PROCEDURE FOR THE PRODUCTION OF MANUFACTURING CHEMICAL SURFACES

The invention concerns a programmable, automated procedure for the production of ready-made chemical surfaces under combinatorial use of specific synthesis modules through in situ synthesis on a carrier. Important areas of application of the procedure according to invention are the molecular diagnostics in the ranges Humanund veterinary medicine (infection diagnostics, Onkologie, Forensik), the development of more again pharmaceutical effective substances, the biological basic research, food analytics, the biotechnology, in particular in the ranges material production and environmental analytics, as well as high throughput procedure for the Wirkstoffscreening in combinatorial-chemical investigations.

Automated systems, which a fast, purposeful and accurate collection and analysis of chemical and/or biological information permit, for example indispensable aids are in life sciences and adjacent fields of activity, like the pharmaceutical active substance development and the medical diagnostics.

An efficient system for collection and analysis of large information capacities, how they result during the collection of genetic information or the analysis of products of combinatorial chemistry, are fluid microprocessors. Among them one understands miniaturized, hybrid functional elements with chemical and technical components, e.g. poorly reactive, at whose surface receptors, e.g. bio molecules are immobilized, which serve as specific interaction partners for appropriate Analyten. Since thousands potential biological and biochemical interaction partners can be arranged in a poorly reactive , these must be applied with micro-technical methods.

Immobilized after that present state of the art the receptors through kovalente or not kovalente reciprocal effects or synthesized in situ on the fixed phase. If necessary a modulating of the physical (e.g. doping density), physicochemical (e.g. redox potential), chemical (e.g. catalytic activity) and biocompatible characteristics (e.g. controlling of molecular recognition processes) of the manufactured surfaces is experimentally very complex and succeeds not always. Optimizations of chemical, biological and biochemical interactions, which are to be proven at surfaces, are from there only under substantial Arbeitsund expenditure of time, if at all, to reach. Since as interaction partners natural substances are applicable such as DNA, RNA, PNA, Oligonukleotide, saccharides, coal hydrates, Peptide, proteins, in addition, derivatives of combinatorial chemistry, the range of optimally suitable surface configurations is very large and so far neither analytically, nor synthetically sufficiently systematically seizes.

The components of a chemical polymerization process are generally called “Synthone”. The functional groups of a Synthons permit a purposeful chemical reaction with suitable other functional groups at a second Synthon. In all rule these reactive centers are masked by so-called groups of protection, which can be removed in a suitable chemical environment purposefully, whereby a controlling of the synthesis process becomes possible, since only such functional groups are converted, which do not carry a group of protection. Dependent on the respective synthesis strategy groups of protection can generally by a change chemical or physikochemischer environment parameters like the redox potential, for which pH value or the temperature, in addition, by the entry of electromagnetic energy, is removed e.g. by irradiation with light of a certain wavelength.

For the sequence-specific synthesis of Oligomeren, like Oligonukleotiden, Oligonukleotidderivaten, Peptiden, or coal hydrates, which are composed of different, however finally many monomer units, the introduced, the sequential polymerization by Merrifield by condensation at a firm phase worked satisfactorily (R.B. Merrifield (1963), J. to. Chem. Soc. 85:2149 - 2154; R.B. Merrifield (1965) Science 150:179 - 185). The synthesis after Merrifield begins at a carrier-bound starting monomer, which can be activated by the distance of one or several groups of protection. Thus the addition in solution of a supplied, next monomer is permitted, which is available after polymerization by condensation for his part as starting point for a further polymerization step. The gradual repetition of activation and following polymerization by condensation leads finally to the synthesis of the desired Oligomeren.

