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Patentsuche

  1. Erweiterte Patentsuche
VeröffentlichungsnummerWO2009080766 A2
PublikationstypAnmeldung
AnmeldenummerPCT/EP2008/068056
Veröffentlichungsdatum2. Juli 2009
Eingetragen19. Dez. 2008
Prioritätsdatum20. Dez. 2007
Auch veröffentlicht unterDE102007062154A1, WO2009080766A3
VeröffentlichungsnummerPCT/2008/68056, PCT/EP/2008/068056, PCT/EP/2008/68056, PCT/EP/8/068056, PCT/EP/8/68056, PCT/EP2008/068056, PCT/EP2008/68056, PCT/EP2008068056, PCT/EP200868056, PCT/EP8/068056, PCT/EP8/68056, PCT/EP8068056, PCT/EP868056, WO 2009/080766 A2, WO 2009080766 A2, WO 2009080766A2, WO-A2-2009080766, WO2009/080766A2, WO2009080766 A2, WO2009080766A2
ErfinderManfred Auer, Hubert Gstach, Guenter Roth, Karl-Heinz Wiesmueller
AntragstellerNovartis Ag
Zitat exportierenBiBTeX, EndNote, RefMan
Externe Links:  Patentscope, Espacenet
Method for manufacturing and application of random ordered arrays of chemical compounds
WO 2009080766 A2
Zusammenfassung
The presented invention describes a method for the efficient manufacturing and application of random ordered arrays of chemical compounds. The method involves the generation of a random ordered array of beads, each bearing exactly one chemical compound. The chemical compounds from the beads of the bead array, called master, are transferred to another surface, the assay plate, generating a chemical copy of the bead array onto this surface. The assay plate could be used like any microarray.
Ansprüche  (OCR-Text kann Fehler enthalten)
Claims
1. Method for the manufacturing and application of random ordered arrays of chemical compounds, which allows the transfer from a master plate onto a assay plate
2. Method according to claim 1, designated by a preservation of spatial information and allocation between master and assay
3. Method according to claim 1 , designated by using of microparticles random ordered on the master plate bearing the chemical compounds
4. Method according to claim 1 , designated by the possibility to make one or more assay plate copies from one master plate
5. Method according to claim 1 , designated by using a "printing like" step to transfer all chemical compounds within one printing step
6. Method according to claim 1 , designated by the following points/process steps
6.1. the chemical compound bears the chemical residues that comprises the attributes to get released from the master plate, transferred to the assay plate and anchor there
6.2. the chemical compound is bound to beads,
6.3. the beads with the chemical compounds are immobilised on a master plate
6.4. the chemical compounds are transferred once or more from the master plate to the assay plate
6.5. biochemical, biomedical, biological, chemical or physical assays could be performed on the assay plate
6.6. a one-to-one allocation between chemical compound dot on the assay plate could be made to each bead on the master plate
7. Method according to claim 6, designated by the fact that the chemical compounds described in 6.1 are applicable to the following principles
7.1. synthesis orthogonal cleavable linker for release of chemical compound
7.2 transfer principle from master plate to assay plate
7.3 chemical spacer which enables biocompatibility Each point in single or in combination
8. Method according to claim 7, designated by any technique that allows the release of the chemical compounds by a synthesis orthogonal cleavable linker. The release is used to start the transfer from master plate to assay plate.
9. Method according to claim 7, designated by where this linker is photo cleavable, preferably 4-Bromomethyl-3-nitrobenzoic acid
10. Method according to claim 7, designated by which the transfer principle anchors on the assay plate via a functional group represented by adhesive, electrostatic, ionic, kovalent, adsorptive, absorptive or ligand-receptor interaction
11. Method according to claim 7, designated by which the transfer principle anchors on the assay plate via hydrophobic interactions, preferably realised with Pam3Cys-OH.
12. Method according to claim 7, designated by the fact that the biocompatible spacer separates spatially the active, preferably the biological active part of the chemical compound, from the chemical part which provides the transfer principle.
13. Method according to claim 7, designated by a biocompatible spacer that prevents spatially non-specific interactions between the chemical transfer principle and/or the assay plate surface.
14. Method according to claim 7, designated by that the biocompatible spacer is a peptidic structure
15. Method according to claim 7, designated to the principles 7.1 to 7.3 could be implemented before, whilst and after the synthesis of the chemical compound.
