DE102004008241B4 - Enzyme-based nanolithography - Google Patents

Abstract

method for the targeted coating of a probe with enzyme or effector molecules, comprising contacting the probe with a flat support the enzymes or effector molecules by means of a polyvalent binding partner A to a binding partner B on the carrier are bound, wherein at least a portion of the probe the same binding partner B, and wherein the molecule, that from the enzyme or effector molecule and the polyvalent binding partner A exists in the state not bound to the binding partner B at least two free binding sites for having the binding partner B.

Description

The The present invention relates generally to an enzyme-assisted method for nanolithography and apparatus for carrying out such methods. Especially The present invention relates to a method for targeted coating a probe with enzyme or effector molecules as well as a lithographic Method for structuring a surface of a carrier, wherein by a localized enzymatic activity in the immediate vicinity of the surface of these chemically or physically modified by positioning an immobilized on a probe enzyme or effector molecule in the immediate proximity the surface. By the inventive method for the targeted coating of a probe with enzyme or effector molecules ensures that the probe enzymes or effector molecules, preferably in a range of less than 100 nm, preferably less than 50 nm, and more preferably in a range of 10 nm or less having. The present invention further relates to probes which according to the inventive method available are, and in particular scanning probe microscopes, the probes according to the invention contain. The methods according to the invention, Surfaces, Carrier, Devices, probes and scanning probe microscopes can be used for Production of, for example, nanochips and microarrays as well as for Storage and / or analysis of data for use in cell biology or to carry out of measurement and investigation methods such as in forensics and chemical or biological analysis.

In The present application is within the text to various Publications with indication of author and date reference. Complete references to these publications also found at the end of the description immediately before the claims become. The disclosures of these publications are hereby incorporated by Reference in its entirety to this application complete to describe the state of the art to the professionals at the time the invention described and claimed herein was known.

Background of the invention

conventional Lithography techniques, such as photolithography, are used around structures of a size of a few hundred nanometers, even under such conditions, as they prevail in the production lines of the semiconductor industry. There is a constant need for increasing the resolution at such procedures. Possible solutions are the use of shorter ones wavelength (UV light) or other media, as in electron lithography. However, it is not clear how feature sizes of tens of nanometers or even just a few nanometers through a mere improvement the present common Technologies are routinely produced can.

A possible alternative technique that is successful under laboratory conditions has proved, is the surface modification through grid-end techniques. For example, soft polymer films may pass through modified mechanical interaction of an atomic force microscope tip (Wendel, Lorenz et al., 1995). The dip-pen technology (Piner, Zhu et al. 1999), in which a suitable ink on an AFM tip adsorbed and then applied locally to a sample, has great interest pulled yourself.

In The future will not only be a need for surface structuring in very small (nanometer) scale, as put forward by the semiconductor industry, but also for the chemical surface modification e.g. in biotechnological applications. The miniaturization of Assays such as DNA chips or protein chips become smaller, chemical require exactly defined structures. An overview of the most popular biochip systems will be available by Bowtell in Nature Genetics Supplement 21 (1999), 25-32.

It There is also a strong need for miniaturization of diagnostic Devices, of biosensors or the production of chemical "nanoreactors" (local areas, that can perform certain chemical tasks). If so, too is that conventional techniques such as UV lithography resolutions in the Tens of nanometers, it is not clear how to meet the need for chemical modifications.

Enzymes have also been described for chemical surface modification, eg, by applying a nanofountain pen to a suitable surface where a substrate capable of being digested by the enzyme has been fixed (Ionescu, Marks et al., 2003). Here, however, already by the process conditions surface changes were observed by independent change of the substrate. Furthermore, the enzyme was used rather in the sense of an "etching", ie the classical etching of the surface, while the enzymatic activity played a minor role.In another application, phospholipase present in the medium was used to modify a lipid film in the order of the lipid film, that of the mechanical Interaction of a scanning probe microscope tip was used to locally alter the sample (Grandbois, Clausen-Schaumann et al., 1998). However, methods for the targeted enzyme-based structuring of a surface, which satisfy the requirements of, for example, the chip industry and nanobiotechnology, are not yet available.

The publication US 5,824,470 A discloses a process for coating a tip with enzyme wherein the tip is contacted with a carrier and the enzyme is bound to the tip by means of a spacer.

Of the present invention, the technical problem underlying surfaces and To provide processes for their preparation, which are the aforementioned To fit in requirements.

This technical problem is marked with those in the claims as well in the following description explained embodiments of the invention solved.

