replaces the pipette. Patch-clamp-on-a-chip enables simultaneous patch-clamp recordings from many liposomes or cells. The decisive factor in this system is the formation of a high-resistance gigaohmic seal between the chip and the lipid membrane. Lower resistances would lead to a low signal to noise ratio. In all systems difficulties with seal formation were ob¬ served, presumably because of non-sufficient contact area (nar¬ row sidewalls, sharp corners of the etched aperture) .
It is an object of the present invention to provide a sub¬ strate for a device for electrophysiological measurements which allows an accurate positioning of the membranous sample on the substrate and supports the formation of a high-resistance gigaohmic seal between a substrate and a membranous sample.
Therefore the present invention provides a substrate for a device for electrophysiological measurements comprising at least one perforation, wherein the substrate is covered at least in part with at least one protein and/or glyoprotein layer.
According to the present invention "the at least one perfor¬ ation" can exhibit different three dimensional shapes. For in- stance the perforation has the form of a frustum of a cone or a pyramid, of a cylinder , of a wedge, of a prism or of a cube.
According to the present invention "substrate" is defined as a structural component of a device for electrophysiological measurements whereon a membranous sample can adhere. Furthermore the substrate may function additionally as a barrier between two electrode compartments of a patch clamp device.
The "protein layer" according to this invention refers to all kind of proteins capable to be immobilised on the surface of a substrate to be used in apparatus for electrophysiological measurements. Examples for suitable proteins are S-layer pro¬ teins, antibodies, other proteins and glycoproteins and frag¬ ments thereof. Of course also polypeptides and peptides may be immobilised on the surface of a substrate.
The substrate according to the present invention may be used in apparatus for electrophysiological measurements employing the path clamp technique. Such apparatus are disclosed for example in the WO 96/13721 Al, WO 99/66329 Al and US 2002/0108869 Al. The presence of more than one perforation enables the substrate to be used also in high throughput screenings. Furthermore the invention enables standardized and reproducible spatial binding properties of membranous samples for electrophysiological meas¬ urements.
The protein layer (glyosylated or non-glycosylated) can be bound covalently or non-covalently, e.g. by hydrophobic or hy- drophilic interactions, to the surface of the substrate. Fur¬ thermore the substrate can be covered entirely or partially with a protein layer, whereby the protein layer may comprise more than one protein species.
The at least one protein layer provides anchoring/adhesion sites for membranous samples, e.g. cells, cell fragments, lipo¬ somes and other lipid membrane aggregates, on a substrate which can be used in patch-clamp devices known in the state of the art. Furthermore the protein layer plays an important role in the high-resistance gigaohmic seal formation between the lipid membrane and the chip surface. Moreover the proteins on the sur¬ face of the substrate can be used as a pre-blocking inert coat¬ ing, in order to prevent interfering effects and unspecific ad¬ sorption of cells, liposomes and other lipid aggregates. The in¬ vention enables a sensitive electrical acquisition of transmem- brane proteins and is a further step toward high-throughput screening for drug discovery efforts.
According to a preferred embodiment the at least one protein layer is modified with at least one functionality. A modifica¬ tion of the protein layer with at least one functionality en¬ ables the substrate to interact with e.g. the membranous sample. The interaction of the at least one protein layer with the mem¬ branous sample is helpful in stabilizing the membrane on the substrate and furthermore to create a gigaohmic seal. The modi¬ fication of the protein layer can occur chemically (by covalent or by non-covalent binding) or by genetic engineering (by creat¬ ing e.g. fusion proteins).
The possibility to create a broad spectrum of recombinant proteins which can be employed in the formation of a protein layer (e.g. S-layer fusion proteins)- enables specific interac¬ tions between the protein and specific groups (ligands, recept¬ ors) on the liposome, lipid bilayer or cell surface. Thus, a protein layer, in particular an S-layer, allows a tight and spe¬ cific binding of unlimited types of liposomes, lipid bilayer ag¬ gregates, whole cells or cell fragments in an extremely gentle way. Thereby gigaohmic seal formation (with or without a gentle suction) is greatly facilitated.
The binding mechanisms and functionalities exhibited by the protein layer are the following (e.g. S-layer protein(s)):
*) Lattice of native or recombinant S-layer proteins, or a mixture of native and recombinant S-layer proteins (co-re- crystallization) :
1) unmodified S-layer lattice; utilization of the intrinsic binding affinities (non-covalent interactions) .
2) chemically modified S-layer lattice providing specific physico-chemical properties and binding affinities.
3) Attachment of cells/liposomes/lipid aggregates due to to¬ pographical features of the S-layer lattice (edge sharp¬ ness of zone/chip boundaries and geometry) .
*) Reactive groups/receptors on the S-layer recognize ligands in the lipid membrane; reactive groups/receptors in the lipid membrane recognize an S-layer-bound ligand; e.g. antigen-antibody, ligand-receptor, lectine-carbo- - A - hydrate-interactions, metal-affinity-tags (e.g. histidine) ; S-layer/streptavidin fusion protein lattice - specific binding via biotinylated molecules in cells/lipo- somes/lipid aggregates.
