DE102007002913A1 - Chip for measurement of characteristics of molecule present in diaphragm, particularly in channel or pore-forming molecule, and for optical single transporter recording technology in biophysical basic research, comprises substrate molecule - Google Patents

Abstract

The chip (5) comprises a substrate molecule, a diaphragm (30) on a part of the surface of a layer (10), nanoscale compartment covered by the diaphragm. The layer is made of a fluorine polymer, silicon, hydrogel or a metal, and is applied on a carrier. The diaphragm is a biological diaphragm, which is a lipid membrane. Independent claims are included for: (1) a test device for the measurement of characteristics of a molecule, which has a chip, and liquid substances, which are contained in the substrate molecules; and (2) a method for production of a chip, which involves stamping nano-base recesses in a layer with the help of embossing die, and attaching a membrane on a part of the surface layer of the layer after stamping the recesses.

Description

State of the art

proteins, transport the substances actively or passively through membranes and briefly referred to here as "transporter" play a central role in the function of biological cells and the Emergence of many economically significant diseases such as z. Heart disease, epilepsy, HIV and cystic fibrosis. Already In 2002, transporters accounted for 11% of all therapy goals in the future an immense potential for drug discovery. Characterization of channel proteins and active transporters However, it is still a big challenge for us biomedicine and biotechnology. Currently available methods are elaborate and slow. That's why new procedures are needed with which the functional properties of transporters and their Influenced by potential drugs in high throughput measured can be. In particular, new single molecule techniques will be used needed to do the analysis of transporters in non-synchronized and to enable heterogeneous molecular populations.

The currently the most commonly used method for are the analysis of functional properties of individual transporters the so-called "patch-clamp" method and the "BLM" (black lipid membrane) method. Both methods are based on electrical Conductivity measurements. With this patch-clamp procedure can transporters that have inorganic ions active (transporter molecules) or passive (ion channels) transport very well Single molecule level are analyzed. The variety of transporters but, the small, electroneutral molecules or macromolecules How proteins and nucleic acids can transport, only in exceptional cases and then only indirectly analyzed with the patch-clamp method become.

Further Disadvantages of the patch-clamp method are a relatively low sensitivity, the impossibility of multiple transport substrates at the same time measure and the difficulties of automating such measurements, parallelize and in high-throughput (High Throughput Screening, HTS).

Because of The great advances in genomics are transporters and their pathophysiological mutants today predominantly through Expression of transporters in microorganisms, common in bacterial or eukaryotic cell systems or by their isolation and reconstitution in artificial lipid vesicles, the more typical Have a diameter of 100-500 nm. For electrical measurements, it is necessary to reconstituted the nanoscopic To fuse lipid vesicles with macroscopic lipid membranes, an experimentally complicated and uncontrollable process.

One inherent disadvantage of electrical measurements is that only electrically charged substrates, such as salt ions or ionized molecules, can be measured. moreover usually affects those used to measure ion transport applied electrical voltage measuring us it must be a high achieved electrical resistance between both sides of the membrane will reach to a sufficient measuring sensitivity.

The Problems of electrical measurements can be confirmed by fluorescence microscopy Measurements are overcome. With high resolution Fluorescence measurements of single molecules are possible single molecules to locate, the transmembrane transport the individual molecules can be effected but usually not be observed. Transport measurements with the one introduced by us Technology of "Optical Single-Carrier Analysis" (OSTR = Optical Single Transporter Recording) are on vans of all Kind possible. OSTR measurements are still due to single molecule sensitivity, Substrate multiplexing and high throughput characterized. The OSTR measurements also have when using reconstituted lipid vesicles Benefits, because they directly on nanoscopic lipid vesicles, d. H. without fusion with macroscopic planar lipid membranes can be.

In previous works, R. Peters "Optical Single Transporter Recording: Transport Kinetics in Microarrays of Membrane Patches," Annu. Rev. Biophys. Biomol. Struct. 2003; 32: 47-67 ; R. Peters "The Nanopore Connection to Cell Membrane Unitary Permeability", Traffic 2005; 6: 199-204 ; Hemmler et al. "Nanopore Unitary Permeability Masured by Electrochemical and Optical Single Transporter Recording", Biophysical J., Vol. 88, June 2005, pp. 4000-4007 ; Kiskin et al. "Optical Microwell Assay of Membrane Transport Kinetics", Biophysical Jouranl, Vol. 85, Oct. 2003: 2311-2322 , whose content is hereby incorporated, so-called OSTR chips were produced by laser ablation of polycarbonate films. This allowed the generation of arrays with a limited number of relatively homogeneous test compartments (TC). However, the diameter of the TCs was limited to> 5 μm. Also, the TCs were not completely cylindrical, but slightly distorted.