The The present invention relates to a method for coating a probe with enzyme or effector molecules and a Method for structuring a surface of a carrier, wherein With the procedure that is achieved by a locally limited enzymatic activity close the surface, this is chemically or physically modified. According to the invention that essential feature of local enzymatic activity at the surface to be structured by the controlled coating of a probe with few or even only one enzyme or effector molecule can be achieved, wherein the enzyme activity close the surface preferably can be switched on and off.

The The present invention involves the targeted coating of a probe with enzyme or effector molecules, comprising contacting the probe with a flat support the enzymes or effector molecules by means of a polyvalent binding partner A such as streptavidin a binding partner B such as biotin is bound to the carrier, wherein at least a part of the probe has the same binding partner B, in this case Biotin.

The The invention is based on the finding that, with the aid of biological or biologically derived enzymes a method of targeted local, chemical or physical modification of a suitable surface receive. The enzyme is thereby on the tip of a scanning probe microscope fixed, whereby the chemical or physical modification with very high resolution can, ultimately based on individual molecules.

The The method described herein can be applied to a wide range of enzyme molecules be applied and enabled therefore a technology platform suitable for various applications can be used. Enzymes are common tools in the Diagnostics and biotechnology. The most famous applications are PCRs (polymerase chain reactions) or ELISAs (enzyme linked immunosorbent assays). They even have in everyday products Home use, e.g. as detergent additives. According to the invention, in future Applications the huge knowledge about Used enzymes from these areas and available enzymes and appropriate Substrate used become.

The The present invention further relates to probes which are obtained by the method according to the invention available are, and in particular scanning probe microscopes, the probes according to the invention contain.

Finally, concerns the present invention uses the methods, surfaces, supports, devices described herein, Probes and scanning probe microscopes for the production of, for example Nanochips and microarrays as well as for storage and / or analysis data, for use in cell biology or for performing measurement and investigation procedures such as in forensics as well in chemical or biological analysis.

The pictures show:

1 : A. An atomic force microscope tip (scanning electron micrograph, width of pyramidal tip about 4 microns) is coated at its foremost end with an enzyme molecule (light gray) by interaction of the enzyme-bound groups (medium light gray, in this case streptavidin, shown as a plug button) with the the top surface bound groups (dark gray, in this case biotin, double concave).

Fig.B. The protein, ie enzyme is connected to a streptavidin molecule (double concave complex), which has four binding sites (edges) for the ligand biotin (shown as a plug button). The enzymes are fixed on a biotinylated carrier (I). The biotinylated atomic force microscope tip is also brought into contact with the enzyme-coated sample. Enzyme molecules are then bound to the tip and to the carrier (II). After separating the top from the Carriers will retain about 50% of the double-bound enzyme molecules at the tip (III). This approach allows only the foremost end of the atomic force microscope tip to be coated.

2 : Possible reaction schemes. The figures exemplify some reaction schemes. By switching on additional effector molecules, such as coenzymes or other molecules, it is, of course, possible to obtain arbitrarily complicated reaction schemes.

Fig.A. The substrate (S) is fixed on the surface and is passed through the enzymatic activity soluble. S is fixed on the sample and the enzyme cleaves S, leaving product P soluble and the remains of S that are still on the sample surface, have other (chemical or physical) properties than S.

Fig.B. The substrate (S) is present in the medium and is affected by the activity of the enzyme insoluble. S is resolved in the medium and the product P immobilized on the surface by chemical or physical Interaction.

Fig.C. The substrate is present in the medium. The soluble Product P then chemically modifies those present on the surface molecules D. The substrate is present in the medium. The soluble Product P reacts with a cofactor B, leaving a complex PB arises. The complex PB can now physically modify the surface, for example, by attaching itself to the surface (analogous to Scheme A).

Fig.E. The substrate is present in the medium. The soluble Product P reacts with a cofactor B, leaving a complex PB arises. The complex PB can now chemically surface (analogous to Scheme A) modify.

Fig.F. The substrate is present in the medium. The soluble Product P reacts with a cofactor B, leaving a modification of the cofactor to B *. The modified cofactor B * can now chemically or physically modify the surface.

3 : The enzyme alkaline phosphatase was fixed to an atomic force microscope tip. The substrate BCIP was present in the surrounding medium.

Fig.A. The product of the enzymatic reaction forms one with NBT water-insoluble Complex on the surface fails.

Fig.B and C. By slowly approaching the atomic force microscope tip to direct contact with the surfaces can any features are generated on the surface.

Detailed description the invention

The The present invention includes a method of targeted coating a probe according to claim 1 and a lithographic process for structuring a surface of a Support according to claim 11 using a targeted coated probe, available after the method according to claim 1, where by a localized enzymatic activity in the immediate Near the surface, this is chemically or physically modified.