As substrates for Merrifield syntheses in particular glass surfaces worked satisfactorily. In addition, silicon or metals such as gold, high-interlaced polystyrene and other plastics like conductive Polypyrrol or copolymers such as N-Vinylpyrrolidon/N-Acryloxysuccinimid, as well as “natural substances” like paper (cellulose) is used than carrier materials. The functionalization of the materials mentioned after the specialist, chemical effected usually admitted (e.g. hydrogenation with following substitution) (Immobilized Affinity ligand Techniques, Academic press 1992, eds. G.T. Hermason, A.K. Mallia, P.K. Smith) or physical standard techniques (e.g. plasma coating), with which the result of the functionalization reaction is empirically stopped and examined. The range between the functional groups on the surface of the carrier and the actual, connection-active functional receptors (e.g. immobilized bio molecules) is bridged by so-called “Linkern”. It acts over arbitrarily modified molecule remainders, which obtain functional groups contained and over these groups of connection of receptors (e.g. bio molecules such as Oligonukleotide or coal hydrate remainders) with the carrier surface. The physiko chemical and the sterisch spatial characteristics of the Linkers affect both the implemented the Merrifield syntheses at them (J. Katzhendler, S. Cohen, E. Rahmim, M. Weisz, Ith Ringel, J. Deutsch (1989) Tetrahedron 45:2777 - 2792; A.v. Aerschot, P. Herdewijn, H. Vanderhaeghe (1988), Nucl. Nucl. 7:75 - 90), and the connection of the potential Analyten to the manufactured connection-active surface (M. Beier, J.D. Hoheisel (1999) Nucleic Acids Res. 27:1970 - 1977). The selection and variance of the used left components basedly usually on empirical values and must be verified empirically.

For a number of carrier-bound, nucleic acid-supported analysis methods detection one takes place via a fluorescent coloring material molecule marked Analyten (e.g. a DNA or a RNA) via hybridizing to a matrix from many different Oligonukleotiden, submitted on the carrier. According to experience thereby the density of the Oligonukleotide submitted on the carrier has a relevant influence on the strength of the measurable signal and on the relationship from signal to background. First beginnings for the optimization of the carrier surface for an improvement of the detection process within the range DNA DNA reciprocal effects are descriptive. In a beginning chemistry from the “Starburst” was used - Dendrimerbreich. Here it acts structures branched out over strongly, which become generationsweise around a Initationspunktaufgebaut. With increasing generation number the number of the bypasses and thus the number at reaction places increases, whereby it finally for an increase of the functional density on the surface and indirectly to an improvement of the detection process (M. Beier, J.D. Hoheisel (1999) Nucleic Acids Res. 27:1970 - 1977) come.

In another beginning amino acids were integrated into the left molecules, in order to be able to steer both for the synthesis, and for the detection process (hybridizing), the charge density on the surface pH-dependently, and so a highly specific hybridizing with the Analyten to ensure (M.S. Shchepinov, S.C. Case Green, E.M. Southern (1997) Nucleic Acid Res. 25:1155 - 1161).

The invention is the basis the task to make a procedure available with which the introduction of functional groups can be modulated “in situ”, thus during the functionalization process, and be adapted to the intended purpose of the surface. A further task consists of it, in the procedure suitable left molecules according to kind of a building block principle of a given number of chemically reactive basic modules selects and automates to condense to be able. Further progressing and the result of the synthesis reaction should be in a rule process supervisable and controllable.

In addition the task which can be solved is the supply a procedure, that with the synthesis or with the immobilization of connection-active structural components in or on a poorly reactive into situ the flexible attitude of the physical (e.g. density), the physikochemischen (e.g. polarity), which chemical (e.g. photochemical instability) and/or the biocompatible (e.g. controlling of molecular recognition processes) characteristics permits, independently of the default by the primary functionalization of the reaction carrier.

This task solved by the structure of a liaison vehicle (“left one”) between a funktionalisierten fixed phase surface and the analytbindenden structural components (receptors). That left ones is developed in or on the poorly reactive systematically from one or more same or different monomers of various chemical substance classes according to kind of a Merrifield strategy, itself in the sense the one

Unit construction system freely to combine leave. For the exact attitude of the physical, physikochemischen, chemical and biocompatible characteristics of the developing active surface a control can do several by an actual value/a goal value alignment over or in or at the poorly reactive implemented Meßzelle (n) to take place, which permits a freely selectable, structure accurate to size and reproducible that left ones from the given monomer units.