According to another preferred embodiment the at least one functionality is selected from the group consisting of proteins, polypeptides, peptides, ligands, chemical groups and mixtures thereof. The functional groups of the at least one functionality may comprise all functional groups knwon in the state of the art, which can be used for protein modifications. For instance these groups comprise proteins (e.g. avidin, streptavidin, lect- ine, antibodies and fragments thereof), ligands (e.g. carbo¬ hydrates, biotin) and chemical groups (e.g. -OH, -COOH, -NH2) . For further details see Wong (1991) and Schalkhammer (2002) .
Preferably the at least one protein layer is composed of at least one S-layer. The chemical and structural features of S- layer proteins turned out to be advantageous for a substrate to be used in patch clamp applications.
S-layer proteins are routinely utilised in several technical applications. For instance the EP 0 306 473 Bl discloses the use of S-layer proteins as a carrier for haptens, immunogenic or im- munostimulant substances. The immobilisation of molecules, par¬ ticularly of proteins, on S-layer proteins is described in the EP 0 362 339 Bl. The WO 01/81425 Al discloses the use of a structure comprising carbohydrates (secondary cell wall polymer) as an anchoring means for S-layer proteins in order to bind them to a solid support. In the WO 02/097118 Al the production of a layer of functional molecules on a substrate using S-layer pro¬ teins is disclosed. Therein the protein layer on the surface of a carrier is formed by creating an electrochemical potential difference between the solution and the surface. The EP 0 189019 Bl discloses the making and the use of an ultra¬ filtration membrane employing S-layer proteins.
S-layer protein species from different organisms can be used. A protein species is used as a native, a recombinant, or a recombinant protein optionally with distinct functional domain (s) , e.g. streptavidin, ligand, affinity tag. The S-layer is composed of identical S-layer proteins, a mixture of at least two species, or different protein species with each comprising one or more specific functionalities. The ratio of the distinct proteins can be varied. The S-layer can be native or chemically modified. Furthermore S-layers can be recrystallized as mono-, bi- or multilayers.
Dependent on the S-layer protein species employed, distinct surface properties are obtained after recrystallization:
*) intrinsic functionalities of native isolated S-layer pro¬ teins and recombinant S-layer proteins with no fused functional domain: e.g. charge density and distribution. In many cases the recombinant S-layer proteins that are predominantly expressed in E. coli show a different charge distribution than the native protein isolated from the bacterial cell wall.
With regard to their orientation in vivo, a common fea¬ ture of S-layers is their smoother charge-neutral outer surface and a more corrugated, net-negatively charged in¬ ner surface (Pum et al., 2000) . Hence, the orientation of the S-layer proteins on the substrate defines the physico-chemical properties.
*) functionalities after chemical surface modification of native S-layers; e.g. change in charge and charge dens¬ ity, introduction of chemically reactive groups.
*) artificially inserted functional domains : genetically en¬ gineered S-layer fusion proteins, chemically fused func¬ tional domains .
*) inert surface character: neutrally charged surfaces. These types of S-layers show no binding affinity or unspecific binding.
Two-dimensional crystalline bacterial surface layers (S-lay¬ ers) can be recrystallized in vitro into coherent lattices on a great variety of surfaces and interfaces, including silicon or glass (Sleytr et al., 2002, 2003; Pum et al., 2000) . Lab-on-a- chip-technologies were facilitated with the patterning of S-lay¬ ers on solid supports by microlithography (Neubauer et al., 1997, 1998; US 6296700 Bl) or micromoulding in capillaries (Gyδrvary et al., 2003a) . Moreover, functional groups of the at least one functionality on S-layers interact with the lipid head group region of phospholipids, leading to a stabilization of - S - lipid membranes. Stabilization was demonstrated on lipid mem¬ branes (Schuster et al., 1999) (Wetzer et al., 1997, 1998; Gu- fler et al., 2004) and on liposomes (Kϋpcϋ et al., 1995; Mader et al., 1999) . S-layers do not penetrate the hydrophobic region of mono- (e.g. tetraetherlipid) or bilayer membranes; therefore they have no impact on the integrity of the lipid aggregates, cells or cell membranes. S-layers provide a natural environment for lipid membranes and biological materials: e.g. in many ar- chaeal organisms the S-layer is the only component external to the cytoplasmic membrane (Baumeister & Lembcke, 1992) . A broad range of S-layer proteins with different physico-chemical sur¬ face properties and functionalities are available. The imple¬ mentation of recombinant S-layer- (US 2002/0168728 Al) and S- layer-fusion proteins exhibiting functional domains, enables the application of specific binding mechanisms (Moll et al. , 2002) . Due to the high regularity of the crystal lattice, functionalit¬ ies repetitively arranged in well defined positions and orienta¬ tions can be obtained. Furthermore a significant reduction in surface roughness of the substrate is observable after recrys- tallization (Gufler et al., 2004) .
