DE10322414A1 - Immobilizing proteins on polymeric film substrates, useful in preparation of diagnostic and screening arrays, where the polymer has been modified with chromatographically active functional groups - Google Patents

Abstract

Immobilizing at least one protein (18) on a substrate (1) on which a polymeric film (12) is immobilized, and where the surface of (12) has been modified to introduce functional groups (15). (15) have at least one of the following chromatographically active functions: ion-exchange; hydrophobic or protein affinity, is new. An independent claim is also included for a sample carrier for immobilization of (18) comprising (10) on which (12) is immobilized.

Description

Territory of invention

With the increasing importance of array-based diagnostic and screening methods Methods for immobilizing biomolecules at defined positions of substrate surfaces more and more weight.

In particular the immobilization of proteins proves to be in the native state difficult to reach here, depending on the surface properties of the proteins different strategies for immobilization can be chosen have to, the native state and thus enzyme activities and steric properties to obtain.

• 1. Direkte kovalente Kopplung von Seitenketten der Proteinoberfläche an passende chemische Funktionen des Substrats. Die am häufigsten verwendete Kopplungsmethode benutzt die auf der Proteinoberfläche vorhandenen, Aminofunktionen tragenden Seitenketten der Aminosäuren Asparagin, Glutamin, Lysin und Arginin. Durch Aufbringen auf Aldehydfunktionen tragende Substratoberflächen ergibt sich eine Kopplungsreaktion zwischen Amin- und Aldehydfunktionen zur Schiff'schen Base, die nachfolgend durch eine Reduktion mittels Chemikalien wie z. B. NaCNBH₃ in ein stabiles sekundäres Amin überführt werden kann. Ähnlich lassen sich die auf der Proteinoberfläche am häufigsten vertretenen Carboxylfunktionen an Substrate mit Aminofunktionen koppeln: Mittels EDC [1-Ethyl-3-(3-dimethylaminopropyl) carbodiimid] umgesetzte Carboxylgruppen der Proteine bilden ein aktiviertes Ester-Zwischenprodukt, das mit primären Aminogruppen auf der Substratoberfläche reagiert und dabei Amidbindungen zwischen Protein und Substrat ausbildet.
• 2. Andere Formen kovalenter Anbindung von Proteinen an Substratoberflächen ergeben sich durch chemische Modifizierung der zu immobilisierenden Proteine vor der endgültigen Kopplung. Hier lassen sich mit den unter 1. beschriebenen chemischen Kopplungsmethoden in einem ersten Schritt lange, bifunktionelle Linker, wie z. B. Glutaraldehyd oder langkettige Diamine an die Proteine koppeln. In einem zweiten Schritt werden dann die freien Enden dieser Linker an die Substratoberfläche gebunden.
• 3. Weitere, auf Proteinmodifikation beruhende Immobilisierungsverfahren ergeben sich durch die Erzeugung von Fusionsproteinen [Nath N, Chilkoti A, 2003. Fabrication of a Reversi ble Protein Array Directly from Cell Lysate using a Stimuli-Responsive Polypeptide. Anal. Chem. 75: 709–715]. Fusionsproteine aus dem gewünschten Protein mit z. B. Streptavidineinheiten besitzen Andockstellen für Biotinmoleküle. Damit kann die äußerst stabile, nichtkovalente Biotin-Streptavidin-Bindung zur Verankerung eines Proteins ausgenutzt werden. Umgekehrt lassen sich Biotin-tragende Liganden auch über N-Hydroxysuccinimidfunktionen an Proteine koppeln und dadurch die Proteine auf Streptavidin-beschichteten Oberflächen verankern.

Previous methods for immobilizing proteins on substrate surfaces are essentially based on covalent coupling and covalent modification of the proteins to be immobilized. The protein-substrate coupling methods currently used can be divided into three groups:

• 1. Direct covalent coupling of side chains of the protein surface to suitable chemical functions of the substrate. The most frequently used coupling method uses the side chains of the amino acids asparagine, glutamine, lysine and arginine which are present on the protein surface and carry amino functions. Application to substrate surfaces carrying aldehyde functions results in a coupling reaction between amine and aldehyde functions to form Schiff's base, which is subsequently reduced by means of chemicals such as. B. NaCNBH _{3 can be converted} into a stable secondary amine. Similarly, the carboxyl functions most frequently represented on the protein surface can be coupled to substrates with amino functions: carboxyl groups of the proteins converted by means of EDC [1-ethyl-3- (3-dimethylaminopropyl) carbodiimide] form an activated ester intermediate which is linked to primary amino groups on the Substrate surface reacts and forms amide bonds between protein and substrate.
• 2. Other forms of covalent attachment of proteins to substrate surfaces result from chemical modification of the proteins to be immobilized before the final coupling. Long, bifunctional linkers such as e.g. B. couple glutaraldehyde or long chain diamines to the proteins. In a second step, the free ends of these linkers are then bound to the substrate surface.
• 3. Further immobilization methods based on protein modification result from the production of fusion proteins [Nath N, Chilkoti A, 2003. Fabrication of a Reversible Protein Array Directly from Cell Lysate using a Stimuli-Responsive Polypeptide. Anal. Chem. 75: 709-715]. Fusion proteins from the desired protein with e.g. B. Streptavidine units have docking sites for biotin molecules. This enables the extremely stable, noncovalent biotin-streptavidin bond to be used to anchor a protein. Conversely, biotin-bearing ligands can also be coupled to proteins via N-hydroxysuccinimide functions, thereby anchoring the proteins on surfaces coated with streptavidin.

A covalent immobilization basically includes one chemical modification of the proteins to be examined, i.e. H. it leaves no more reliable predictions can be made as to whether the modified Protein is still in its native state or whether a possible enzymatic Activity or certain steric properties are still available.

task the invention is to immobilize proteins on a substrate in such a way that their enzymatic activity preferably preserved.

solution

This The object is achieved by the inventions with the features of the independent claims. advantageous Further developments of the inventions are characterized in the subclaims.

to solution the task becomes a procedure and one created by the procedure sample carrier specified.

• – Ionenaustausch-Funktion,
• – hydrophobe Funktion oder
• – proteinaffine Funktion.

The method serves to immobilize at least one protein on a substrate. A polymer film is immobilized on the substrate. The polymer film is modified in such a way that it has functional groups on its surface facing away from the substrate, the functional groups having at least one of the following functions (chromatographically active functions):

• - ion exchange function,
• - hydrophobic function or
• - Protein-affine function.

The at least one protein is on the polymer film prepared in this way applied.

• 1. auf die Verwendung von Anionenaustauscher-Funktionen tragenden makroskopischen Oberflächen zur Immobilisierung von Proteinen, wie z. B. Oberflächen mit Aminofunktionen und DEAE-Funktionen (DEAE = O-[2-(Diethylamino)ethyl] als funktionelle Gruppe);
• 2. auf die Verwendung von Kationenaustauscher-Funktionen tragenden makroskopischen Oberflächen, wie z. B. Oberflächen mit Carboxylfunktionen;
• 3. auf die Verwendung von makroskopischen Oberflächen, die hydrophobe Funktionen tragen, wie z. B. Oberflächen mit kovalent gebundenen langkettigen aliphatischen oder aromatischen Funktionen; und
• 4. auf die Verwendung von makroskopischen Oberflächen, die chelatbildende Funktionen oder Heparin-Funktionen tragen.

The invention preferably relates to:

• 1. on the use of anion exchanger-bearing macroscopic surfaces for the immobilization of proteins, such as. B. Surfaces with amino functions and DEAE functions (DEAE = O- [2- (diethylamino) ethyl] as a functional group);
• 2. on the use of cation exchanger-bearing macroscopic surfaces, such as. B. Surfaces with carboxyl functions;
• 3. on the use of macroscopic surfaces that carry hydrophobic functions, such as. B. Surfaces with covalently bonded long chain aliphatic or aromatic functions; and
• 4. on the use of macroscopic surfaces that carry chelating functions or heparin functions.

According to the invention soft, chromatographically effective polymer films on macroscopic surfaces for the purpose of non-covalent immobilization of biomolecules, especially proteins, generated.

This includes the creation and covalent fixation of polymer films on solid substrates with subsequent derivatization of the polymer towards chromatographically effective surfaces.

The The present invention thus relates to immobilization of proteins on substrate surfaces based on electrostatic, hydrophobic and protein affine principles, the - in Contrary to the previously used methods of covalent immobilization - only one Minimum protein changes and impairments result in [Deutscher MP (ed), 1990. Methods in Enzymology: Guide to protein purification. Vol. 28, Academic Press Inc., San Diego, CA, USA]. allows this is achieved through the formation of the chromatographically active functional Groups on the surface of the sample carrier.