• – eine geeignete Oberfläche direkt oder indirekt durch die chemische Aktivität eines Enzyms modifiziert wird; und
• – das Enzym relativ zur Probe so positioniert wird, dass die Modifikation nur lokal stattfindet; siehe beispielsweise 3.

As mentioned above and illustrated in the examples, a method of modifying a suitable surface by means of biological or biologically derived enzyme molecules is provided. The enzyme molecules are brought into close proximity to the sample and modify it by material deposition, material removal or modification of sample molecules. The essential features of the method are that

• A suitable surface is modified directly or indirectly by the chemical activity of an enzyme; and
• - The enzyme is positioned relative to the sample so that the modification takes place only locally; see for example 3 ,

Of the primary Advantage of this idea lies in the use of the chemical activity of a Enzyme of the variety available Enzymes whose properties are the inventive method makes use of. This allows the creation of a technology platform for many different applications.

The Method is preferably characterized in that a resolution of less than about 200 nm, preferably less than 100 nm, more preferably less than 50 nm, and more preferably less than about 10 nm to let.

Suitable supports and surfaces are the materials customary in array and chip technology, such as glass, gold and plastic membranes, which can also be the surface itself. Particularly suitable metals, for. As copper, titanium, chromium, gold and especially platinum. Usually, the carrier surface is derivatized. Glass surfaces, for example, can be treated with silane reagents such as aminopropyltriethoxysilane (APTES), 3-mercaptopropyltrimethoxysilane (MPTS), glycidoxypropyltrimethoxysilane (GPTS), bis (hydroxyethyl) aminopropyltriethoxysilane (HE-APTS), hydroxybutyramide-propyltriethoxysilane (HBPTES) and (perfluorooctyloxy) propyltriethoxysilane (POPTS) , Preference is given to using gold, Ni-NTA (nitrilotriacetic acid), avidin or biotin-coated carriers of glass, silicon, silicon oxide or silicon nitride.

Especially preferred are gold coated glass slides. The gold surface can for example, by differently functionalized alkanethiol monolayers (SAMs = self assembled monolayers) such as biotin, N-oxysuccinimide ester, Epoxy groups, maleimide groups, amino groups, and oligo (ethylene glycol) be coated.

For the production or modification of protein chips, in particular soft carriers such as PVDF, nitrocellulose and polystyrene are used.

There on glass surfaces A variety of different types of molecules can be fixed, glass becomes in the inventive method preferably used.

Suitable surfaces are, in particular, surfaces already loaded with biomolecules, such as DNA chip formats, which are also commercially available. The production of arrays of immobilized biomolecules, which can also be modified in the sense of the present invention, are for example in the German patent application DE 100 41 809 A1 described.

Out the above explanation it turns out that a carrier at the same time also represent the surface can. Accordingly, the terms carrier and surface possibly used synonymously.

Carrier in mind The registration is usually materials with rigid or semi-rigid surface and in that sense as firm. Common are planar surfaces. The Shape of a carrier can be varied and may vary according to the type of use judge. These may be, for example, slides, chips, dipsticks, stamps, caps, Particles, spherical Body, like spheres or globules, Membranes, leaves, Disc, foil or plate act. Even wafer formats can as carrier serve, if desired are singular. The carriers can further with further expedient Be provided components, for example, you can encapsulate chips.

Included in the invention the term "modification" in particular the Embodiments described herein such as deposition, removal or actual modification of the substrate. The modification of the surface can be done with very high resolution, where ultimately an enzyme molecule is always only one molecular Carrying out modification at a single point. The modification of the surface can directly through the activity of the enzyme, or indirectly, so that the enzymatic products even the surface modify.

If Unless otherwise stated, the term enzyme is used in the conventional Senses used. Under an enzyme, i.a. a chemically active Molecule understood, that is a substrate molecule modified and thereby produces one or more product molecules. An enzyme can be the substrate molecule can chemically modify it into several sub-molecules or a combination of both. The chemical modification of the substrate molecule becomes directly or indirectly a chemical or physical Modification of the sample lead. There this modification takes place only locally, this process according to the invention is enzyme-based nanolithography called.

Under effector become common Understood substances that stimulate or inhibit both the enzyme reaction can. Effectors are usually not involved in the reaction itself, they can after understanding for the but trigger the present invention. You can also referred to as activator or inhibitor depending on their effect become.

According to the invention under the term "effector molecule" also cofactors like NADPH, as well as in general molecules that have an enzymatic reaction trigger can, for example, by binding to the enzyme, whereby this activation learns i.e. becomes enzymatically active. Accordingly, the effector molecule may be a cofactor Substrate, a catalyst or also an enzyme. cofactors are also called coenzymes.