Contrary to the state of the art, where surface properties by trial and error be only adjusted and modified can, reproducibly a ready-made chemical surface can programmed be developed with the help of the invention be automated and.

In a first aspect the procedure according to invention for the production of a coated carrier covers the steps

• a) Make available a carrier, which exhibits reactive groups at its surface, and
• b) Develop a funktionalisierten surface on the carrier by gradual synthesis of left molecules, which contain functional groups, from Synthon components,

For the monitoring of the synthesis process for one or more physicochemical parameters of the funktionalisierten surface of goal values are preferably given, and during the synthesis an alignment takes place from measured actual values with the given goal values or the parameters. Dependent on the result of the alignment on ISTund goal values following synthesis steps can be modulated. The monitoring can take place after in each case one or more synthesis steps. Preferably after each synthesis step a monitoring is accomplished. Goal values can be given e.g. by the experimenter as a function of application.

The last actual value, which corresponds ideally to the preset in each case goal value, can consider itself as actual value for the following coupling of the connection-active element, and/or receptor to that left ones and/or as internal standard for the quality assurance. For a later qualitative and/or quantitative collection of the signal of a chemical, biochemical or biological reaction at the connection-active elements the surface properties of the reaction carrier are in each experiment well-known and can than parameters into the analyses flow.

As measuring methods to the monitoring are possible for example: Absorption, emission, conductivity, pH value, NMR, mass spectrometry, radioactivity, Plasmonenresonanz, refraction of light, light scattering, heat of reaction, electron diffraction, neutron diffraction or Ellipsometrie. The measurement takes place in a Messzelle, which is integrated preferably in the poorly reactive or into the surrounding fluid elements. With fluid separated reaction areas within the reaction carrier the measurement takes place separately according to reaction areas preferably. Preferred goal values and/or physicochemical parameters, which can be supervised, e.g. are the density of functional groups on the surface, the distance of functional groups from the surface, the surface polarity, optical, magnetic, electronic, dielectric and catalytic characteristics, thermal stability, the photochemical and enzymatic activity in addition, spatially structural characteristics (molecular recognition).

In a further aspect the procedure according to invention concerns the use of Synthon components, which serve the modelling of ready-made chemical surfaces. Are used a set of components , which can be assigned different chemical connecting compounds, as for instance amino acids, nucleosides, Glykosiden. Usually such Synthon components are used in protection-group-modified form. According to their chemical, physicochemical and biological characteristics these components can take over different functions for manufacturing the surface: Spacers (“Spacer”) like alkyl residues, serve the variation of the vertical distance between funktionalisiertem substrate and connection-active element. Spacers with sterisch fastidious groups can be used for the variation of the space fulfillment, i.e. the horizontal distance between individual left molecules connection-active elements (“Spacern”). During introduction of polar or loaded remainders to the spacers (e.g. use of Oligoethylenglycol in place of alkyl residues) the polarity of the surface can be modulated. Dielectric characteristics can be e.g. varied for example by introduction of loaded groups, amino acid residues to that left ones.

The components can be thus for example selected from Synthonen of nucleic acid, Peptidoder coal hydrate chemistry and/or from Spacermolekülen.

The left molecules synthesized on the carrier contain functional groups, which make the coupling possible of further left Synthon components or - after completion of the left synthesis - from receptors or receptor components. The functional groups of the left molecules can be selected for example out - OR, - NR2, - SR, - PO3R2, - CN, - SCN, - COR' and - OCOR', where R H or a group of protection means and R' H or a group of protection or - OR, - a NR2 or - SR means. Further R and R' can stand for alkyl, aryl, alkenyl and/or allyl remainders and/or further useful organic remainders.