16. Method according to claim 6, designated to method step 6.2 where a coupling of the chemical compound is realised before whilst, or after the synthesis of the chemical compound.
17. Method according to claim 16, designated by an orthogonal cleavage of the chemical residue by which the chemical compound is bound synthesis beads
18. Method according to claim 6, designated by the beads are immobilised to a monolayer in a random order on the master and each bead bearing chemical compounds
19. Method according to claim 18, designated by the beads which are a mixture of different synthesis procedures and bear different chemical libraries.
20. Method according to claim 18, designated by a compartmentation of the beads thus forming a patterned master and leading to spatially separated compartmented subarrays on the assay plate.
21. Method according to claim 18, designated by the working steps by which the bead monolayer is formed
21.1. Individualization of the beads
21.2. coating procedure to form mainly monolayer (dip coating, powder coating, centrifugal coating, likewise) and immobilise beads
21.3. combing to remove multilayer beads
21.4. removal of dust
22. Method according to claim 18, designated by a master plate which is pre-coated by a adhesive layer.
23. Method according to claim 6, designated where step 6.4 is realised by the following substeps
23.1. cleavage of orthogonal linker
23.2. diffusion from bead to assay plate
23.3. interaction of the transfer principle with the assay plate surface
23.4. alignment of the chemical compounds due to transfer principle to the assay plate surface
23.5. binding or anchoring of the chemical compounds onto the assay plate surface
24. Method according to claim 23, designated to any method that modifies the transfer principle or the surface of the assay plate to ensure the transfer, preferably hydrophobic modifications of the assay plate
25. Method according to claim 23, designated where the transfer process is performed in a media enclosing the beads and providing itself as diffusion media to transfer the chemical compounds, preferably water or biological buffers.
26. Method according to claim 23, designated that the transfer is performed over single contact zones, by which each bead is generating only one contact zone.
27. Method according to claim 23, designated to the generation of one dot on the assay plate per each bead on the master plate, so that each dot is containing the chemical compounds the bead is bearing.
28. Method according to claim 23, designated to step where multiple assay plate copies of a single master plate are generated
29. Method according to claim 6, designated where the activity of the chemical compound could be tested directly on the assay plate
30. Method according to claim 6, designated to the allocation between bead and spot made by pattern recognition..
31. Method according to claim 30, designated where a marker system is used, to generate an inter pattern which could be used as coordinate system.
32. Method according to claim 30, designated to a marker system which is implemented in the beads and is generating a random ordered pattern that could be used as coordinate system.
33. Method according to claim 31 , designated to any application with any coordinate system which allows the one-to-one allocation between any dot and its progenitor bead and vice versa.
34. Method according to claim 31 , designated to any coordinate system which could be established within a random pattern of dots.
35. Method according to claim 31 , designated to be able to remove or recalculate transfer errors, artefacts and/or geometrical alterations like torsions, sheering or shrinking effects between the master plate and the assay plate to allow again a one-to-one allocation between master plate and assay plate.
36. Method according to claim 6, designated where the analysis of the chemical structure could be made directly from the master plate on the beads which are allocated to positive dots on the assay plates.
37. Method according to claim 1 , designated for the extensive screening of test series for the analysis of chemical or biological interactions between chemical compounds and another given molecule of interest, preferably called target
38. Method according to claim 1 , designated to print several copies from a master plate onto a assay plate.
39. Method according to claim 1 , designated for extensive test series to analyse the biological activity of chemical substances, preferably used in drug-screening, antibody screening or epitope mapping.
40. Method according to claim 1 , designated for applications in the field of biological or biochemical diagnostics.
41. Method according to claim 1 , designated to use centrifugal coating to generate the bead array, either complete or in sub compartments.
Beschreibung  (OCR-Text kann Fehler enthalten)