According to a preferred embodiment of the present invention the substrate comprises an electrically insulating material. It is a basic requirement of devices for electrophysiological meas¬ urements, e.g. patch clamp devices, that the current can only pass the susbstrate through the pores, transporters and channels part of a membranous structure present in the vicinity of a per¬ foration of the substrate. Therefore substrates used for patch clamp experiments may comprise electrically insulating material. The surface of the substrate or the entire substrate may com¬ prise an electrically insulating material.
Preferably the electrically insulating material material is selected from the group consisting of silicon, glass, solid state polymers and mixtures thereof. Microfabricated chips made of silicon, glass, solid state polymers or other materials are preferably used. Transparent chips, made e.g. of glass or syn¬ thetic polymers, can be used for simultaneous electrophysiolo¬ gical and optical or spectroscopical measurements (e.g. fluores¬ cence measurements, microscopical methods) . According to another embodiment part of the substrate may also comprise an electrode. In this case part of the substrate is electroconductive. The perforation is preferably 100 ran to 10 μm, preferably 500 nm to 9 μm, more preferably 1 μm to 8 μm, particularly 2 μm to 7 μm wide. The perforations of the chips are fabricated e.g. by nano- and micro-lithographic methods, laser or micromoulding techniques. The dimension of the perforation is adjusted accord¬ ing to the size of the membranous sample, whereby different per¬ forations of the substrate can possess differing dimensions. This demonstrates once more the flexible use of the substrate according to this invention.
"Wide" according to this invention refers to the physical dimensions of the perforations. For instance the term "wide" for a circular perforation refers to its diameter and "wide" for a quadratic perforation is referred to the distance between the nooks of such a perforation.
According to a preferred embodiment the substrate is sub¬ stantially planar. The geometry of a substrate can be manifold. For instance the substrate can be planar like chips or exhibit a spherical form.
According to another preferred embodiment the substrate is chemically or physically modified. Not only the protein layer on the surface of the substrate can be modified, but also the sur¬ face of the substrate itself. Such modifications allow creating zones in which e.g. no protein layer can adhere and other zones where an interaction between surface of the substrate and the membranous sample is prevented. Other modifications may prevent an unspecific binding of the membranous sample on the surface of the substrate, where no protein layer is present.
Such a modification is preferably selected from the group consisting of ionizing radiation, atomic radicals, corona treat¬ ment, silane groups, chemical groups and mixtures thereof. Chem¬ ical groups may comprise carbohydrates (e.g. secondary cell wall polymer), biotin and reactive groups. Other chemical groups and modification methods are disclosed in several publications and textbooks (Schalkhammer, 2002; Wong, 1991) .
According to another aspect of the present invention the substrate can be used for electrophysiological measurements, preferably for patch clamp measurements. Especially apparatus for patch clamp experiments can employ a substrate' as disclosed herein.
Preferably the substrate according to the present invention can be used for a device for simultaneous electrophysiological measurements, preferably for patch clamp measurements, and op¬ tical or spectroscopical measurements, if the substrate com¬ prises a transparent electrically insulating material.
Another aspect of the present invention relates to a method for measuring an electrical property of a membranous sample us¬ ing a substrate as disclosed.
According to a preferred embodiment the membranous sample is selected from the group consisting of cells, cell fragments, cell membranes, liposomes, artificial lipid membranes, lipid layers and mixtures thereof. The membranous sample comprises next to lipids and other cell membrane components particularly transporters and ion channels.
The substrate of the present invention may be preferably used in a method for the simultaneous electrophysiological and optical or spectroscopical measurements of a membranous sample.
Another aspect of the present invention relates to kit for the electrophysiological measurement of a membranous sample com¬ prising a substrate according to the present invention and a device for electrophysiological measurements, preferably a patch clamp device.
The present invention is further illustrated by the follow¬ ing example and figures, yet without being restricted thereto.
Fig.l shows two examples of substrate - and perforation (a)- structures (sections: IA and IB; top view: 1C) . The substrate is provided with a plurality of perforations.
Fig.2 shows S-layer coatings. The S-layer adhesion site (a) for the positioning of cells/liposomes/artificial membranes is recrystallized around the aperture (section: 2A, top view: 2B) . An inert coating (b) of the residual chip surface can be .per¬ formed by using another S-layer protein species (section: 2C, top view: 2D) . The S-layer adhesion sites (Fig. 2, marked with a) can be structured or non-structured. The S-layer patterns can be performed simple, economical, reproducible and versatile. Structuring can be performed by (i) nano- and micromoulding techniques (Gyδrvary et al. , 2003a), (ii) microcontactprinting or (iii) local depletion of previously generated coherent S-lay¬ er lattices (microlithography, e.g. ion beam, deep UV) (U.S. Patent: US 6296700 Bl) . The mould is placed onto the chip sur¬ face. The mould can either be prefilled with the distinct S-lay- er solution(s) or the protein solution(s) is (are) injected af¬ terwards into the cavity(ies) .