After terminated left synthesis takes place the coupling of receptors, which can likewise take place via gradual synthesis from synthesis components depending upon used synthesis strategy, e.g. peptid, Oligonukleotidoder coal hydrate synthesis at the fixed phase or via localspecific and/or not-localspecific immobilization from complete receptors.

In still another further aspect the available invention concerns the use from doping Synthonen to the chemical and physikochemischen, in addition, for the biocompatible modulation of the surface properties. Here it concerns on the one hand branched Synthone, their linking to a multiplication of the number of initial reaction places leads itself (signal amplification) and by blind Synthone, which lead to a decrease of the reaction places, since they do not carry functional groups (signal reduction). All Synthone is characterised by it that they are applicable in the sense of a Merrifield synthesis, is called the functional groups of the Synthone carries groups of protection, which can be removed under suitable chemical or physical conditions selectively. The attitude of the Oberfächeneigenschaften takes place via polymerization, e.g. via condensation of a suitable combination of spacers and doping Synthonen. The surface property is modulated by selection of suitable doping Synthonen, as after each polymerization step the actual value is compared with the goal value, and Synthone or mixtures from suitable Synthonen, suitable by the system, to be supplied so long, until the surface exhibits the desired characteristics.

The first addition of Spaceroder doping Synthone can have followed from a blocking step, with which all not used functional groups of the surface are inactivated. In case of of glass surfaces this preferably happens with Hexamethyldisilazan, Trüsobutylchlorsilan or tri methyl chlorosilane. Then the group of protection is selectively removed from this first Synthon at the surface of the reaction carrier. Subsequently, - according to the result of a monitoring of the surface properties - second or a further synthesis step with a selection modulated if necessary is accomplished by Synthon components regarding the portions of Blindoder/and bypass Synthonen, whereby also the length, which space fulfillment and/or the polarity of the Synthone can be varied. Additionally or alternatively the Synthonmenge can be varied and/or - concentrations or also the reaction conditions for individual or several synthesis steps.

Preferably the synthesis process takes place automated and the attitude of a characteristic profile of the funktionalisierten surface is programmable.

In still another further aspect the procedure according to invention the localspecific structure of the funktionalisierten surface on the carrier betriftt, i.e. the funktionalisierte surface extends not over the entire carrier, but only over pre-determined surface ranges, whereby array structures to be produced can.

The localspecific structure of the surface can by spatial and/or temporally limited exposure or through spatially and/or temporally limited fluid supply to take place. Also at least two differently funktionalisierte surfaces on a carrier can be developed. The differences between individual surface ranges can be thereby in the kind of the functional groups and/or in the density of the functional groups within individual ranges.

Still another further aspect of the available invention is that the combination of different synthesis procedures, e.g. Peptidsynthese and/or DNA synthesis or hybrid synthesis procedures permits, different strategies for adjustment the surface properties. As a variant different surface properties can be realized on a carrier. When using localspecific synthesis procedures also the attitude of localspecifically different surface properties is possible. Such a procedure is for example the light-steered synthesis with according to photo-unstable groups of protection, those over a suitable exposure matrix (e.g. see PCT/EP99/06316 and PCT/EP99/06317, to whose revealing in this connection expressly is referred) localspecifically to be removed to be able.

The carrier can a surface in principle at will be selected, e.g. from particles, in particular magnetic particles, micro titer plates and micro-fluid carriers (like e.g. fluid microprocessors) and can selected from glass, metals, metalloids, metallic oxides or plastic exhibit. The micro particle revealed in PCT/EP 99/06315 and in PCT/EP the 99/06316 are particularly preferential and PCT/EP 99/06317 revealed carrier with planar and/or with micro-channels (cross section e.g. 10 -1000 µm) provided surfaces. To revealing the documents mentioned expressly is referred.