With Help remain with the inventive method native and thus functional proteins even in the immobilized state obtained, whereby following the immobilization enzymatic Tests and protein recognition reactions will be possible.

A possibility the formation of the chromatographically active groups on the sample carrier in that the polymer film is chosen such that it is active or has activatable functional groups for binding molecules. The polymer film is modified by binding bifunctional molecules. The bifunctional molecules are chosen such that they carry a functional group with which they can reach the active or can bind activated functional groups of the polymer film. Wear additionally a chromatographically active functional group.

at The polymers used can be both biopolymers are also synthetic copolymers, their soft physical It is particularly suitable for the immobilization of proteins suitable.

Instead of The polymer film can also be used to bind bifunctional molecules himself chosen that way be that it is chromatographically active or activatable functional Has groups without the need for subsequent modification. The In this case, polymer film has already been modified.

On a macroscopic substrate the non-covalently bound proteins in a spatially defined Arrangement applied and bound. Let it this way to make protein chips.

in the The invention is explained in more detail below on the basis of exemplary embodiments which: are shown schematically in the figure. In detail shows:

1 a schematic representation of a sample holder for proteins which are bound to the sample holder via chromatographically active ligands.

1 shows a substrate 10 on which a polymer film 12 is covalently bound. There are ligands on the polymer film 14 bound to a chromatographically active group 15 exhibit. The group 15 mediates z. B. via ion exchange 16 a bond between the ligands 14 and proteins 18 ,

Example I:

On The first method consists in the covalent fixation of an oxidized one Form of the biopolymer agarose [Afanassiev V, Hanemann V, Wölfl S, 2000. Preparation of DNA and protein micro arrays on glass slides coated with an agarose Movie. Nucleic Acids Res 28: e66] on aminated substrate surfaces and the conversion of the agarose-side aldehyde groups into chromatographic active chemical functions by coupling corresponding amino functions Ligands. With ligands, those molecules or molecular groups referred to, which can bind to proteins.

The Agarose oxidation and the coating of aminated substrate surfaces with The finished oxidation product can be made using the following procedure to reach:

a. Agarose oxidation specification: 0.4 g of an agarose, preferably of type IIa, is dissolved in 25 ml of water by boiling, then cooled to 45 ° C. and stirred. 20 ml of a 0.4 M meta-NaJO ₄ solution in water are added and the reaction solution is stirred at 40 ° C. for a further hour. The oxidation reaction is stopped by adding 20 ml of 100 glycerol and stirring. The reaction solution is poured into a mold to form a gel in order to obtain the largest possible surface and solidified by a cooling period at 4 ° C. The gel is then freed from low molecular weight compounds by dialysis against 10 l of distilled water for several hours.

b. Coating of aminated glass slides with oxidized agarose: Because a film made of agarose is very sensitive to mechanical stress , a one-sided coating of a slide is recommended Agarose to prevent damage to avoid the agarose layer. That is why it makes sense before the Coating of the slide one side of the slide mask with self-adhesive film. Serve as substrates for the slide commercial aminated ("silylated") glass slides (Telechem Inc., Sunnyvale, CA, USA; Quantifoil GmbH, Jena, Germany).

To the dialysis of the gel from oxidized agarose is melted, to a temperature of approx. 70 ° C brought and the solution on the prepared slides cast. After 10 min at 70 ° C the excess agarose solution from the slides knocked off and the carrier at least Dried for 1 h at room temperature. Storage in air and Room temperature before the subsequent modification reaction for the agarose should not exceed 24 hours around Degradation effects of the aldehyde groups in air as little as possible hold.

The final stabilization of the Schiff bases formed in the aldehyde-amine reaction between agarose and substrate by means of NaCNBH ₃ is expediently carried out only after incubation of the agarose surface with the corresponding ligands, because the ligands with the agarose in turn have Schiff bases form, which can then be reduced to the secondary amine in a common reaction.