Fig.3 shows possible architectures of S-layer adhesion sites. Areas of tight contact and local specific interactions (= adhesion sites) between the cells/liposomes/artificial membranes may be designed next to each other (e.g. bands, rings, patches etc., hereafter referred to as "zones") . Some examples of pos¬ sible patterns of S-layers around the chip aperture are illus¬ trated in Fig. 3. The rings, squares/rectangles and bands can be thin (a,f) or broad (b,g) . The apertures can be surrounded by a single S-layer zone (a,b,f,g) or by multiple zones (c,d e, h , i , j) . The distance between two zones (d,i) as well as the thickness of the different zones (e, j) can be varied. The rings, rectangles or bands can be continuous (a-e, f-j ) or non- continuous (k-n) . Different zones may be covered with different types of S-layers.
A further structuring embodiment is obtained by time-depend¬ ent control of the recrystallization process. Starting from a few randomly distributed nucleation sites, 2-dimensional growth of S-layer domains takes place in all directions, until neigh¬ bouring areas meet (Gyδrvary et al., 2003a) . Thus, the dimension and the number of crystallites generated per unit area (and hence, the area devoid of S-lattice) can be controlled by the time of incubation (== recrystallization time) : Fig. 3; (o) short recrystallization time, (p) longer recrystallization time. When different types (mixtures) of S-layer proteins are used for coating (recrystallization) , individual crystallites will as¬ semble next to each other (Sleytr, 1975) .
Fig.4 shows the composition of the S-layer lattice (protein type, lattice symmetry, functionality) within the adhesion site (A, B, C, D) and within one zone (E, F, G) . Variable architec¬ tures (as shown in Fig.3) exhibiting zones of S-layer lattices composed of proteins from either the same (Fig. 4A) or different (Fig. 4B) organisms (native or recombiant; single or variable functionalities) can be used. Different zones of an S-layer ad¬ hesion site can be composed of one functionality (Fig. 4C) or variable functionalities (Fig. 4D) . One zone can be composed of an S-layer lattice with one single functionality (Fig. 4E) or a mixture of at least two functionalities (same S-layer proteins, but different fused functionalities) (Fig. 4F) . One zone can be composed of S-layer proteins from different organisms exhibiting varying lattice symmetry (pA, pB, pC) and/or functionalities (Fig. 4G) .
Fig.5 shows the fabrication of an S-layer adhesion site by using the micromoulding technique (e.g. PDMS mould) .
Fig.6 shows additional modification of the substrate surface devoid of S-layer lattice within the adhesion site.
Fig.7 shows S-layer proteins as means for smoothing the sharp corners of the chip perforations.
Fig.8 shows the closing- and opening-behaviour of single gramicidin pores reconstituted in anchored and clamped giant liposomes.
EXAMPLE: The application of 2-dimensional protein crystals for gramicidin ion channel recordings from giant liposomes placed on a planar chip-device
Two-dimensional bacterial surface layer (S-layer) proteins were applied for coating of planar silicon chips and subsequent binding of giant unilamellar vesicles (GUVs) that were function- alized with gramicidin. The GUVs contained a low amount of biot- inylated lipids. A genetically engineered S-layer protein that exhibited a fused heterotetrameric streptavidin domain was re- crystallized around the aperture of the microstructured silicon chip forming an S-layer adhesion site. Successful binding via biotin-streptavidin interactions between GUV and the S-layer- streptavidin fusion protein layer (one GUV per aperture) was ob¬ served. Gigaseal formation was monitored by resistance measure¬ ments. The closing- and opening-behaviour of single gramicidin pores reconstituted in the anchored and clamped giant liposomes was observed. The additional coating of the residual chip sur¬ face (around the S-layer streptavidin fusion protein adhesion site) by an inert coating of wildtype SbpA, significantly dimin¬ ished unspecific adsorption and/or fusion processes of GUVs.
1. MATERIAL AND METHODS
1.1 Chemicals
The phospholipids EggPC and Biotin-cap-DPPE (Avanti Polar Lipids, Alabaster, AL) were dissolved and stored at -200C in chloroform (Merck, Darmstadt, Germany) at a concentration of 0.1M. As a standard buffer an aqueous solution of IM KCl was used. The buffer was prepared in MilliQ-water (Millipore, minim¬ um resistance >18M0hms cm) and thoroughly degassed and filtered through a 0.2-μm filter (Sartorius AG, Gδttingen, Germany) be¬ fore use. All chemicals were purchased from Sigma Aldrich (Vi¬ enna, Austria) .
1.2 Chip (substrate) preparation and treatment
A silicon wafer (NMRC, Cork, Ireland, 1x1 cm2, contact angle 64°) was structured by standard lithographic techniques and an¬ isotropic ion etching, resulting in an aperture with a diameter of ~ 1-7 μm. The aperture was placed in the centre of the wafer (one pore/chip) . The chips were cleaned by ultrasonic treatment twice for 20 min in 2% Hellmanex (Hellma, Mϋllheim, Germany) and twice for 20 min in absolute ethanol. After each sonication step, the slides were rinsed thoroughly in MilliQ-water and fi¬ nally dried in a stream of dry nitrogen. Cleaned chips were stored in clean air until usage.