Summary of the invention

By contrast, the invention described here makes it possible to produce dense and very large arrays of TCs. The diameter of the TCs ge According to the invention can be freely selected in the range of about 50-3000 nm. The TCs are very homogeneous and have an almost perfectly cylindrical geometry. Examples are in 2 and 3 shown. The TCs can be cylindrical depressions. The TCs may also be holes in a first material and be covered on one side by a second material that may be different from the first.

The invention described here is ideal for the cost-effective production of OSTR chips in industrial Scale. This allows the OSTR chips as To produce disposable items and to cumbersome cleaning to renounce.

The Availability of very small TCs makes it possible reconstituted lipid vesicles (diameter 100-500 nm) directly, because the diameter of the TCs is smaller than the diameter of the lipid vesicles can be chosen. Ie. a fusion with macroscopic bilayers is not necessary in the invention.

So far The polycarbonate OSTR chips were used. This material is birefringent with refractive indices in the range of 1.52-1.55. By the refractive index difference between the OSTR chip and the physiological aqueous medium (refractive index about 1.35) it comes to optical distortion, which is a high-resolution microscopic Illustration of the membranes spanning the test compartments to make impossible.

The OSTR chips described herein are made of materials whose refractive index is substantially identical to the physiological aqueous media. As materials, a fluoropolymer, silicon, hydrogel or a metal can be used. This avoids optical artifacts due to differences in refractive index and enables high resolution fluorescence microscopic analysis on the TC spanning membranes. The optical properties of the TC arrays described herein are in 3 shown.

The material used for the OSTR chips is biocompatible and harmless to membranes and transporters.

So far Membranes were cleaned on cleaned but unmodified polycarbonate surfaces bound. For the OSTR chips described here, first small organic molecule (linker) to the fluoropolymer surface bound. These linkers carry chemical groups that contain sulfhydryl groups (SH groups) quickly form a strong, covalent bond can. More cellular on the surfaces Membranes are often free SH groups. In the Surface of artificial lipid vesicles can free SH groups by addition of appropriately modified lipids be inserted at will. At contact cellular Membranes or SH lipid-doped lipid vesicles with the ones here described surface-modified OSTR chips is formed practically immediately a firm, irreversible bond. It is because of that possible, membranes and membrane vesicles whose diameter is> 100 nm, with the To analyze OSTR procedures.

at OSTR technology binds membranes to OSTR chips. these are planar and transparent platelets, which are a lot smaller Wells, here called test compartments (TC) included. Of the Transport of fluorescent substrates through the membranes in the TCs are measured by confocal microscopy. It can also change the fluorescence of a substance present in the TC by a through the membranes transported substrate are measured, for. B. by Fluorescence quenching, change in local Ca ++ concentration, the local pH value or similar.

The The invention described herein is intended to enable the OSTR technique to a routine procedure of biophysical, physiological, pharmacological and pharmaceutical basic research and the to make industrial pharmacological drug development.

The OSTR chips described herein are made up of thin layers a transparent fluoropolymer such as Teflon FEP, Teflon AF or Cytop, which are applied on planar glass saws and a dense array of microscopic or nanoscopic wells (Test compartments, TC). The structuring of the polymer layers done by a nanoimprint technique. The surfaces the nanostructured chips are modified in a special way, so that cellular and artificial membranes become firm tied and stretched over the TCs.

The The invention will now be explained with reference to the following drawings. Show it

1 the principle of optical single-transporter analysis

2 the wells with different diameters

3 Fluorescence images of the chips

4 Presentation of the stamping process

Detailed description the invention

The OSTR chip 5 ( 1 ) is used to analyze transport rates of substrates by transporters. Transporters may be passive transporters such as pore-forming or channel-forming peptides or proteins, such as nuclear pro-complex (NPC), α-hemolysin, porins, ligand- or voltage-gated ion channels, or other proteins. Transporters may also be active transporter molecules that transport substrates from one side of the membrane to the other, such as ATP-ADB translocators, protein translocase or the like.