• 1. Erzeugung von Anionenaustausch-Funktionen: Inkubation der Agarose-Oberfläche mit Verbindungen, die einerseits eine primäre Amino-Funktion und andererseits sekundäre oder tertiäre Aminofunktionen, wie z. B. eine Diethylaminoalkyl-Funktion umfassen, um entsprechende Liganden auszubilden. Beispiele solcher Verbindungen sind 3-Diethylamino-1-propylamin oder 2-Diethylamino-ethylamin.
• 2. Erzeugung von Kationenaustausch-Funktionen: Inkubation der Agarose-Oberfläche mit Verbindungen, die einerseits eine primäre Amino-Funktion und andererseits Carboxylfunktionen umfassen, um entsprechende Liganden auszubilden. Beispiele solcher Verbindungen sind alle natürlich vorkommenden Aminosäuren außer Prolin, insbesondere die sauren Aminosäuren Asparaginsäure und Glutaminsäure.
• 3. Erzeugung von Oberflächen mit hydrophoben Funktionen: Inkubation der Agarose-Oberfläche mit Verbindungen, die einerseits eine primäre Amino-Funktion und andererseits hydrophobe aliphatische oder aromatische Reste umfassen, um entsprechende Liganden auszubilden. Beispiele solcher Verbindungen sind 2-(Trifluormethyl)benzylamin oder Octylamin.
• 4. Erzeugung von Oberflächen mit proteinaffinen Funktionen, z. B. mit chelatbildenden Gruppen oder Heparin-Gruppen: Inkubation der Agarose-Oberfläche mit Verbindungen, die einerseits eine primäre oder sekundäre Amino-Funktion und andererseits Chelatbildner oder Heparin-Gruppen umfassen, um entsprechende Liganden auszubilden. Beispiele solcher Verbindungen sind Iminodiessigsäure oder Heparin-Albumin. Die Chelat-Funktionen müssen im Anschluss an die Bindung bzw. Inkubation noch mit Salzen zweiwertiger Metalle, wie z. B. Nickel oder Zink, komplexiert werden, bevor sie spezifisch für die Bindung von Proteinen mit Histidin-Funktionen eingesetzt werden können.

c. The modification of oxidized agarose surfaces to chromatographically active surfaces can be achieved by incubation and subsequent reduction of the agarose-coated substrates with the substances listed below (one or two examples are given here to produce each species of a chromatographically active surface):

• 1. Generation of anion exchange functions: Incubation of the agarose surface with compounds which on the one hand have a primary amino function and on the other hand secondary or tertiary amino functions, such as, for. B. include a diethylaminoalkyl function to form corresponding ligands. Examples of such compounds are 3-diethylamino-1-propylamine or 2-diethylamino-ethylamine.
• 2. Generation of cation exchange functions: Incubation of the agarose surface with compounds which on the one hand comprise a primary amino function and on the other hand carboxyl functions in order to form corresponding ligands. Examples of such compounds are all naturally occurring amino acids apart from proline, in particular the acidic amino acids aspartic acid and glutamic acid.
• 3. Generation of surfaces with hydrophobic functions: Incubation of the agarose surface with compounds which on the one hand comprise a primary amino function and on the other hand comprise hydrophobic aliphatic or aromatic residues in order to form corresponding ligands. Examples of such compounds are 2- (trifluoromethyl) benzylamine or octylamine.
• 4. Generation of surfaces with protein-affine functions, eg. B. with chelating groups or heparin groups: incubation of the agarose surface with compounds which, on the one hand, comprise a primary or secondary amino function and, on the other hand, comprise chelating agents or heparin groups in order to form appropriate ligands. Examples of such compounds are iminodiacetic acid or heparin albumin. After binding or incubation, the chelate functions must also be treated with salts of divalent metals, such as. As nickel or zinc, are complexed before they can be used specifically for the binding of proteins with histidine functions.

For the modification recommend the films of oxidized agarose on the solid substrates by incubation yourself if possible concentrated solutions of mostly water-miscible compounds bearing amino groups. For connections, which are present as solids, it is therefore advisable to use saturated, aqueous solutions. For in liquid form existing connections are solutions with 10% water content makes sense. The addition of water facilitates later all the mixing of the solution with the aqueous fixative solution to be applied subsequently. The pH values of the solutions should be between 6.5 and 8.5 using HCl or NaOH can be set.

Approx. 300 μl of the ligand solution are required for a slide; For a uniform covering of the surface with reagent it is advisable to cover the solution with cover glasses in object slides size. Times of 60 min at 37 ° C are suitable as incubation parameters.

d. Fixation or reduction of the Schiff bases to the secondary amine by means of NaCNBH ₃ : The reduction with NaCNBH ₃ directly follows the respective modification reaction. 300 μl of a fresh, aqueous 0.25 M NaCNBH ₃ solution in a fixation buffer (0.1 M Na-PO ₄ buffer, pH 7.4) per slide are used as the reduction solution.

The excess modification reaction solutions will previously knocked off the agarose substrate supports, and the reducing solution becomes direct applied. The incubation period is 15 minutes at room temperature.