1.3 Preparation of S-layer protein solutions
Growth, cell wall preparations, and extractions of the S- layer protein SbpA from the gram-positive strain B. sphaericus CCM 2177 (Czech Collection of Microorganisms) were performed as described elsewhere (Sleytr et al., 1986) . The wildtype SbpA stock solution was diluted with to a final concentration of 0.1 mg/ml. Molecular cloning, gene expression, isolation, refolding and purification procedures of S-layer streptavidin fusion pro¬ teins (rSbpA/strp) were described elsewhere (Moll et al., 2002) (Huber et al. , submitted) . rSbpA/strp proteins recrystallized on hydrophobic silicon as a monolayer with the streptavidin domain exposed to the aqueous solution. An appropriate amount of rSbpA/strp was desintegrated in 4M GHCl for 5 min. The protein was dialysed against MilliQ-water for 2 h. The protein solution was adjusted to a concentration of lmg/ml with MilliQ-water, centrifuged in an Eppendorf Centrifuge and stored at 4°. For re- crystallization rSbpA/strp was diluted with recrystallization buffer (0.5 mM Tris (pH = 9.0) containing 1OmM CaCl2) to a final concentration of 0.1 mg/ml. Recrystallization was performed from 2 to 4h.
1.4 Preparation of giant unilamellar liposomes (GUVs) func- tionalized with gramicidin
Preparation of giant unilamellar functionalized liposomes was performed by a slightly modified dehydration-rehydration procedure according to Riquelme et al. (1990) . In a first step unilamellar proteoliposomes were prepared. Chloroformic solu- tions of EggPC and Biotin-cap-DPPE (molar ratio 98:2) were added into a glass flask. An ethanolic solution of Gramicidin D, which predominantly (80%) consists of gramicidin A (Sigma, Deisen- hofen, Germany) was added (molar lipid/protein-ratio = 200:1) . The organic solvent was removed in a rotary evaporator for at least 4h. The dried film was hydrated to a final lipid concen¬ tration of 1% (w/v) by adding a standard buffer containing 2% CHAPS (Sigma, Vienna, Austria) . The detergent was either removed by dialysis or by addition of Bio Beads SM-2 (Sigma, Vienna, Austria) . Glass microscopic slides (ELKA, Germany) were cleaned in Hellmanex and absolute ethanol (ultrasonic treatment) , thor¬ oughly rinsed with MilliQ-water and dried in a stream of nitro¬ gen. Small drops (~ 20 μl) or wide stripes (for larger amounts) of the vesicle suspension were deposited onto the glass slides. The suspension was partially dehydrated for 3 h/4°C in a desic¬ cator. Giant liposomes exhibiting a size distribution from 20 μm to 80 μm were obtained by rehydration: an appropriate amount of standard buffer solution was deposited on the top of the dehyd¬ rated drop. Size and size distribution of GUVs were examined by transmission electron microscopy (Philips, Eindhoven, The Neth¬ erlands) of negatively stained samples. Formvar/carbon coated EM copper grids (Athene old/300 mesh) were deposited on a drop of the sample (5min) . Afterwards the sample was fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.0 and stained in 2% uranyl acetate.
1.5 Recrystallization of S-layer proteins on the chip An S-layer adhesion site composed of rSbpA/strp was recrys- tallized around the micromachined aperture on the chip by a soft-lithographic technique, micromoulding in capillaries, as previously described (Gyδrvary et al., 2003a) . For mold forma¬ tion, mesastructures mold masters in photoresist (AZ 9260; Clariant) on 4-in. silicon wafers were fabricated by photolitho¬ graphic methods. A master pattern of two horseshoe-like struc¬ tures was chosen. The horseshoe structures were arranged to each other such that an almost continuous ring with two inlets was formed. Poly(dimethylsiloxane) , PDMS (Sylgard 184; DOW corning), is used to fabricate the molds from these masters using the standard procedure (Kumar et al., 1994) (Kim et al., 1995) . The mold was carefully placed onto the manufactured silicon chip (microscopical control) such that the aperture was circularily surrounded by the two horseshoe-like capillaries. Starting from inlets the microchannels (20 μm channel width) were filled with the rSbpA/strp recrystallization solution. After self-assembly and recrystallization of rSbpA/strp proteins the mold was re¬ moved under MilliQ-water, leaving the patterned rSbpA/strp ar¬ rays behind. Additionally, in some experiments the residual chip surface was coated with wildtype SbpA, which exhibited low un- specific binding surface properties. SbpA formed a monolayer on hydrophobic silicon. The recrystallization quality and integrity of the S-layer was controlled by Atomic Force Microscopy (AFM) analysis. AFM images were recorded in contact mode in liquid with a Nanoscope III AFM (Nanoscope III, Digital Instruments, Santa Barbara, CA) . Oxide-shaped silicon nitride tips (Nano- Probes, Digital Instruments) with a nominal spring constant of 0.06 N/m were used. A 100 mM NaCl solution was used for imaging.