In In this context come as effector molecules and vitamins as ingredients of coenzymes, for example thiamine (vitamin B1) or Thiamine diphosphate as a coenzyme of oxidative decarboxylation, riboflavin (Vitamin B2) with nicotinic acid, Nicotinamide, folic acid and pantothenic acid forms the B2 complex, pyridoxine (vitamin B6), which in transaminations and decarboxylations needed cobalamin (vitamin B 12), which plays a role in isomerases, ascorbic acid (Vitamin C) as H-donor in oxidoreductases, and biotin (Vitamin H) as a CO group carrier.

in the Case of using immobilized on a probe effector molecules in the process preferably allosteric enzymes, for example in the medium on the surface, used that in addition to their catalytic center still have binding sites on those the Effektormolekül reversible can bind.

Some Points are essential for the implementation of the procedure. For example, must Enzyme and substrate brought into close proximity to the surface (Distance in the range of nm or tenth-nm) and relative to each other be positioned. This can be achieved by the Enzyme or an effector molecule is immobilized on a probe.

The Enzyme or effector molecule can chemically on a bead (Glass, latex or other materials) or at the top of a scanning probe microscope (Scanning Tunneling Microscope, Atomic Force Microscope, SNOM or others) be fixed. The bead or the tip can be positioned by external forces, e.g. electrical, magnetic, optical control forces (magnetic or optical traps) or others, or by a micropositioning device (piezo converter, microtechnological manufactured electromechanical transducers and so on) relative to Sample. Immobilization of the enzyme or effector molecule can by covalent bonding, by chemical bonding or by physical Adsorption done.

covalent Bindings can For example, be achieved with silane or thiol chemistry, chemical Bindings could specific biological interactions such as ligand-receptor interactions for Use (e.g., biotin-streptavidin, antibody-antigen and the like). Physical fixation can be electrostatic forces, van der Waals forces, hydrophobic personnel and the like. Fixation can also be through combination the method described above can be achieved.

Therefore is in one embodiment the method according to the invention the probe is a micro or nanoparticle and preferably comprises Latex, glass, polystyrene or silica beads. Such beads can For example, be coated with biotin or streptavidin. These beads can with a so-called optical tweezers are positioned locally. This technique is based on so-called optical traps; see for example Ashkin 1970, 1997. A newer type of three-dimensional trap (single-beam gradient force trap) has been described in Ashkin, 1986.

Optical tweezers are based on the fact that small transparent dielectric objects (with n _object > n _environment ) are conveyed by a force into the focus of a focused laser beam; see, for example, Hegner, 2002. As dielectric objects, as mentioned above, microparticles of polystyrene, glass or silica beads are suitable, which in turn can be carriers for biomolecules, ie enzymes or effector molecules according to the invention. The structure of a single-jet optical tweezers and of mechanical accessories such as a liquid cell and a mechanical adjustment table are known in the art. This also applies to control programs, for example in LabView, to move the piezoelectric table relative to the optical trap and to calibrate the optical trap; see, for example, the diploma thesis "Construction of Optical Tweezers" by Andy Sischka, Faculty of Physics, Bielefeld University (June 2002).

In another preferred embodiment the method according to the invention the probe is the tip of a scanning probe microscope.

all Scanning probe microscopes in common is the need for precise control of the Distance between probe and sample surface and the lateral grid movement with a resolution in the nanometer range and much better. This movement precision can be realized by piezo elements. These consist of piezoceramics, which has the property that it contracts when voltage is applied or elongated.

Scanning probe microscopes be in many cases for local surface modification used. The most well-known cases are the scratching or hammering of holes into a soft polymer film by atomic force microscopy (helix, Lorenz et al., 1995), dip-pen technology (Piper, Zhu et al., 1999), in which one adsorbed to a tip Ink is slowly applied to a sample, or local electro-oxidation a conductive sample through the electron stream in a scanning tunneling microscope.

In a recent publication were described experiments in which one at a atomic force microscope tip fixed enzyme was used to modify a suitable surface. Here, the specific binding of the substrate molecule to the enzyme molecule used to remove the substrate from the sample (Takeda, Nakamura et al., 2003). In contrast to the present invention was in These experiments, however, exploited only the specific enzyme-substrate binding and, so to speak, the substrate (peptide) from the surface down brushed. In contrast, the inventive method based on the use the chemical, i. enzymatic activity of the enzyme. In other words, the product of the chemical reaction that accelerates the enzyme becomes used around the target surface to modify.

Another major difference with respect to the present invention is that in the Takeda et al. used force microscope, no targeted coating of the tip surface with enzyme and insufficient by the large number of bound enzyme molecules sufficient de local limitation of enzyme activity has been achieved. Furthermore, the authors did not point out the need for a local limitation of enzyme activity, let alone recommended measures on how to achieve this.

