METHOD FOR PRODUCING STRUCTURED, SELF-ORGANIZED MOLECULAR MONOLAYERS OF INDIVIDUAL MOLECULAR SPECIES, IN PARTICULAR SUBSTANCE LIBRARIES.

Abstract

The invention relates to a method for producing structured, self-organized molecular monolayers of individual molecular species. The invention is preferably used to create solid-phase bonded substance libraries. To this end, a) a substrate (2) suitable for the first monolayer is provided; b) a microstructured polymer mask (1) having openings is applied to the subtrate (2) at least once in a defined direction. Said polymer mask (1) is sufficiently thin and flexible for it to adhere to the substrate (2) by adhesion, and the material selected for the polymer mask (1) is chemically stable; c) the sandwich constituted by the substrate (2) and polymer mask (1) is flooded with a first agent corresponding to the intended use, so as to create at least one first molecular monolayer in the areas defined by the mask openings; c1) depending on the monolayer structure to be created step c) is repeated using the first agent or one or more additional agents; and d) after carrying out the step described in c) or the steps described in c) and c1), the polymer mask is pulled off after modification of the substrate surface areas which were left uncovered by the mask openings and were subjected to the agent or agents.

Description

BACKGROUND OF THE INVENTION

[0001] The invention relates to a method for producing structured, self-organizing molecular monolayers of individual molecular species. The invention is preferably applied in the formation of solidphase-coupled substance libraries or in other assays for testing molecular interactions. Libraries of this kind which are used for fast detection of interacting reactants in the molecular field are known, in principle. Such substance libraries are generated in different ways according to the known prior art nearest to the invention. Thus, it is known to use so-called “microbeads” under which small beads are to be understood which are provided with a molecular reactant on their surface. Furthermore, a so-called “mix&split” procedure is known for producing such libraries [Erb, E.; Jander, K. D.; Brenner, S.: Recursive Deconvolution of Combinatorial Chemical Libraries, Proc. of Natl. Acad. Sci. USA 91, pg. 11422-6 (1994)]. Solidphase-coupled libraries are particularly advantageous, the components of which are uniquely determined by their position (x-y-direction). For the series production of such libraries modified jet printers are employed, for example, which by point-spraying apply the substances to be brought into connection on a preselected substrate (Schober, A. Guenther, R.; Schwienhorst, A. Doering, M.; Lindemann, B. F.: Accurate high-speed Liquid handling of very small biological samples, Biotechniques 15: pg. 324-9, 1993). So-called “applicators” have to be used for the parallel generation of such libraries, whereby a head being provided with a plurality of thin channels is set upon a surface, the head being adapted to simultaneously supply a plurality of micro-reaction cavities; refer, for example, to U.S. Pat. No. 5,429,807. Furthermore, to the same end, the application of light-activatable protective groups (U.S. Pat. No. 5,489,678) and of print-techniques has already been described (DE 195 43 232.0).

[0002] One or several basic disadvantage/s is/are inherent in these known methods and devices: Thus the use of a jet-printer does not permit to simultaneously supply a greater number of surface elements with a substance all at once, since the process concerned is a serial one. The devices known as “applicator” border to their technically feasibility with respect to their miniaturization. The printing syntheses, which utilize prints having structurized and profiled surfaces for transferring substances, cause considerable problems with respect to an exact positioning; hence, reproducible multiple syntheses are practically excluded. Known methods of the complex protective group chemistry under use of light-activatable protective groups require long exposure times when a high step efficiency is aimed at, whereby the entire process for producing a library is rendered very time-consuming. Moreover, the light sensitivity of the last mentioned method necessitates a particularly expensive execution of the process. In Proc. Natl. Acad. Sci. USA, vol. 93, Nov. 96, pg. 13555-13560 such a method for generating substance libraries is described in which at first a substrate is provided with a suitable protective group or with a monomer supporting a first protective group. Then a substrate surface is coated with a light-activatable photoresist layer, partial areas are exposed and structurized according to a preselected synthesis algorithm. In the structurized areas, the splitting-off of the protective group is carried out, then the bonding to a second polymer can be carried out in the uncovered ranges. After removal of the remaining photo-resist layers the entire process can be repeated with an accordingly adapted photo-resist layer which has to be structurized anew. The main disadvantage in this proceeding is, apart from the disadvantages mentioned hereinbefore, the non-complete removal of the residuals of the photo-resist layers from the library matrix, whereby the output per synthesis step and, hence, the entire synthesis output decreases.