2.6 Experimental set-up
The voltage-clamp setup was mounted on a steel core bread¬ board (CS-11-4, Newport, Darmstadt, Germany) . The headstage of the patch clamp amplifier (EPC 9, HEKA Electronic, Lambrecht/Pfalz, Germany) was fixed on a translation stage (M- 426, Newport, Darmstadt, Germany) which could be moved vertic¬ ally (Motorizer 860 A, Newport, Darmstadt, Germany; regulated DC power supply: Kenwood PR 18-1.2, Tokyo, Japan) . The headstage of the amplifier, together with measurement chamber were placed within a grounded Faraday cage. The S-layer covered chip was placed horizontally into a home-made teflon-chamber. The chip was sealed into the chamber by a silicone paste (Bayer AG, Leverkusen, Germany) und thus divided the chamber into two com¬ partments. Ag/AgCl redox electrodes were used for voltage-clamp recordings. The reference electrode was placed in the lower, the working electrode in the upper compartment. The lower compart¬ ment comprised a vacuum device in order to apply a suction to the clamped liposome. The S-layer covered surface was facing the upper compartment. During this procedure, desiccation of the S- layer was carefully prevented. After placing the chip, the cham¬ ber was filled with the standard buffer solution. A small amount of the freshly prepared biotinylated gramicidin-GUV suspension was pipetted onto the S-layer. A single liposome was bound to the rSbpA/strp adhesion zone. After application of a gentle suc¬ tion, seal formation was monitored by resistance measurements. 2.7 Signal recording and data acquisition
The current response of a given voltage function was meas¬ ured at 250C to calculate the electrophysiological parameter of the clamped liposome patch. Conductance was measured via rectan¬ gular voltage pulses of up to 20OmV across the membrane. All the data handling (voltage stimulation, data acquisition, data' stor¬ age, data analysis) was performed on a Power Macintosh 7600/120 computer (Apple Computer GmbH, Ismaning, Germany) . The setting of the two built-in Bessel filters of the EPC 9 amplifier for the current-monitor signal was 10 and 1.5 kHz, respectively. The data analysis was performed by the Pulse + PulseFit software 7.89/Quadra (HEKA Elektronik, Lambrecht/Pfalz, Germany) . Correl¬ ations of conductance vs. time and statistical analysis (ANOVA) were performed by using Jandel SIGMA PLOT program for Windows.
2. RESULTS AND DISCUSSION
A prototype of a planar S-layer-based patch-clamp-chip was implemented. GUVs functionalized with biotinylated lipids and gramicidin were chosen as an appropriate system for single chan¬ nel recordings. The size of GUVs varied from ~20-80 μm, the size distribution was fairly balanced. In some cases, multilamellar vesicles were obtained, which were not used.for the experiments. The GUVs were used immediately after preparation or for later experiments devided into aliquots and stored at -800C until us¬ age.
The recrystallization of rSbpA/strp and wildtype SbpA was monitored by Atomic Force Microscopy. All types of S-layers used in this example recrystallized on silicon chip as monolayered S- layers with the functional (for rSbpA/strp) or the inert surface (for wildtype SbpA) exposed to the aqueous solution. Recrystal¬ lization of S-layers on silicon as well as the microstructuring of S-layers by using the micromoulding technique (PDMS moulds) are well-established (Gyδrvary et al., 2003a, 2003b) . Diameters of microstructured S-layer adhesion sites ranging from 60-100 μm were obtained. Optionally, the residual chip surface around the rSbpA/strp adhesion site was coated with wildtype SbpA. The out¬ er surface of SbpA is charge-neutral. On chips without a wild¬ type SbpA layer, adhesion and/or fusion of GUVs to larger lipid aggregates or bilayered structures were observed. A significant decrease in unspecific adsorption and/or fusion processes of GUVs was achieved. Since liposomes only adsorbed slightly and at a very low amount, they could be washed away easily by gentle rinsing of the upper compartment of the measurement cell. In the majority of experiments a single GUV was bound per S-layer adhe¬ sion site. In some experiments either the GUV was not centered and thus no gigaseal was obtained or more smaller GUVs per adhe¬ sion site were bound.
Functional studies of single membrane-active peptides and membrane proteins like e.g. ion channels require the reduction of electrical background noise and consequently a high electric¬ al insulation of the surrounding membrane patch, the so-called Λλgiga-seal" (R>109 Ω) . The arrangement of the giant liposomes on the perforation is achieved by the linkage of biotinylated lip¬ ids within the giant liposomes with the patterned S-layer-strep- tavidin fusion protein. The tight seal, however, is obtained manually by suction. Stable membranes on the chip with seal res¬ istances of R>109 Ω were obtained in 17 out of 19 experiments.
The challenge of preparing lipid membranes on silicon chips is the recording of single ion carriers and channels. The gener¬ al functionality of this concept was demonstrated with gramicid¬ in, a peptide known to self-integrate into one leaflet of a lip¬ id bilayer and to form single channels if dimerization occurs. The closing- and opening-behaviour of single gramicidin pores reconstituted in the anchored and clamped giant liposomes was observed (Fig. 8) . As a driving force for ion flux, a voltage typically around 200 mV was applied across the bilayer. Gram¬ icidin is selective for monovalent cations and as KCl solution was used, K+ currents were recorded. There were clearly five different levels (on horizontal) visible, the lowest represent¬ ing all channels closed, and each additional level the opening of a channel unit (corresponding to a peptide dimer) . Also most channels had the same amplitude of about 8.4 pA, some channels were smaller, i.e., carried less current or transports fewer ions per unit time. This may be due to impurities in Gramicidin D preparation as this preparation is known to contain also a few proportions of other peptides.