Scanning probe microscopes, the for the inventive method can be used Scanning Tunneling Microscope (STM), Scanning Near - field Optical Microscope (SNOM), and most preferably the atomic force microscope (AFM) used for the in The experiments described in the examples were used.

At the AFM probes a probe the atomic hilly landscape of the examined Sample object. The probe consists of a tip, which consists of few Atoms is formed (ideally sitting at the top of the top a single atom), which is mounted on a movable thin "beam".

If one approaches the surface with this fine tip, a Lennard-Jones potential between the foremost atom of the tip and the surface atom acts in a first approximation, ie, depending on the distance, an attracting and then a repulsive effect acts on the tip at a greater distance first Force. When scanning the surface, the force of the tip is kept constant. The thus changed bending of the "beam" is registered with a sensor. As a sensor, an atomic force microscope, but usually a deflected laser beam can be used. Scanning probe microscopes are known in the art. Recent developments are, for example, in the European patent application EP 0 896 201 A1 described.

As in the pictures of 2 As shown, the substrate molecule that is chemically modified by the enzyme can be present on the surface or in the surrounding medium. In one embodiment of the method according to the invention, the substrate of the enzyme is immobilized on the surface. In an alternative embodiment, the substrate is in a medium on the surface. But it is also possible to combine both embodiments, if necessary, with different weighting.

usual Way is the substrate molecule present in the surrounding medium, a constant amount of available To ensure substrate. to Removal of material from the surface will preferably be the substrate presented on the sample, and the enzyme can cleave off portions of the substrate molecule (e.g., lysozyme can split off part of a fixed polysaccharide). Other embodiments are but also acceptable. For example, glucose oxidase can be local Produce hydrogen peroxide, which then chemically a suitable surface modified.

As mentioned above, if the enzyme in the process can be present in a medium on the surface, especially when the effector molecule is bound to the probe.

In a preferred embodiment In the process, the enzyme is bound to the apex of the tip; please refer also the examples.

As already mentioned above, It is important that the enzymatic activity is local and possibly limited in time becomes. For example, the activity of the enzyme molecule via a external control on and off. In this embodiment may, although less preferably, the entire probe, for example the entire atomic force microscope tip is coated with enzyme or effector molecules be the activity of the enzyme on the surface controlled by an external signal. A suitable external Signal, for example, by an electric or magnetic Field, surface charge, pH change, Ion gradients or optical signal are mediated; see also the above statements to the optical tweezers above.

Thus, any signal capable of allowing only enzyme molecules in the immediate vicinity of the sample to be modified to be active is suitable. One possible approach is to create these activating conditions only near the surface. For example, a photoactivatable enzyme molecule can be activated by the evanescent field of total internal light reflection at the sample / medium borderline. In another embodiment, local surface charges (or surface potential) may provide a surface pH that is different from the pH in the medium. Since most enzymes have pH-dependent activity, this method can be adapted so that the enzyme is active only near the sample surface. A simple way to turn on and off the activity of the enzyme is, for example, availability or non-availability of the substrate. This can be achieved simply by a substrate dissolved in the medium, which can be replaced by a medium without a substrate. However, the activity of the enzyme can also be modified by external stimuli (electric or magnetic fields, irradiation with light or electromagnetic waves, temperature, pH). The same applies to the activity of the substrate or to molecular groups that are to be modified on the surface. A standardized approach to making the substrate available after an external stimulus is the use of transiently inactive compounds by the irradiation activated with light (for example, "caged compounds" such as caged-ATP, caged-CA, etc.).

Alternatively, or additionally, the local activity of the enzyme in the vicinity of the surface can be achieved by coating the probe only with a specific site, in the case of the scanning force microscope tip only at the foremost end, with active enzyme or effector molecules, see, for example 1 , Accordingly, it is preferred that only the apex of the probe tip is provided with enzyme or effector molecules. Most preferably, the apex of the probe tip carries only one enzyme or effector molecule.

As already mentioned above, structuring, ie modification of a surface, is based on the enzymatic activity of an enzyme, ie an enzyme molecule transforms a substrate molecule S into a product molecule P. In order to make use of this chemical reaction for surface modification, several concepts are conceivable; see for example the figures in 2 ,

In an embodiment of the method results in the modification of the surface the precipitation a product of the enzymatic reaction, while in another embodiment the modification of the surface resulting from the modification of the substrate on the surface.

It it is obvious that other reaction schemes are used can. The common basic idea is that a local enzymatic reaction in their vicinity a sample surface Add of molecules or modified by removal or modification of molecules on the sample surface. This can be done directly from the enzyme or indirectly from Product of the enzymatic reaction. The enzyme or the active product can additional Coenzymes or helper molecules to fulfillment need this task.