According to the invention the transport of the substrates or the corresponding transport rate be analyzed by fluorescence. These can be the substrates for example, directly with fluorescent molecules, fluorescent Nanoparticles are fluorescently labeled in a manner known to those skilled in the art. However, it is also possible to introduce fluorescence sensors into the ICs be their fluorescence depending on the pH, a Change ion concentration, temperature or other parameter.

The fluorescence intensity or a change in the fluorescence intensity can be determined individually for each of the test compartments (TCs) using confocal laser scanning microscopy and the transport kinetics and permeability of individual transporters can be determined therefrom. C _TC , F _TC , C _C and F _C are concentration and fluorescence intensity in the test compartments ( 20 ) and the measuring chamber ( 50 ). Here are transparent platelets, so-called OSTR chips 5 used dense arrays with a variety of microscopic or nanoscopic pits 20 (so-called test compartments) included. Cellular or reconstituted artificial membranes 30 , the transporters 40 contained, are firmly tied to the chips. The transport of substrates which can either fluoresce themselves or be indicated by fluorescence indicators is measured by confocal microscopy. On a slide 10 with a variety of nanoscopic test compartments 20 becomes a lipid membrane 30 with transporters 40 applied. A fluorescent transport substrate (•) and a control substrate (o) are placed in a measuring chamber 50 given.

The Fluorescence can also be determined using zero-mode waveguide methods become. In doing so, the structure or a section of the structure used as waveguide (waveguide). For example, light can be routed into the lower section of the OSTR chip, so that Light is scattered into the ICs (evanescent light). This can the background radiation by excitation of fluorescent molecules be avoided outside the ICs.

The following describes the production of the OSTR chip. First, a stamping tool 300 produced.

One Silicon substrate was coated with one for electron beam lithography coated resist to be used. The structuring of this resist was carried out by electron beam lithography, d. h., the desired Structures were designed and dimensioned by CAD and incorporated into CAD fed the plant. This can be both as electron beam microscope as well as electron beam writer. In this example The resist was exposed directly by electron beam. Compared to other exposure methods is the illumination of the resist very time consuming, because the entire silicon substrate does not come with a is illuminated, but the individual structures in succession to be written in the paint. The process pays for that but by the high possible resolution. In a further process step, the development will be the previously exposed Removed areas and the structures are in the resist in front.

The actual structuring of the silicon substrate was carried out by means of reactive ion etching (RIE), the previously structured Resist served as an etching mask. Since the erosion is both chemical As well as physically, the method was both due to its high selectivity as well as anisotropy.

After this the residual resist has been removed, lies the finished microstructured Embossing tool in front.

The Structures are then formed by hot stamping.

Hot stamping is one of the standard processes for the microstructuring of plastics in addition to injection-compression molding and injection molding. The basic idea of hot stamping is that both the structured stamping tool 30 as well as a plastic substrate to be heated above the glass transition temperature (T _G ) of the polymer before the power or path controlled stamping process takes place. For the final step, the demolding, substrate and tool are cooled to a temperature just below T _G and separated from each other.

The embossing of a polymer layer is now based on the 4 described.

Attempts to apply a polymer 310 on a substrate 320 With spincoating of various liquid fluoropolymers were successful, making imprints in the nanoimprint process of arrays with cylindrical holes 330 with 3 μm, 500 nm and 250 nm could be performed. However, only layer thicknesses of up to 5 μm could be achieved by means of spincoating, whereby due to the precipitation of the material during embossing too little material for homogeneous embossing of holes 330 with ~ 3 μm depth was available.

For this reason, in another example, a thicker embossable fluoropolymer film was made 310 used. The fluoropolymer film 310 is on the intended substrates 320 laminated from glass in a separate process step before the actual embossing. To achieve maximum adhesion, the substrate becomes 320 previously degreased and cleaned. In addition, an etching of the substrate takes place 320 using a dry etching process to increase the surface area. The lamination of the polymer film 310 occurs at a temperature well above T _G. The resulting low viscosity of the polymer allows filling of the smallest voids on the roughened substrate surface. In this case, arrays of wells with diameters of 250 nm, 500 nm and 3 μm and a depth of 2.5 μm were successfully shaped (cf. 4 ).