To In the reduction reaction, the slides are washed thoroughly in water and air dried.

Example II:

A second method consists in the covalent fixation of a polyacrylamide film on surfaces with aldehyde functions, such as. B. aldehyde-modified glass surfaces, and subsequent reduction of the resulting Schiff bases by NaCNBH ₃ to stable secondary amines. The acid amide functions of the polyacrylamide can then be converted into chromatographically active chemical functions or ligands by transamidation with amino functions of corresponding compounds.

Usually the polyacrylamide film is thereby modified on the aldehyde Substrate immobilized that a solution of - at least partially - polyacrylamide monomers is applied to the substrate. The polymerization reaction for the monomer solution is started a few seconds before application. During this reaction comes there is a covalent bond between the monomers. The transamidation reaction, with a covalent bond between the polyacrylamide film and the Aldehyde functions of the substrate is subsequently generated during a minute 30 Incubation at 90 ° C.

• 1. Erzeugung von Anionenaustausch-Funktionen: Inkubation des Polyacrylamid-Films mit Verbindungen, die einerseits eine primäre Amino-Funktion und andererseits sekundäre oder tertiäre Aminofunktionen, wie z. B. eine Diethylaminoalkyl-Funktion umfassen, um entsprechende Liganden auszubilden. Beispiele sol cher Verbindungen sind 3-Diethylamino-1-propylamin oder 2-Diethylamino-ethylamin.
• 2. Erzeugung von Kationenaustausch-Funktionen: Inkubation des Polyacrylamid-Films mit Verbindungen, die einerseits eine primäre Amino-Funktion und andererseits Carboxylfunktionen umfassen, um entsprechende Liganden auszubilden. Beispiele solcher Verbindungen sind alle natürlich vorkommenden Aminosäuren außer Prolin, insbesondere die sauren Aminosäuren Asparaginsäure und Glutaminsäure.
• 3. Erzeugung von Oberflächen mit hydrophoben Funktionen: Inkubation des Polyacrylamid-Films mit Verbindungen, die einerseits eine primäre Amino-Funktion und andererseits hydrophobe aliphatische oder aromatische Reste umfassen, um entsprechende Liganden auszubilden. Beispiele solcher Verbindungen sind 2-(Trifluormethyl)benzylamin oder Octylamin.
• 4. Erzeugung von Oberflächen mit proteinaffinen Funktionen, z. B. mit Heparin-Gruppen: Inkubation des Polyacrylamid-Films mit Verbindungen, die einerseits eine primäre Amino-Funktion und andererseits Heparin-Gruppen umfassen, um entsprechende Liganden auszubilden. Ein Beispiele einer solchen Verbindung ist Heparin-Albumin.
• 5. Erzeugung von Oberflächen mit proteinaffinen Funktionen, z. B. mit chelatbildenden Gruppen: Inkubation des Polyacrylamid-Films mit Verbindungen, die einerseits eine primäre Amino-Funktion und andererseits Chelatbildner umfassen, um entsprechende Liganden auszubilden. Ein Beispiele einer solchen Verbindung ist Iminodiessigsäure.
• 6. Erzeugung von Oberflächen mit proteinaffinen Funktionen, z. B. mit chelatbildenden Gruppen: Inkubation des Polyacrylamid-Films mit Verbindungen, die die Säureamid-Funktionen des Poly acrylamids zu Chelat-Funktionen modifizieren, wie z. B. Bromessigsäure.

The transamidation of polyacrylamide films to chromatographically active surfaces can be achieved by incubation with the substances listed below (one or two examples are given in turn to represent each species of a chromatographically active surface):