For the procedure are basically all types of enzymes usable, such as oxidoreductases, transferases, Hydrolases, lyases, isomerases and ligases. In one embodiment the method according to the invention the enzyme is selected from the group consisting of oxidoreductases, glucose oxidases, transferases, Kinases, hydrolases, carboxylic ester hydrolases, Lipases, ribonucleases, hyaluronidases, invertases, amylases, beta-galactosidases, Proteinases and polymerases. Particularly preferred are the enzyme alkaline phosphatase, glucose oxidase, DNA or RNA polymerase, or a catalytic antibody used.

The Substrates and possibly necessary cofactors of enzymes are those skilled in the art known and include, for example, nucleotides, amino acids, or Precursors to dyes, especially when it is metabolizing Enzymes, i. Product "building" enzymes acts. For example, in the case of anabolic reactions, the substrate may nucleic acids, Peptides, polypeptides, sugars, or dyes include.

By way of example, the following enzymatic reactions may be mentioned:
The surface is coated with phospholipid. The enzyme phospholipase cleaves the phospholipid into two separate molecules that become soluble and are removed from the surface.

The Enzyme alkaline phosphatase dephosphorylates p-nitrophenyl phosphates, and the product binds due to electrostatic interactions a charged surface.

The Enzyme glucose oxidase oxidizes glucose and produces hydrogen peroxide. The hydrogen peroxide oxidizes suitable molecules on the surface.

The Enzyme alkaline phosphatase dephosphorylates the substrate BCIP / NBT, the product becomes insoluble and falls on a suitable surface; see also the examples.

As described above, the enzyme molecule can be chemically bound by the toolkits available in biotechnology. In the following example, for example, a phosphatase molecule was used, to which a streptavidin molecule was covalently bound. Streptavidin has four binding groups for biotin. The atomic force microscope tip was covalently coated with a biotinylated silane. According to a method of the invention, dipping the atomic force microscope tip into a streptavidin-enzyme complex solution resulted in the atomic force microscope tip being coated with enzyme molecules only at the foremost end of the tip; see Example 1 and 1 ,

According to one The above-described methods are available carriers which, as above described and shown in the examples, in the nanometer range exactly are structured.

Furthermore, devices are available which contain these carriers. Corresponding devices include chips, microarrays, diagnostic devices, biosensors or nanoreactors. Of course, depending on the intended application, these carriers or their surfaces can be further modified, for example, incorporated in housings and / or loaded with molecules, for example biomolecules, by conventional methods. Preferably, the aforementioned devices comprise a DNA or protein chip. So-called gene chips can be used for example for forensic applications. Furthermore, it is for example possible with the method to produce so-called PISA (protein in situ arrays), which can be used today only in the conventional multi-well order.

As already described above, is in a particularly preferred embodiment the method according to the invention used a probe in which only a small controlled area, for example only the foremost part of an atomic force microscope tip or the Tangential area of a bead with enzyme or effector molecules is coated to a resolution to reach in the nanometer range. This can not be done with standard techniques such as pipetting or masking and the like can be achieved.

According to the invention was found a new method of how to make enzymes or effector molecules of one flat carrier can collect by contacting the probe with this carrier; see also Example 1. In a second step, the probe is then added to the to be modified sample surface emotional.

The The present invention therefore relates to a method for targeted Coating a probe with enzyme or effector molecules comprising contacting the probe with a flat support the enzymes or effector molecules by means of a polyvalent binding partner A to a binding partner B on the carrier are bound, wherein at least a portion of the probe the same binding partner B has.

Basically any probe as already described above may be used. Particularly preferred in the method according to the invention is that only the apex of the probe tip is coated. Of course, it is whole particularly preferred that the probe with only a single Enzyme or effector molecule is occupied.

Usually be in the process of the invention Binding partner A, each with at least two free binding sites for binding partners B used. Typical binding partners are streptavidin and Biotin or antibodies or antibody fragments as well as appropriate antigens.

The The present invention also relates to probes according to a The above-described methods are available, preferably thereby Probes from scanning probe microscopes. Accordingly, the concerns The present invention also relates to scanning probe microscopes comprising a probe according to the invention contain. Inventive probes, in particular those which have only one enzyme or effector molecules, can for example, in cell biology, e.g. to study the activity of enzymes or other molecules on the membrane of cells.