In a further aspect the procedure according to invention covers a coupling of receptors to the functional groups of the left molecules, whereby coupling the receptors can take place via gradual synthesis from Synthon components. Also here the synthesis process of the receptors can be supervised and modulated if necessary. Receptors are biological and/or chemically functional molecules, to be e.g. selected can from nucleic acids, nucleic acid analogues, Peptiden, proteins and coal hydrates. Alternatively however receptors also prefabricated can be coupled to the left molecules. Also coupling receptors on the carrier can localspecifically take place.

So e.g. at least two can be developed concerning kind of receptor and/or receptor sequence and/or receptor density different receptor ranges.

In the procedure according to invention of manufactured carriers e.g. in a procedure for the determination of a Analyten, if necessary in an integrated device for the carrier synthesis and Analytbestimmung as revealed in PCT/EP a 99/06317 can be used. The carrier according to invention differs from well-known carriers by its for the respective application purpose optimized surface finish and its high reproductibility.

In a further aspect the invention concerns a device to the steered synthesis of liaison vehicles (Linkern) between reactive groups on the carrier surface and connection-active receptors. These liaison vehicles are modular condensates from several same or different Synthon components. The synthesis of the left molecules on the entire surface of the carrier can take place, or however localspecifically take place at selected reaction places.

The device to the steered synthesis of liaison vehicles can be a programmable source of light matrix, which permits a localspecific light-dependent synthesis. Another possibility is a device separate to the fluid synthesis, which permits a steered supply of fluids, and concomitantly the synthesis reagents, and this alternatively according to reaction ranges or uniform.

In a preferential execution form the device according to invention covers for the production of coated carriers:

• a) at least one carrier, which exhibits reactive groups at its surface,
• b) several reservoir, which contain solutions with Synthon components to the gradual synthesis of left molecules,
• c) Means for the supply of the Synthon component solutions on the carrier and for the derivative of used up solutions of the carrier, e.g. pumps and lines and/or capillaries,
• d) Means for supervising the synthesis process for the left molecules, e.g. Meßzellen or - elements for regulation one or several physicochemical parameters,
• e) Means for modulating the synthesis process for the left molecules, e.g. an appropriate software for the alignment of the actual values for one or more parameters with pre-determined goal values,
• f) if necessary means for coupling receptors to left molecules on the carrier surface and
• g) if necessary means for the execution of a Analytbestimmung on the carrier, e.g. means for the supply of a sample and for the measurement of a signal caused by the Analyten.

The following figures and remark examples are meant for explanation and are to limit the scope of protection of the procedure according to invention in no way.

Show:

Illustration 1

an example of a trifunctional left molecule, e.g. see Burmeister et al. (Tetrahedron Lett. 4995,3667-3668).

Abbilung 2

Examples of fluorescence molecules on Tritylbasis and

Illustration 3

Examples of fluorescence molecules on Nitrobenzylbasis.

On a poorly reactive, a fluid microprocessor made of glass, primary alcohol functions are preferably introduced. The coating takes place via a process, which consists of two indexing steps. After an activation a Silanisierung lasting several hours takes place via Bespülen of the reaction carrier with a solution from 1 - 10% (v: v) Hydroxyalkyltriethoxysilan in an alcohol. For the feasibility of the invention-moderate procedure a constant reproducible coating is important. This can being analyzed with different procedures, like e.g. with Ellipsometrie, glow discharge spectroscopy, conductometric analysis or another suitable analysis method, partially also for the detection steps during the procedure is suitable. The developing primary alcohol functions serve among other things as starting points for a carrier-bound Oligonukleotidsynthese after the Phosphonatbzw. Phosphoramiditmethode or a Peptidsynthese modifies after Merrifield.

As target for a ready-made chemical surface the distance between the OH-functions on the surface after of the fluid microprocessor and one is the Phosphonatbzw in the specified example. Phosphoamiditmethode to synthesizing DNA Oligomer (“connection-active structural component”) of 50 atoms amount to. The parking bay density of these Oligonukleotide is to be stopped to approx. 10 nmol/cm2, the polarity of the Linkers to a medium size.