• 1. Generation of anion exchange functions: Incubation of the polyacrylamide film with compounds which on the one hand have a primary amino function and on the other hand secondary or tertiary amino functions, such as, for. B. include a diethylaminoalkyl function to form corresponding ligands. Examples of such compounds are 3-diethylamino-1-propylamine or 2-diethylamino-ethylamine.
• 2. Generation of cation exchange functions: Incubation of the polyacrylamide film with compounds which on the one hand comprise a primary amino function and on the other hand carboxyl functions in order to form corresponding ligands. Examples of such compounds are all naturally occurring amino acids apart from proline, in particular the acidic amino acids aspartic acid and glutamic acid.
• 3. Generation of surfaces with hydrophobic functions: Incubation of the polyacrylamide film with compounds which on the one hand comprise a primary amino function and on the other hand comprise hydrophobic aliphatic or aromatic residues in order to form corresponding ligands. Examples of such compounds are 2- (trifluoromethyl) benzylamine or octylamine.
• 4. Generation of surfaces with protein-affine functions, eg. B. with heparin groups: incubation of the polyacrylamide film with compounds which on the one hand comprise a primary amino function and on the other hand heparin groups to form appropriate ligands. An example of such a compound is heparin albumin.
• 5. Generation of surfaces with protein-affine functions, eg. B. with chelating groups: Incubation of the polyacrylamide film with compounds which on the one hand comprise a primary amino function and on the other hand chelating agents to form corresponding ligands. An example of such a compound is iminodiacetic acid.
• 6. Generation of surfaces with protein-affine functions, eg. B. with chelating groups: Incubation of the polyacrylamide film with compounds that modify the acid amide functions of the poly acrylamide to chelate functions, such as. B. bromoacetic acid.

The Chelation functions need after the binding or incubation even more divalent with salts Metals such as As nickel or zinc, are complexed before they specific for the binding of proteins with histidine functions can be used can.

Example III

A third method consists in the covalent fixation of a polymer film with hydroxy functions such as e.g. B. the copolymer of 2-hydroxyethyl methacrylate and ethylenedimethylacrylate on surfaces with amino functions. Activation of the hydroxyl groups of the polymer can e.g. B. done by means of CNBr. Then chromatogra phically active chemical functions are generated by coupling corresponding ligands via their amino functions.

• 1. Erzeugung von Anionenaustausch-Funktionen: Inkubation des aktivierten Hydroxy-Polymerfilms mit Verbindungen, die einerseits eine primäre Amino-Funktion und andererseits sekundäre oder tertiäre Aminofunktionen, wie z. B. eine Diethylaminoalkyl-Funktion umfassen, um entsprechende Liganden auszubilden. Beispiele solcher Verbindungen sind 3-Diethylamino-1-propylamin oder 2-Diethylamino-ethylamin.
• 2. Erzeugung von Kationenaustausch-Funktionen: Inkubation des aktivierten Hydroxy-Polymerfilms mit Verbindungen, die einerseits eine primäre Amino-Funktion und andererseits Carboxylfunktionen umfassen, um entsprechende Liganden auszubilden. Beispiele solcher Verbindungen sind alle natürlich vorkommenden Aminosäuren außer Prolin, insbesondere die sauren Aminosäuren Asparaginsäure und Glutaminsäure.
• 3. Erzeugung von Oberflächen mit hydrophoben Funktionen: Inkubation des aktivierten Hydroxy-Polymerfilms mit Verbindungen, die einerseits eine primäre Amino-Funktion und andererseits hydrophobe aliphatische oder aromatische Reste umfassen, um entsprechende Liganden auszubilden. Beispiele solcher Verbindungen sind 2-(Trifluormethyl)benzylamin oder Octylamin.
• 4. Erzeugung von Oberflächen mit proteinaffinen Funktionen, z. B. mit Heparin-Gruppen: Inkubation des aktivierten Hydroxy-Polymerfilms mit Verbindungen, die einerseits eine primäre Amino-Funktion und andererseits Heparin-Gruppen umfassen, um entsprechende Liganden auszubilden. Ein Beispiele einer solchen Verbindung ist Heparin-Albumin.

The substances listed below can be used as ligands for coupling to polymer films with activated hydroxy functions (one or two examples are given in turn for the generation of each species on a chromatographic surface):

• 1. Generation of anion exchange functions: Incubation of the activated hydroxy polymer film with compounds which on the one hand have a primary amino function and on the other hand secondary or tertiary amino functions, such as. B. include a diethylaminoalkyl function to form corresponding ligands. Examples of such compounds are 3-diethylamino-1-propylamine or 2-diethylamino-ethylamine.
• 2. Generation of cation exchange functions: Incubation of the activated hydroxy polymer film with compounds which on the one hand comprise a primary amino function and on the other hand carboxyl functions in order to form corresponding ligands. Examples of such compounds are all naturally occurring amino acids apart from proline, in particular the acidic amino acids aspartic acid and glutamic acid.
• 3. Generation of surfaces with hydrophobic functions: Incubation of the activated hydroxy-polymer film with compounds which on the one hand comprise a primary amino function and on the other hand comprise hydrophobic aliphatic or aromatic residues in order to form corresponding ligands. Examples of such compounds are 2- (trifluoromethyl) benzylamine or octylamine.
• 4. Generation of surfaces with protein-affine functions, eg. B. with heparin groups: Incubation of the activated hydroxy polymer film with compounds which, on the one hand, comprise a primary amino function and, on the other hand, heparin groups, in order to form corresponding ligands. An example of such a compound is heparin albumin.