SUMMARY OF THE INVENTION

[0003] It is an object of the present invention to provide a method for producing structured, self-organizing molecular monolayers of individual molecular species which permits the reproducible and multiple repeatability of the formation and locally defined bonding of molecular units to molecular monolayers, and ensures a high output. In particular, the inventional method to be provided is adapted to ensure the formation of test-assays and of solid-phase-bonded substance libraries.

[0004] The object is realized by the characteristic features of the patent claims. According to the invention specially prepared micro-structurized polymeric masks are employed which are designed to be deposited upon an adapted substrate and which, in preselected ranges, are provided with apertures which permit the generation of monomolecular monolayers on the substrate.

DETAILED DESCRIPTION OF THE INVENTION

[0005] The invention will be explained hereinafter in more detail by virtue of embodiments schematically illustrated. There is shown in:

[0006] FIGS. 1 a to 1e a feasible embodiment for producing the required polymeric masks,

[0007] FIG. 2 a schematic flow scheme for producing a solid-phase-bonded substance library by means of a specific example,

[0008] FIG. 3 an example for an inventional surface nano-structurized in z-direction,

[0009] FIG. 4 a part, indicated, of a set of different polymeric masks for generating substance libraries.

[0010] In FIGS. 1a to 1d there is shown an example of a feasible embodiment of the polymeric masks as required within the scope of the invention, wherein a 4″-silicon wafer 10 represented in FIG. 1a is the starting point, said wafer being coated on both of its sides with a thermal SiO2-oxide layer 11 of a thickness of 500 nm. The SiO2-oxide layers 11 are covered with a photo-resist layer 12, as commonly used in micro-structuring, applied by spin-coating. One of said photo-resist layers 12 is provided with a not shown mask which is embodied in such a way that, as has to be described in the further course of proceeding, a frame 14 remains after completion of the polymeric mask structuring. Said frame 14 is adapted in its dimensions to the substrate 2 which will be inserted later. The respective residual photo-resist layer areas 120 are shown in FIG. 1b, whereby windows 121 are rendered uncovered. In accordance with the presetting by the mask, windows are etched into the first SiO2-oxide layer 11 and the photo-resist layers are removed; the result of these steps, with the residual SiO2-mask areas 110, is represented in FIG. 1c. Furthermore, FIG. 1c shows a newly deposited photo-structurable polymeric layer 13 which, in the example, is made of PDMS (polydimethylsiloxane) under addition of a crosslinking agent such as a diazido compound soluble in organic solvents, such as xylol or chloroform, or 2,2 dimethoxy-2-phenyl-acetophenone. It lies within the scope of the invention, also to use, depending on later cases of application and of the employed agents for this polymeric layer 13, other materials, for example, polyvinyl alcohol, PMMA, polyvinylpyrollidon etc., provided with a suitable crosslinking agent such as a salt of the diazidostilbenesulfate. Said polymeric layer 13 is provided with a further, not shown mask. The structure of said mask corresponds to those structures which by the structures to be generated in the polymeric layer 13 have later to be transmitted onto a substrate 2, still to be described. FIG. 1d shows the result of the structure generation in the polymeric layer 13 with the produced mask opening areas (windows) 130. At the same time FIG. 1d shows how the silicon areas, that have not been covered by the SiO2-masking areas 110, are removed under use of known etching means in a further processing step. Subsequently, the entire non-covered SiO2-areas are removed in a further etching bath. In FIG. 1e there is shown the result of an individual silicon frame which is overlapped by a self-supporting, structured polymeric layer 13. This unit designated hereinafter as a micro-structured polymeric mask 1 finds a single to a multiple application within the scope of the invention. In order to produce substance libraries, further polymeric masks 1 of this kind are required, which hereinafter are all designated by the reference numeral 1. The predetermined mask opening areas (windows) 130 of said polymeric masks are principally designed congruently to one another. They are, however, adapted to a synthesis and modification algorithm, respectively, partially executed non-opened, as schematically indicated in FIG. 1f by an arrow 131. The kind of producing the structures in the polymeric layers 13 mentioned is irrelevant within the scope of the invention; thus it is feasible to employ the so-called dry-etching procedures, when respective small openings up to the submicrometer range are required. Essentially is only that the produced polymeric mask is embodied thin and flexibly enough to hold on, when deposited upon a substrate 2, to be described in the following, by adhesion only, and that said mask is chemically stable towards agents which find application in the further procedure. Without illustrating the same in more detail, respective adjusting marks are given to the entire polymeric masks 1 being used in the procedure and to the substrates 2 employed, said marks permitting a precise alignment, for example, by aid of mask-aligners conventionally used in micro-structuring.