Method for manufacturing and application of random ordered arrays of chemical compounds

The following invention describes a method for efficient generation and application of random ordered arrays of chemical compounds.

The methods significance is that an array of random ordered chemical substances (print) is generated by a transfer (printing step) from an array of random ordered array of particles (master). The particles are polymer beads which are bearing these chemical substances. Thus the "printed" array is mirror symmetrical to the array bearing the beads. The spatial allocation between the master and the print could be therefore made and each structure on the print could be allocated to its "progenitor" particle on the master.

The methods significance is also that the generation of the print, is independent from the number of transferred chemical substances. This is due to the parallel transfer of all compounds within one transfer step (instead of sequential transfer of chemical compounds in other methods). The printed arrays are mainly being used for biomedical, diagnostic or biochemical assays for biological activity.

The protocols to prove that a chemical compound is biological active and detect it within an assay were changed to the needs and purposes of the random ordered array. The spatial allocation between each chemical compound and its progenitor particle allows the identification of the particle which bears the biological active chemical compound. And thus the identification of the identity of the chemical compounds itself. The allocation is made via intelligent pattern recognition.

The allocation itself is an old technique which first was derived by Riemann 1849 and is used widely with nearly any pattern recognition.

The main claim of this patent is not this pattern recognition. The main claim is the parallel transfer of all chemical compounds for the purpose to generate an array, time independently of the number of chemical compounds.

State of the art

Roughly 20 years microarrays (Ekins, R.P.: Multi-analyte immunoassay. J.Pharm.Biomed.Anal. 1989, 7:155-168. Fodor, S.P.; Read, J. L; Pirrung, M. C; Stryer, L; Lu1 AT. und Solas, D.: Light- directed, spatially addressable parallel chemical synthesis. Science 1991, 251 :767-773) have been derived as logical miniaturisation and parallelisation step from already used assays and arrays. Today microarrays, also named biochips, are used to simplify and parallelise biomedical, biochemical, pharmaceutical or biological tests, processes and assays. The used chemical compounds are manifold like nucleic acids, proteins, peptides, small chemical compounds or sugars, which are interacting either with other molecules, biochemical compounds, cells, microorganisms or whole organs or species. Mainly the activity of a chemical compound is detected as molecular binding event on microarrays. Microarrays consist of a multitude of different spots with each spot bearing one single chemical compound. This high density order of spots allows the parallel detection of activity with a small amount of sample. To prepare such arrays each compound has to be deposited on a predefined position, such that in case of activity from the position of the active spot one could conclude which chemical compound is active.

The preparation or manufacturing of such an array with each chemical compound deposited on a predefined position is, especially for a vast number of chemical compounds quite time and material consuming and is also connected to a high technical effort. Mainly it could be said that the time and material consumption is more than doubling if the number of transferred compounds is doubled.

Today the widely spread techniques to generate such microarrays are the direct synthesis of the chemical compounds on the array surface (US 5,591 ,646), the transfer and immobilisation or synthesis of compounds via photolithography (US 5,412,087, US 5,489,678, WO 03/089900) or the transfer via printing techniques derived from desk jet printers(US 6,079,283, US 6,083,762, US 6,094,966). An overview about the actual state of the art and science is given by the publications of Ng/llag, Jainund Mϋller/Rode. (Ng, J. H. und Hag, L.L.: Biochips beyond DNA: technologies and applications. Biotechnol.Annu. Rev. 2003, 9:1-149. Jain, K.K.: Applications of biochips: From diagnostics to personalized medicine. Curr.Opin.Drug Discov.Devel. 2004, 7:285-289. Mϋller, H.-J. und Rδder, T: Der Experimentator: Microarrays. Spektrum Akademischer Verlag, Heidelberg 2004).

The methods of the combinatorial solid phase synthesis opened the possibility to synthesis millions of different chemical compounds within several days. The basic idea of this method is to synthesise all compounds on microparticles (beads). According to the spit-and-mix synthesis method after the synthesis each bead is bearing exactly one chemical compound. Actual this beads were separated from each other an the chemical compounds are cleaved of the bead with each bead in another vial, thus ensuring that each vial contains only one compound in solution (stock solution). This means a high technological and time consuming effort. From this vials than the chemical compounds are either tested for activity direct within the vial or for performance of a multitude of experiments instead of only one the chemical compounds are transferred onto a microarray or a microtiter plate, and tested there. The transfer of the chemical compounds is made by pipetting the stock solutions of the chemical compounds onto a mircoarray of into a well of a microtiter plate. For both, microarray and microtiter plates, pipetting robots with automatic xyz-pipetting devices are used to transfer the millions of compounds. But still it is a technique which is time consuming and quite a lot of effort.

For a rational and highly efficient performance of a large number of biomedical or diagnostic assays miniaturisation is an ongoing process. Actual for these tests and assays of chemical compounds microtiter plates with 1536 or 3456 wells are available. But this structures are so tiny that a manual dispensing of chemicals isn't practicable any more. And as more physical effect the surface to volume ratio is increasing with the miniaturisation of the wells, which is also increasing the background signal of the assay and also increasing the non-specific binding of the chemical compounds to the surface. These effects undermine the usability of the assays. Therefore the 3456 well plate isn't used so much for assays.