The present example shows that stable gigaohm seals over mi¬ crometer-sized holes can be obtained. The self-positioning of biotinylated giant liposomes can easily be achieved by the high binding affinity of biotin to the streptavidin hetrotetramers which are presented in a well-defined and oriented way by the S- layer lattice. Thus, no suction is necessary for the positioning of the target molecules, which is always problem as this leads often to clogging of the aperture. If the target molecule is on the right position, suction guarantees the giga-seal formation between the liposome and the aperture. The application potential of this new technique (positioning by the S-layer fusion - lipid anchor molecule interaction and subsequent seal formation by suction of giant liposomes) was clearly demonstrated by measur¬ ing the transmembrane current through single ion channels, or in other words by tracing single molecules.
References:
Baumeister,W. and G.Lembcke. 1992. Journal of Bioenergetics and Biomembranes 24:567-575.
Brϋggemann,A. , George,M., Klau,M., Beckler,M., Steindl,J., Beh- rends,J.C, Fertig,N. 2003. Assay and Drug Development Technolo¬ gies 1 (5) :665-673.
Fertig,N., M.Klau, M.George, R.H.Blick, and J.C.Behrends. 2002. Applied Physics Letters 81:4865-4867.
Gufler,P., D.Pum, U.B.Sleytr, and B.Schuster. 2004. Biochimica et Biophysica Acta 1661:154-165.
Gyorvary,E.S. , A.O'Riordan, A.J.Quinn, G.Redmond, D.Pum, and U.B.Sleytr. 2003a. Nano Letters 3:315-319.
Gyorvary,E.S. , O.Stein, D.Pum, and U.B.Sleytr. 2003b. Journal of Microscopy 212:300-306.
Kim, E; Xia, Y. and Whitesides, G. M. 1995. Nature 376:581-584. Klemic,K.G. , J.F.Klemic, M.A.Reed, and F.J.Sigworth. 2002. Bio¬ sensors and Bioelectronics 17:597-604.
Kumar,A., H.A.Biebuyck, and G.M.Whitesides. 1994. Langmuir 10:1498-1511.
Kϋpcϋ,S., M.Sara, and U.B.Sleytr. 1995. Biochimica et Biophysica Acta 1235:263-269.
Mader,C, S.Kϋpcϋ, M.Sara, and U.B.Sleytr. 1999. Biochimica et Biophysica Acta 1418:106-116.
Moll, D., C.Huber, B.Schlegel, D.Pum, U.B.Sleytr, and M.Sara. 2002. Proceedings of the National Academy of Sciences of the United States of America 99:14646-14651.
Neubauer,A., S.Pentzien, S.Reetz, W.Kautek, D.Pum, and U.B.S¬ leytr. 1997. Sensors & Actuators B 40:231-236.
Neubauer,A., W.Kautek, S.Pentzien, S.Reetz, M.Sahre, T.Solomun, R.Korntner, D.Pum, and U.B.Sleytr. 1998. PTB-Berichte F-34:75- 81.
Pantoja,R., D.Sigg, R.Blunck, F.Bezanilla, and J.R.Heath. 2001. Biophysical Journal 81:2389-2394.
Pum, D., A.Neubauer, E.Gyoervary, U.B.Sleytr. 2000. Nanotechno- logy 11:100-107.
Riquelme,G., E.Lopez, L.M. Garcia-Segura, J.A. Ferragut, and J.M.Gonzalez-Ros. 1990. Biochemistry 29:11215-11222. Sakmann, B., and E.Neher. 1995. Single channel recording (2nd edition) . New York, London:Plenum Press. Schalkhammer, T.G.M. (ed.) 2002. Analytical Biotechnology. Basel, Boston, BerlinrBirkhauser Verlag.
Schmidt,C, M.Mayer, and H.Vogel. 2000. Angewandte Chemie Int. Ed. Engl. 39:3137-3140.
Schuster,B., ϋ.B.Sleytr, A.Diederich, G.Bahr, and M.Winterhalter. 1999. European Biophysics Journal 28:583-590. Sigworth,F.J. and K.G.Klemic. 2002. Biophysical Journal 82:2831- 2832.
Sleytr,U.B. 1975. Nature 257:400-402.
Sleytr,ϋ.B., M.Sara, Z.Kϋpcϋ, and P.Messner. 1986. Archives of Microbiology 146:19-24.
Sleytr,U.B., D.Pum, E.Gyδrvary, B.Schuster, S.Kϋpcϋ, and M.Sara. 2002. Progress in Colloid and Polymer Science 121:57-61. Sleytr,U.B., B.Schuster, and D.Pum. 2003. IEEE Engineering in Medicine and Biology Magazine 22:140-150.