[0011] In the following, the preparation of a substrate 2 according to the invention will be described by means of example in FIG. 2. A silicon wafer coated with gold will be used as a substrate 2. The same should be freshly prepared and can be stored in double-distilled water. A polymeric mask 1 produced according to the foregoing example is superimposed upon the substrate 2 and is flooded with a reaction solution 3 which, by the way, is to react with the gold coat, which is not shown in more detail. In the example, NHS-SS-biotin dissolved to 10 μg/ml in phosphate buffer, 0.01 M, pH 8,5 is selected as reaction solution 3. After about 15 minutes incubation at room temperature, the surface is washed with pure buffer and distilled water and the polymeric mask 1 is stripped off. Subsequent thereto streptavidin is given over the entire surface of the substrate 2, said streptavidin with an affinity of approximately 10−15 M binds to the biotin immobilized in the areas preselected by the opening areas 130 of the polymeric mask 1. Thus, the defined areas 20 are produced on the gold surface, only three of which are provided with reference lines in FIG. 3, which areas are exclusively occupied by streptavidin. It is feasible to employ a thus produced monomolecular mono-layer, which is subdivided into several, uniformly distributed ranges 20, as, for example, an immobilizing matrix for biotinized molecules, but also as a substrate having a spatially selective adhesion or a defined boundary face energy.

[0012] In a further embodiment the preparation of a substrate is described which is adapted to serve as a parent substrate for the formation of a substance library. Herein, for example, a cleaned silicon wafer is used as a substrate 2, the entire surface of the same being, in the example, at first modified by treatment with 3-glycidoxypropyltrimethoxysilane. The substrate 2 is flooded by the 3-glycidoxypropyltrimethoxysilane (10% in toluene, 80° C., about 8 h). A two-hour incubation of the substrate in 0.05 M HCl leads to a hydrolysis of the mentioned epoxide, hence, there are free hydroxy-groups present. The latter spontaneously react, for example, with phosphoramidite. To this end, in the example, a C18 spacer-phosphoramidite, which is activated with tetrazole in 0.1 M of solution in acetonitrile, reacts for 10 minutes with the modified substrate surface. Subsequently thereto the surface is washed thoroughly with acetonitrile. The surface of the substrate is now occupied by dimethoxytrityl-groups, the latter being known standard protective groups in the chemistry of phosphoramidite protective groups. A polymeric mask 1, produced for example, according to FIG. 1 is deposited upon the substrate 2, prepared as described hereinbefore in analogy to FIG. 2, and respectively positioned by means of a mask-aligner. After positioning, the sandwich arrangement, constituted of the substrate 2 and of the polymeric mask 1, can be taken from the holder, since the polymeric mask, due to its design, sticks by adhesion to the surface of the substrate. 2% trifluoroacetic acid in H2O is given over the sandwich arrangement, wherein a splitting-off of the protective groups, produced in accordance with the above example, takes place in the opening areas 130 of the polymeric masks. Subsequently, the polymeric mask 1 is stripped off the substrate and ranges 20, distributed in analogy to FIG. 3, are obtained which are freed from a predefinedly selected protective group. The substrate can be used as test-assay already in this state. The respectively specific kind of preparation of the substrate is various and can be adapted to a great number of later applications. Said substrate prepared in such a way can subsequently be subjected, for example, to a phosphoramidite cycle as generally known, in order to add a further oligonukleotide-monomer to the designated areas 20 by synthesizing.