As can already be seen from the prior art, the methods, carriers or surfaces, devices, probes and scanning probe microscopes according to the invention provide a wealth of possible uses and uses, for example for the production of nanochips or microarrays, to name only two. Furthermore, the following applications are mentioned by way of example:
Diagnosis, treatment choice and therapy control of tumor and metabolic diseases;
Investigation of genetic predisposition (SNPs), in particular the detection of mutations, also in the forensic field; Gene expression control; Sequencing, in particular sequencing by hybridization; microbiological applications such as in bacteriology and virology, for example for the differentiation of different strains; ELISA applications with immobilized antibodies, antigens, receptors and ligands in clinical chemistry to food chemistry and environmental analysis; Allergy diagnostics with immobilized allergens; High throughput screening of compound libraries; Information and communication technology; and more.

The inventive method, Carrier or Surfaces, Devices, probes and scanning probe microscopes can in particular for storage and / or analysis of data, cell manipulation, or to carry out be used by measuring or inspection procedures. Further come the use in recent Techniques like bioMEMS (see for example "Fundamentals of Microfabrication: The Science of Miniaturization, Second Edition, Marc J Madou, University of California, Irvine, California, USA, (2002) ISBN: 0849308267), uTAS (see for example the "uTAS Virtual Journal", which is published exclusively in electronic format available is and micro-total analysis systems (uTAS) has the subject, and about the Website of the publisher ELSEVIER is available).

The The present invention will be illustrated in the following examples.

EXAMPLES

Example 1: Targeted coating a probe of an atomic force microscope.

For the exclusive coating of only the foremost end of the atomic force microscope tip we prepared a flat sample, which also with biotinylated silane was coated. To this sample, we added enzyme so that the sample was coated. Upon contacting the biotinized atomic force microscope tip with this sample, the biotin groups at the foremost end of the atomic force microscope tip may also bind to streptavidin molecules because of the versatility of streptavidin. Due to geometric limitations, it is actually conceivable that streptavidin binds to a maximum of two biotin groups on the surface and consequently the two other binding sites must face the atomic force microscope tip.

The Immobilization of streptavidinized proteins at the apex of a Scanning probe tip and preparing a suitable surface can For example, be made as follows:

cleaning

• 1. Cleaning the surface area of a piece of oxidized silicon wafer (CrysTec S3012) in a mixture of conc. Sulfuric acid (Riedel de Haen - C8029) and 30 percent hydrogen peroxide solution (Merck 8.22287.1000) (ratio 3: 1) in the ultrasonic bath for 15 minutes.
• 2. Rinse the wafer with deionized water (MilliPore ^® quality).
• 3. Irradiate an AFM tip (Veeco Nanoprobe NP-STT) with UV light to break up organic dirt on the surface.

silanization

• 4. Applying a mixture of: - 9 ml of methanol reagent grade (Riedel de Haen - 32213) - 80 μl (microliters) conc. acetic acid (Fluka 45731) 370 μl of deionized water 230 μl of N-2-aminoethyl-3-aminopropyltrimethoxysilane (Merck 8.19172.0100)
• 5. Insert the wafer and AFM tip into this mixture for 30 minute
• 6. Remove and wash twice the wafer and AFM tip in methanol.
• 7. Dry the wafer and AFM tip with nitrogen gas.
• 8. Heat the wafer and AFM tip in an oven 120 ° C for 3 min.

Application of the biotin linker

• 9. Prepare a solution of - 4 ml of dimethyl sulfoxide (DMSO) (Fluka 41640) - 1 μg (micrograms) biotin-N-hydroxysuccinimide ester (NHS-biotin) (Sigma H1759) 10. Insert the wafer and AFM tip into this mixture for 2 h.
• 11. Remove and wash twice the wafer and AFM tip in ethanol (Riedel de Haen 32205).
• 12. Dry the wafer and AFM tip with nitrogen gas.

Immobilization streptavidinized alkaline phosphatases on the wafer surface

• 13. The biotinized wafer is placed in a 0.2 nmol solution of alkaline phosphatase (Sigma 52890) for 10 min.
• 14. Rinse with deionized water.
• 15. Incubate in a 5 mmol solution of p-nitrophenyl phosphate (pNNP) (Sigma N4665) in 40 mM TRIS buffer p.A. (Roth 4855.2) and 1 mmol magnesium chloride (Sigma M2670) for 10 min on a stirred table.
• 16. Rinse with deionized water.

Immobility of streptavidinized alkaline phosphatases on the AFM tip

• 17. Installation of biotinized AFM tip and of the prepared Wafers in an atomic force microscope.
• 18. Approach the AFM tip to the surface of the Wafer.
• 19. Slowly move the AFM tip over the surface ~ 100 nm / s) a distance of ~ 50 μm. In this case, individual streptavidinized alkaline phosphatases bind to the AFM tip.