Wetzer,B., D.Pum, and ϋ.B.Sleytr. 1997. Journal of Structural Biology 119:123-128.
Wetzer,B., A.Pfandler, E.Gyδrvary, D.Pum, M.Lδsche, and U.B.S- leytr. 1998. Langmuir 14:6899-6906.
Wong, S.S. 1991. Chemistry of protein conjugation and cross- linking. Boca Raton, Ann Arbor, Boston, London:CRC Press, Inc.

Claims

Claims :
1. A substrate for a device for electrophysiological measure¬ ments comprising at least one perforation, characterised in that the substrate is covered at least in part with at least one pro¬ tein and/or glycoprotein layer.
2. A substrate according to claim 1, characterised in that the at least one protein layer is modified with at least one func¬ tionality.
3. A substrate according to claim 2, characterised in that the at least one functionality is selected from the group consisting of proteins, polypeptides, peptides, ligands, chemical groups and mixtures thereof.
4. A substrate according to any one of claims 1 to 3, character¬ ised in that the at least one protein layer is composed of at least one S-layer.
5. A substrate according to any one of claims 1 to 4, character¬ ised in that the substrate comprises an electrically insulating material.
6. A substrate according to claim 5, characterised in that the electrically insulating material is selected from the group con¬ sisting of silicon, glass, solid state polymers and mixtures thereof.
7. A substrate according to any one of claims 1 to 6, character¬ ised in that the perforation is 100 nm to 10 μm, preferably 500 nm to 9 μm, more preferably 1 μm to 8μm, particularly 2 μm to 7 μm wide.
8. A substrate according to any one of claims 1 to 7, character¬ ised in that the substrate is substantially planar.
9. A substrate according to any one of claims 1 to 8, character¬ ised in that the substrate is chemically or physically modified.
10. A substrate according to claim 9, characterised in that the modification is selected from the group consisting of ionizing radiation, atomic radicals, corona treatment, silane groups, chemical groups and mixtures thereof.
11. Use of a substrate according to any one of claims 1 to 10 for a device for electrophysiological measurements, preferably for patch clamp measurements.
12. Use of a substrate according to any one of claims 1 to 10 comprising a transparent an electrically insulating material for a device for simultaneous electrophysiological measurements, preferably for patch clamp measurements, and optical or spectro- scopical measurements.
13. A method for the electrophysiological measurement of a mem¬ branous sample using a substrate according to any one of claims 1 to 10.
14. A method according to claim 13, characterised in that the membranous sample is selected from the group consisting of cells, cell fragments, cell membranes, liposomes, artificial lipid membranes, lipid layers and mixtures thereof.
15. A method for the simultaneous electrophysiological and op¬ tical or spectroscopical measurements of a membranous sample us¬ ing a substrate according to any one of claims 1 to 10.
16. A kit for the electrophysiological measurement of a membran¬ ous sample comprising a substrate according to any one of claims 1 to 10 and a device for electrophysiological measurements, preferably a patch clamp device.
PCT/EP2004/007878 2004-07-15 2004-07-15 Substrate for a device for electrophysiological measurements WO2006005370A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
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Application Number Priority Date Filing Date Title
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Publications (1)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6315940B1 (en) * 1996-11-16 2001-11-13 Nmi Naturwissenschaftliches Und Medizinisches Institut An Der Universitat Tubingen In Reutlingen Microelement device
WO2002004943A2 (en) * 2000-07-07 2002-01-17 Bristol-Myers Squibb Company Electrophysiology configuration suitable for high throughput screening of compounds for drug discovery
US20020182627A1 (en) * 2001-03-24 2002-12-05 Xiaobo Wang Biochips including ion transport detecting strucutres and methods of use
US20030146091A1 (en) * 1997-12-17 2003-08-07 Horst Vogel Multiaperture sample positioning and analysis system
US20040033576A1 (en) * 2000-04-26 2004-02-19 Sleytr Uwe B Use of secondary cell wall polymer of procaryotic microorganisms

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6315940B1 (en) * 1996-11-16 2001-11-13 Nmi Naturwissenschaftliches Und Medizinisches Institut An Der Universitat Tubingen In Reutlingen Microelement device
US20030146091A1 (en) * 1997-12-17 2003-08-07 Horst Vogel Multiaperture sample positioning and analysis system
US20040033576A1 (en) * 2000-04-26 2004-02-19 Sleytr Uwe B Use of secondary cell wall polymer of procaryotic microorganisms
WO2002004943A2 (en) * 2000-07-07 2002-01-17 Bristol-Myers Squibb Company Electrophysiology configuration suitable for high throughput screening of compounds for drug discovery
US20020182627A1 (en) * 2001-03-24 2002-12-05 Xiaobo Wang Biochips including ion transport detecting strucutres and methods of use

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PUM D ET AL: "The application of bacterial S-layers in molecular nanotechnology", TRENDS IN BIOTECHNOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 17, no. 1, January 1999 (1999-01-01), pages 8 - 12, XP004155527, ISSN: 0167-7799 *

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