Example IV:

On fourth method is the covalent fixation of a glycidyl function containing polymer film [Hermanson GT, Mallia AK, Smith PK, 1992. Immobilized Affinity Ligand Techniques. Academic Press Inc., San Diego, CA, USA], such as. B. the copolymer of 33% glycidyl methacrylate, 60% 2-hydroxymethacrylate and 7% ethylene dimethacrylate, on aminated substrate surfaces and the conversion of the glycidyl functions of the copolymer to chromatographic active chemical functions. This is done by reacting appropriate Amino functions containing ligands with the glycidyl functions with strong heat input.

• 1. Erzeugung von Anionenaustausch-Funktionen: Inkubation des Copolymer-Films mit Verbindungen, die einerseits eine primäre Amino-Funktion als Ankopplungsstelle für die Glycidyl-Funktionen des Copolymers aufweisen, und andererseits Anionenaustausch-Funktionen, wie z. B. eine Diethylaminoalkyl-Funktion. Beispiele solcher Verbindungen sind 3-Diethylamino-1-propylamin oder 2-Diethylamino-ethylamin.
• 2. Erzeugung von Kationenaustausch-Funktionen: Eine Inkubation des Glycidyl-Funktionen tragenden Copolymers in Gegenwart von Wasser erzeugt über eine Ketenzwischenstufe eine Polycarbonsäureoberfläche. Diese kann direkt als Kationenaustauscher eingesetzt werden, für den in der Regel Carboxylgruppen benötigt werden.
• 3. Erzeugung von Oberflächen mit hydrophoben Funktionen: Inkubation des Glycidyl-Funktionen tragenden Copolymers mit Verbindungen, die einerseits eine primäre Amino-Funktion und andererseits hydrophobe aliphatische oder aromatische Reste umfassen nach Aktivierung der Carboxylgruppen des Polymer-Films mittels EDC. Beispiele solcher Verbindungen sind 2-(Trifluormethyl)benzylamin oder Octylamin.
• 4. Erzeugung von Oberflächen mit proteinaffinen Funktionen wie z. B. Heparin-Gruppen: Inkubation des Glycidyl-Funktionen tra genden Copolymers mit Verbindungen, die einerseits primäre Amino-Funktionen und andererseits Heparin-Gruppen umfassen, wie z. B. Heparin-Albumin.

The conversion of films of the copolymer to chromatographically active surfaces can be achieved by incubating at 90 ° C. for 10 minutes with the substances listed below (one or two examples are given in turn for the production of each species of a chromatographic surface):

• 1. Generation of anion exchange functions: Incubation of the copolymer film with compounds which on the one hand have a primary amino function as a coupling site for the glycidyl functions of the copolymer, and on the other hand anion exchange functions, such as. B. a diethylaminoalkyl function. Examples of such compounds are 3-diethylamino-1-propylamine or 2-diethylamino-ethylamine.
• 2. Generation of cation exchange functions: Incubation of the copolymer carrying glycidyl functions in the presence of water produces a polycarboxylic acid surface via a ketene intermediate. This can be used directly as a cation exchanger, for which carboxyl groups are generally required.
• 3. Generation of surfaces with hydrophobic functions: Incubation of the copolymer carrying glycidyl functions with compounds which, on the one hand, comprise a primary amino function and, on the other hand, hydrophobic aliphatic or aromatic residues after activation of the carboxyl groups of the polymer film by means of EDC. Examples of such compounds are 2- (trifluoromethyl) benzylamine or octylamine.
• 4. Generation of surfaces with protein-affine functions such. B. Heparin groups: Incubation of the glycidyl functions-carrying copolymer with compounds which on the one hand comprise primary amino functions and on the other hand heparin groups, such as. B. heparin albumin.

Application of biomolecules such. B. Proteins on the Prepared Substrates:
Proteins that are to be bound by non-covalent immobilization must be dissolved in special application buffers depending on the chromatographic characteristics of the selected polymer surface. While buffers with the lowest possible ionic strength are suitable for the application of proteins to surfaces with anions, cations or protein-related characteristics, buffers with the highest possible ionic strength must be selected for surfaces with a hydrophobic characteristic.