[0013] In order to set up a substance library further polymeric masks 1 are provided within the frame of the invention which, in accordance with the substance group to be synthesized, have to be variably and adaptably embodied in such a manner that the openings 130 in principle congruently correspond to the openings 130 of the first polymeric mask. congruently correspond to the openings 130 of the first polymeric mask. However, the former partially exhibit non-opened mask ranges 131, as indicated by example of FIG. 4. It also lies within the scope of the invention to provide for larger openings, if required, so that adjacent larger ranges on the substrate can be treated. In order to extend the molecular species at the places provided, a new polymeric mask, which corresponds to the synthesis cycle, is superimposed, the respectively to be synthesized substance is deposited, the respective reaction and cleaning steps are carried out, and finally the polymeric mask employed is stripped off. In spite of the thereby resulting different growth of height of the individual molecular species, constituted of a plurality of monomers each, in the different synthesis ranges of the substrate, the adhesive sticking of the respective polymeric masks is maintained, and even with synthesized sandwich parcels of chain lengths of 30-100 or more, there will be no underwashing of the respective polymeric masks by the subsequent synthesis solution. In the manner described it is feasible to set up reliably substance libraries of high reproducibility and output, with the aid of apparatus techniques from the field of microstructuring.

[0014] It is feasible to set-up any desired polymeric and substance libraries such as, for example, peptide, polysaccharide, polyterpene etc. libraries by means of the present invention. It is also feasible to produce mixed libraries, which is to be understood as the application of molecular species from different substance classes on a substrate. Furthermore, there is the feasibility of setting-up libraries with mixed polymers which means polymers consisting of monomers from different substance classes. This, however, requires the compatibility of the protective groups and of the functional coupling groups, respectively. The production of branched out polymers under use of respective multiply functionalized and multiply-protected monomers is also feasible. The described problems in the substance libraries produced according to the prior art are effectively obviated. The openings provided in the employed polymeric masks can be produced up into the submicrometer range. The inventional method offers the basic advantage of in no way restricting the respective chemistry applied. The materials of the employed mechanical parts are generally adaptable to the reaction cycle.

[0015] All features represented in the specification, in the subsequent claims, and in the drawings are substantial for the invention both, individually and in any combination with one another.

List of Reference Numerals

[0016] 1 —polymeric mask

[0017] 10 —silicon wafer

[0018] 11 —SiO2-oxide layer

[0019] 110 —SiO2-masking areas

[0020] 12 —photo-resist layer

[0021] 120 —residual photo-resist layer areas

[0022] 121 —window

[0023] 13 —polymeric layer

[0024] 130 —opening areas in polymeric layer 13

[0025] 131 —non-opened masking ranges in the polymeric layer 13

[0026] 14 —frame

[0027] 2 —substrate

[0028] 20 —defined areas/ranges on substrate 2

Claims

1. Method for producing structured, self-organizing molecular monolayers of individual molecular species, in particular, substance libraries, characterized in that a) on a selected or specially prepared substrate adapted to the intended application and to a first monolayer to be produced b) a microstructurized polymeric mask is deposited, at least once definedly aligned relative to said substrate, said mask being provided with definedly preselected openings corresponding to areas of said substrate to be addressed at will each, wherein the polymeric mask is designed thin and flexibly enough that it sticks to the substrate only by adhesion, and in that the selection of the material employed for the polymeric mask is in accordance with further agents used in the respective step of proceeding in such a manner that the material of the polymeric mask is chemically stable against said agents, c) the sandwich, constituted of said substrate and said polymeric mask, is flooded by a first agent, adapted to the purpose of application, for the formation of at least a first molecular monolayer in the opening areas of the mask, c1) the step according to c) is repeated depending on the monolayer structure to be formed with the first agent or one further agent or a plurality of further agents, and d) the polymeric mask is stripped off after completion of the step according to c) or the steps according to c) and c1) subsequent to the completed modification, corresponding to the opening areas of the mask, of the surface ranges of the substrate having been subjected to the agent and the agents, respectively.

2. Method according to claim 1, characterized in that the steps according to b) to d) can be multiply repeated under use of further polymeric masks and further agents in such a manner that the further polymeric masks are adjusted in a congruent alignment relative to the alignment of said first polymeric mask, the openings provided in said further polymeric masks are stipulated always in congruent analogy to the openings of said first polymeric mask, being, however, partially executed non-opened, in order to match the respective synthesis or modification algorithm.

3. Method according to claims 1 and 2, characterized in that polymeric masks are employed constituted of a frame, the external dimensions of which substantially correspond to the external dimensions of the substrate upon which they are deposited, and in that the frame is overlapped, on the side of the substrate, by a polymeric film, into which said openings are inserted.