There the same streptavidin molecule now to the carrier and the tip is tied, it is 50% when the tip is removed likely that streptavidin remain bound to the top becomes. Since the enzyme is covalently bound to streptavidin we therefore active enzyme at our atomic force microscope tip accumulate. Because of the size of the molecules and the it is possible only in this space involved gaps to coat the foremost end of the atomic force microscope tip. We can estimate that at most the foremost 10 nm of the atomic force microscope tip should be coated.

Example 2: structuring a surface with the atomic force microscope tip of Example 1.

To produce in the 3B and 3C In the structures shown, the functionalized AFM tip was incorporated into an AFM according to the procedure described above (Asylum Research MFP 3D). Glimmer (Plano GmbH, Wetzlar) was used as the surface to be structured.

When surrounding medium was added 40 mM TRIS buffer (Roth 4855.2) with 1 mM magnesium chloride (Sigma M2670) used in the ratio 1: 1 with the substrate BCIP / NBT (Sigma 6404) is mixed.

The alkaline immobilized at the top Phosphatase (Sigma 52890) converts the substrate in solution so that it becomes insoluble. To create the dot structure ( 3C ), the AFM tip was contacted with the mica surface for each 20 seconds for each point. After writing, the same area was mapped in tapping mode. To create the contiguous L-shaped structure ( 3B ), the AFM tip was brought into contact and moved at a speed of 10 nm / s. This area was then also mapped in tapping mode.

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Claims (27)

Method for the targeted coating of a probe with enzyme or effector molecules, comprising contacting the probe with a flat support the enzymes or effector molecules by means of a polyvalent binding partner A to a binding partner B on the carrier are bound, wherein at least a portion of the probe the same binding partner B, and wherein the molecule, the from the enzyme or effector molecule and the polyvalent binding partner A, not in the Binding partner B bound state at least two free binding sites for the Binding partner B has. The method of claim 1, wherein the probe is a microporous or nanoparticles. The method of claim 2, wherein the micro or Nanoparticles comprises latex, glass or silica beads. The method of claim 1, wherein the probe is the tip of a scanning probe microscope. The method of claim 4, wherein the scanning probe microscope a scanning tunneling microscope or atomic force microscope is. Method according to one of claims 1 to 5, in which only the apex of the probe tip is coated. Method according to one of claims 1 to 6, wherein the binding partner A streptavidin and binding partner B is biotin. Method according to one of claims 1 to 7, wherein the probe is occupied by only one enzyme or effector molecule. Probe, available according to a method according to the claims 1 to B. Lithographic process for structuring a surface a carrier, being characterized by a localized enzymatic activity in the immediate Near the Surface, this is chemically or physically modified and one at a Probe according to claim 9 immobilized enzyme or effector molecule positioned in the immediate vicinity of the surface becomes. The method of claim 10, wherein the substrate of the Enzyme on the surface is immobilized. The method of claim 10, wherein the substrate is in a medium on the surface is present. A method according to any one of claims 10 to 12, wherein the enzyme in a medium on the surface is present. A method according to any one of claims 10 to 12, wherein the enzyme is bound on the apex of the probe tip. The method of any one of claims 10 to 13, wherein the effector molecule is a cofactor, a sub strat, an enzyme or a catalyst. A method according to any one of claims 10 to 15, wherein the activity of the enzyme through at the surface an external signal is controlled. The method of claim 16, wherein the external signal by an electric or magnetic field, surface charge, pH change, Ion gradient, or optical signal is mediated. Method according to one of claims 10 to 17, wherein only provide the apex of the probe tip with enzyme or effector molecules is. The method of claim 18, wherein the apex of the probe tip carries only a single enzyme or effector molecule. A method according to any one of claims 10 to 19, wherein the modification the surface from the precipitation a product of the enzymatic reaction results. A method according to any one of claims 10 to 20, wherein the modification the surface resulting from the modification of the substrate on the surface. A method according to any one of claims 10 to 21, wherein the enzyme selected is from the group consisting of oxidoreductases, glucose oxidases, transferases, Kinases, hydrolases, carboxylic ester hydrolases, Lipases, ribonucleases, hyaluronidases, invertases, amylases, beta-galactosidases, Proteinases and polymerases. A method according to any one of claims 10 to 22, wherein the enzyme alkaline phosphatase, glucose oxidase, DNA or RNA polymerase, or a catalytic antibody is. A method according to any one of claims 10 to 23, wherein the substrate Nucleotides, amino acids, or Comprises precursors to dyes. A method according to any one of claims 10 to 23, wherein the substrate nucleic acids, Peptides, polypeptides, sugars, or dyes. Scanning probe microscope containing a probe after Claim 9. Use of a method according to one of claims 10 to 25, a probe according to claim 9 or a scanning probe microscope according to claim 26 for the production of nanochips or microarrays, for storage and / or analysis of data, cell manipulation, or to carry out of measuring or examination procedures.