The embossed structures were determined by fluorescence analysis examined in the laser scanning microscope. The material also showed no autofluorescence after the nanoimprint. After adding a fluorophore solution diffused into the impressed depressions and enabled Fluorescence images. The material shows due to the low Refractive index a slight optical fiber effect, reducing the nanoscopic Wells appear elongated in the z direction.

The Electron micrographs and fluorescence images show that the method described here is suitable, OSTR chips with different Diameters of the individual wells in the range of 250 nm to to reproducibly produce in the μm range.

OSTR chips can also be made from a hydrogel, metal or silicon using other methods known to those skilled in the art can be.

For the OSTR technique, it is necessary to couple biological membranes or lipid bilayers to the surface of the chips. The fluoropolymer film used 320 allows the application of a lipid layer without further surface treatment, allowing the use of OSTR for membrane pores in lipid membranes without further modification of the surfaces. As a biological membrane z. B. the nuclear membrane of Xenopus oocytes used. This adheres to the fluoropolymer film 320 good enough for an investigation in the OSTR procedure.

The Possibility of high throughput of the OSTR method with appropriate Biochips also exists, but the necessary preparation is required correspondingly large stamp in the cost plan of the feasibility study not included.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited non-patent literature

• - R. Peters "Optical Single Transporter Recording: Transport Kinetics in Microarrays of Membrane Patches", Annu. Rev. Biophys. Biomol. Struct. 2003; 32: 47-67 [0007]
• - R. Peters "The Nanopore Connection to Cell Membrane Unitary Permeability", Traffic 2005; 6: 199-204 [0007]
• - Hemmler et al. "Nanopore Unitary Permeability Masured by Electrochemical and Optical Single Transporter Recording", Biophysical J., Vol. 88, June 2005, pp. 4000-4007 [0007]
• Kiskin et al. "Optical Microwell Assay of Membrane Transport Kinetics", Biophysical Jouranl, Vol. 85, Oct. 2003: 2311-2322 [0007]

Claims (15)

Chip ( 5 ) for measuring properties of a membrane ( 30 ), in particular a channel or pore-forming molecule or molecule having a transport mechanism, with a substrate molecule; the membrane ( 30 ) on at least part of the surface of a layer ( 310 ), the nanoscale compartments covered by the membrane ( 209 ). The chip ( 5 ) according to claim 1, wherein the layer ( 310 ) of a fluoropolymer, silicon, hydrogel or a metal. The chip ( 5 ) according to claim 1 or 2, wherein the layer ( 310 ) on a support ( 320 ) is applied. The chip ( 5 ) according to any one of claims 1 to 3, wherein the membrane ( 30 ) is a biological membrane. The chip ( 5 ) according to any one of claims 1 to 4, wherein the membrane ( 30 ) is a lipid membrane. The chip ( 5 ) according to any one of claims 1 to 5, comprising an optical measuring instrument for measuring the substrate molecules which have entered the compartments. The chip ( 5 ) according to any one of the preceding claims, wherein in the membrane ( 30 ) is a protein. The chip ( 5 ) according to any one of the preceding claims, wherein the substrate molecule comprises a fluorescent group. Test device for measuring properties of a Molecule, with a chip according to one of the claims 1 to 8, and liquid substances that are the substrate molecules contains. Method for producing a chip ( 5 ) comprising: embossing nanoscale depressions ( 20 ) in a layer ( 10 ; 310 ) using an embossing tool ( 300 ). - attaching a membrane ( 30 ) on at least part of the surface of the layer ( 10 ) after embossing the wells ( 20 ). The method for producing a chip ( 5 ) according to claim 10, wherein the layer ( 310 ) on a support ( 320 ) is applied. The method for producing a chip ( 5 ) according to claim 11, wherein the layer ( 310 ) by applying a film on the support ( 320 ) is applied. The method for manufacturing a chip according to claim 11, wherein the attachment of the layer ( 310 ) by spin coating a resist. The method for producing a chip according to any one of claims 10 to 13, wherein the layer ( 310 ) is made of fluoropolymer. The method for producing a chip according to any one of claims 10 to 14, wherein the embossing tool ( 30 ) is made of silicon substrate.