For the production of the actual value the functionalization density on the surface of the fluid microprocessor must be intended. This is done preferably via automated coupling of a fluorescent more dimethoxytrityloder a photoactive nitrobenzylgeschützten monomer from the Phosphoramiditchemie to free primary alcohols. In principle different beginnings are applicable for the introduction of fluorescence:

With this substance class trifunctional left molecules are used, at whose a functionality a coloring material (color) and to their different one a group of protection (S1) were coupled.

The fluorescence coloring materials remain in this case during the entire process in the system. The actual Oligonukleotidsynthese takes place after splitting off of the group of protection of S1. In the remark example 3-O (4.4 ' - Dimethoxytrityl) - 1-O (1-pyrenyl) - butyl glycerin (Burmeister et al., supra) Pyren was used as coloring material and Dimethoxytrityl (DMT) as group of protection of S1.

In order secondary reactions back urge, it is to be made certain when using groups of photo protection for position S1 that the excitation wavelengths for the splitting off of the group of protection and the suggestion of fluorescence do not overlap.

Introduction of fluorescence takes place on the one hand via substitution of the Phenylgruppe in 4,4 ' - Dimethoxytriphenylmethylchlorid (DMT-CI) via a suitable coloring material, e.g. Pyren (type I) on the other hand by substitution one or both groups of methyls of the Methoxyfunktionalitäten, e.g. Pyrenbutanol (type II). The ability is not crucial to stabilize the cation developing with the splitting off of the group of protection is lost.

Advantage of this method is it that the fluorescent acid-unstable Tritylschutzgruppen can be integrated easily into the standard Oligonukleotidsynthese.

Beside the acid-unstable DMT Schutzgruppen also strategies are used, which are based on the splitting off of photoactive groups of protection. Usually it concerns thereby Nitrobenzylderivate. By purposeful incorporation of a suitable fluorophoren group, as for instance 7 (Dimethylamino) -, these fluorescent photo-unstable groups of protection can cumarin grouping be integrated into the Oligonukleotidsynthese.

As the further source for fluorescence gold particle serves. If these reversibly immobilized nano-particles under a certain angle are illuminated, Light Scattering of effects arise.

The extent of arising fluorescence gives an indirect measure for the number of the accessible primary OH-functions. The found value serves as actual value for the parking bay density of the desired Linkers. This actual value is compared with the goal value. Depending on whether the default is more überoder fallen below, in the next coupling step a suitable monomer or a mixture from suitable monomers is appliziert (a pallet at monomers is provided with stock in the system. The reactivity data of the monomers are contained of an electronic in data base.), preferably a mixture from a blind monomer, e.g. Methoxy [(2-cyanoethyl) - (N, N-diisopropyl)]phosphoramidit and a fluorescent bypass monomer.

Again a fluorescence measurement is consulted for the determination of the new actual value. This step is repeated gradually, until the desired functionalization density is reached. Subsequently, the attitude of the distance takes place between the Trägeroberlfäche and the DNA Oligonukleotid which can be synthesized via gradual linking of preferably commercially available Spaceramiditen. If a distance of e.g. 40 is - 60, preferably approx. 50 atoms, to the carrier surface realizes, can be begun with the automated synthesis of the desired DNA Oligonukleotids. In place of the Dimethoxytrityl protected monomers can be inserted also photo-unstable, e.g. with 2-Nitrobenzyloxycarbonyl or 2-Nitroethyloxycarbonyl protected monomers. In the case of use of a programmable source of light matrix for exposure on the fluid microprocessor, so localspecifically different left molecules can be developed. Particularly in case of the nano-particles the Ellipsometrie represents a further method for the generation of actual values.

All specified variants, which are based on fluorescence, combine the advantage in itself that they simplify the adjustment of the optical structure in a carrier system in accordance with PCT/EP99/06317 substantially, since the focusing is substantially more exact on discrete layers as the focusing on a diffuse fluorescent solution.