Vice versa can immobilized proteins on surfaces with anion, cation or protein - affine characteristic through buffers with high ionic strength or with a high content of a competitor molecule. Corresponding finds a replacement of proteins on surfaces with a hydrophobic characteristic rather than with buffers with low ionic strength.

To Application of the proteins to the prepared surface of the sample carrier should be used at temperatures between 0 ° C and 4 ° C to the binding equilibria between proteins and surface or Sample carrier if possible to keep far on the side of protein binding.

To the Application of protein solutions inkjet processes are suitable, Drip on or spray on better than needle printing because it hits hard needle tips the soft, mechanically sensitive polymer surfaces are light to damage can.

10

substratum

12

polymer film

14

ligand

15

chromatography active group of ligand 14

16

binding mediating interaction

18

protein

• 1. Deutscher MP (ed), 1990. Methods in Enzymology: Guide to Protein Purification. Vol. 28, Academic Press Inc., San Diego, CA, USA.
• 2. Afanassiev V, Hanemann V, Wölfl S, 2000. Preparation of DNA and protein micro arrays on glass slides coated with an agarose film. Nucleic Acids Res 28: e66.
• 3. Hermanson GT, Mallia AK, Smith PK, 1992. Immobilized Affinity Ligand Techniques. Academic Press Inc., San Diego, CA, USA.
• 4. Nath N, Chilkoti A, 2003. Fabrication of a Reversible Protein Array Directly from Cell Lysate using a Stimuli-Responsive Polypeptide. Anal. Chem. 75: 709–715.

List of literature cited:

• 1. German MP (ed), 1990. Methods in Enzymology: Guide to Protein Purification. Vol. 28, Academic Press Inc., San Diego, CA, USA.
• 2. Afanassiev V, Hanemann V, Wölfl S, 2000. Preparation of DNA and protein micro arrays on glass slides coated with an agarose film. Nucleic Acids Res 28: e66.
• 3. Hermanson GT, Mallia AK, Smith PK, 1992. Immobilized Affinity Ligand Techniques. Academic Press Inc., San Diego, CA, USA.
• 4. Nath N, Chilkoti A, 2003. Fabrication of a Reversible Protein Array Directly from Cell Lysate using a Stimuli-Responsive Polypeptide. Anal. Chem. 75: 709-715.

Claims (7)

Method for immobilizing at least one protein ( 18 ) on a substrate ( 10 ), characterized in that a) that on the substrate ( 10 ) a polymer film ( 12 ) is immobilized; b) that the polymer film ( 12 ) is modified so that it contains functional groups ( 15 ) on its from the substrate ( 10 ) facing away from the surface, the functional groups at least one of the following functions ( 16 ) (chromatographically active functions) have: b1) ion exchange function, b2) hydrophobic function or b3) protein-affine function; and c) that the at least one protein ( 18 ) on the modified polymer film ( 12 ) is applied. Method according to the preceding claim, characterized in that the polymer film ( 12 ) is selected such that it contains active or activatable functional groups ( 15 ) for binding molecules ( 18 ) Has; that the polymer film ( 12 ) by binding bifunctional molecules ( 14 ) is modified; that the bifunctional molecules ( 14 ) be chosen such that they carry a functional group with which they can bind to the active or activated functional groups of the polymer film; and that the bifunctional molecules are chosen such that the other functional group ( 15 ) is chromatographically active. Method according to one of the preceding claims, characterized in that a macroscopic substrate ( 10 ) is selected; that the non-covalently bound proteins ( 18 ) on the macroscopic substrate ( 10 ) are applied and bound in a spatially defined arrangement. Method according to one of the preceding claims, characterized in that as the polymer of the polymer film ( 12 ) Agarose is chosen; and that the agarose is oxidized. A method according to claim 1, characterized in that the polymer film ( 12 ) is chosen such that it contains chromatographically active or activatable functional groups ( 15 ) Has. Method according to one of the preceding claims, characterized in that as functional groups ( 15 ) with protein-affine function groups with chelating functions or heparin functions are selected. Sample carrier for immobilizing at least one protein ( 18 ) a) with one on a substrate ( 10 ) immobilized polymer film ( 12 ); b) the polymer film ( 12 ) is modified in such a way that it contains functional groups ( 15 ) on its from the substrate ( 10 ) facing away from the surface, the functional groups at least one of the following functions ( 16 ) (chromatographically active functions) have: b1) ion exchange function, b2) hydrophobic function or b3) protein-affine function.