As the further method the UV/VIS absorption of the Tritylkations (λ = 498 Nm) of of the coupled Amidits can (preferably 3-Dimethoxytrityloxypropyl-1 [(2-cyanoethyl) - (N, N-diisopropyl)]- phosphoramidit), after-effected Entschützungsreaktion for the determination of the actual value to be consulted. It is a direct measure for the number of the accessible primary OH-functions. The found value serves as actual value for the parking bay density of the desired Linkers. This actual value is compared with the goal value (10 nmol/cm2). Depending on whether the default is more überoder fallen below, becomes in the next coupling step a suitable monomer or a mixture from suitable monomers (a pallet at monomers is provided with stock in the system, which are reactivity data of the monomers contained in an electronic data base), preferably Methoxy [(2-cyanoethyl) - (N, N-diisopropyl)]phosphoramidit (“blind monomer”) and [4.4 ' - Dimethoxytrityloxyl-1,1-bis-ethyl [(2-cyanoethyl) - (N, N-diisopropyl)]- appliziert phosphoramidit (“bypass monomer”). Again the absorption measurement of the Dimethoxytritylkationen is consulted for the determination of the new actual value. This step is repeated gradually, until the desired functionalization density is reached. Subsequently, the attitude of the distance between the carrier surface and the one which can be synthesized the DNA Oligonukleotid takes place via the gradual linking of Spaceramiditen, preferably 3-Dimethoxytrityloxypropyl-1 [(2-cyanoethyl) - (N, N-diisopropyl)]phosphoramidit and/or for the increase the polarity 9-Dimethoxytrityloxy-triethylenglycol, 1 [(2-cyanoethyl) - (N, N-düsopropyl)]phosphoramidit. If a distance from 50 atoms to the carrier surface is realized, can be begun with the automated synthesis of the gewünschen DNA Oligonukleotids.

In place of the Dimethoxytrityl protected monomers also photo-unstable can be inserted, with 2-Nitrobenyloxycarbonyl or 2-Nitroethyloxycarbonyl protected monomers. In the case of use of a programmable source of light matrix for exposure on the fluid microprocessor, so localspecifically different left molecules can be developed.

As an alternative execution that is made available to left ones in accordance with procedure descriptive above. Subsequently, the accumulation places in form of entschützter functional groups serve the immobilization of finished polymer probes. A localdissolved Entschützung can make the accordingly localdissolved immobilization possible if necessary different probes.

In a further execution the technology of group of protection for localdissolved the different doping of the surface is used, so that within different reaction ranges different concentrations and/or densities of same or different polymer probes develop.

Again a uniform defined density at Spacer molecules can be made available with automated collection and attitude. Subsequently, a parliamentary group of the reaction ranges can be activated on use of a localdissolved distance of the group of protection and a polymer probe synthesis be begun. For the density of the probes enstehenden within these reaction ranges the suitable mixture of extendable, extendable or branched Synthonen cannot be admitted as described. Polymer probes of same sequence, but different density can be synthesized in the consequence, as now different reaction ranges are activated and brought with a mixture in contact, which contains the same kind of components, but another mixture of the extendable, non-extendable or branched Synthone. Accordingly also reaction ranges with different concentrations at different polymer probes can be manufactured. Alternatively the density of the probes can be stopped in such a way, as with well-known radioactive half-life_{T1/2 of a} group of protection the exposition is e.g. selected to the entschützenden influence, a source of light, thus that only a defined portion of the groups of protection is removed and the density of the polymer probes manufactured afterwards on the reaction range is thus adjusted. Thus polymer probes of same or different sequence at different places with different density can be synthesized with localdissolved Entschützung, e.g. lichtabhängier Entschützung. This can be used among other things for quantitative determination of the measuring signal, e.g. as the density of the polymer probes affects the number of bound Analytmoleküle and thus the signal strength.