The production of microarrays is mainly limited due to the time of transfer of the chemical compounds. But the printing process takes a minimum limit of time to take up the chemical compound solution and transfer it to the microarrays. To reduce this transfer time multiparallel print heads had been developed with up to 384 printing heads. But still the printing time is increasing with the number of chemical compounds which have to be transferred.

Therefore the idea was raised that the assays could be made directly on bead. The chemical compounds therefore are still bound to the synthesis beads and the assay with small target molecules (other small molecules, proteins, DNA and other bio molecules) were performed by direct mixing of beads with these target molecules. But the polymers of which the synthesis beads are providing also binding sites and unspecific binding to the same molecules which are used for the assay. This leads also to unwanted non-specific binding of the molecules and therefore reduces the yield of the assays. In many cases the non-specific interaction is so much higher than the specific binding of the target molecules that no assay could be performed at all. Only sophisticated techniques circumvent this effect.

For a reduction of the non-specific interaction mixtures of different subcollections of chemical compounds are tested in parallel within one assay. The results of this subcollections could be averaged and recalculated. But the final results are hard to interpret and are only of reduced use for the assay outcome as well as for the conclusion of which chemical compound is how active. Only an averaged activity of the subcollection could be generated. This doesn't lead to a conclusion about which and if there is a special chemical compound with a rather high activity.

Another alternative is the spatial resolved addressing of assays within a liquid-liquid suspension. Within these assays the chemical compounds are coded within the physicochemical attributes of the synthesis beads which could be colour, colour intensity, shape, size or even a barcode system. The most coding systems relay onto a fluorophores (fluorescent colours) which are embedded within the chemical compound carrying beads (US 2005/0106711 , US 2005/0106712). In another method the target molecules are labelled with fluorophores and will also detect special molecules on the bead. Finally both methods have been combined (US 7,011 ,945). But the number of possible combinations and amounts of fluorophores is limited due to resolution of the devices and different colours of fluorophores. Therefore it is possible to code <1 ,000 beads but not to code millions of beads. Normally this assays are performed in a flow-through cytometer and allows the screening of millions of beads, but only could separate into several hundred beads (US 5,981 ,180). Other methods like the use of light-wave guiding cables are also used, but are not so common (US 6,023,540, US 6,266,459). An enhancement of this bead-coded idea is the implementation of fluorescent nanocrystals in a random spatial within the bead. This internal coding allows millions of coding, but lacks the possibility to make a fast screening in a cytometer at the moment. But never the less all these bead based methods have the problem of the non-specific interaction of the bead polymer matrix with the target molecules.

As for all those methods the technical effort is quite high. There have been methods reported to transfer the beads from a liquid phase to a surface via a sticky gelatine surface (WO 2005/016516), electromagnetic fields (US 2007/0231825) chemical or mechanical bindings or forces (US 2002/0051971). In all cases a random order of this chemical compound bearing immobilized beads is made. The identification of the chemical compounds is made in that cases with a bead coding like described before with fluorophores either by CCD-cameras (US 2003/0143542) or spectrometers (US 7,034,941 ) coupled with microscopic readouts. As an application many ultra high throughput and massive parallel sequencing techniques now use such a colour coding (WO 2007/044245). The identification of the bead and the chemical compound is made directly on the surface either before, whilst or after the assay.

Some of these sequencing techniques use a non-bead based method and generate a random distribution of the DNA on the surface.

Except the use in nucleic acid assays (WO 2007/044245), there are no methods described for a random ordered array of beads.

And only for nucleic acids, a method for copying the chemical content of a random ordered array has been described (US 2003/0124594). Similar methods for proteins, peptides and other molecules are not published until now. Until now we don't know any methods that report, the separation of the assay from the beads whilst also separating the identification of the chemical compound on the master whilst making assays on the print. Also a printing of such chemical compounds from a master to the print is not reported. Even if the idea of colour coded beads allows an increasing number of combinations for the coding, this process is still connected with an high effort. All this techniques lack of a simple and easy use and have a high effort of devices. Although that effort they decreased the price per assay. A simplification of the process or the method to generate microarrays would decrease the price dramatically.

This invention therefore comprises to solve the need for an efficient method for the production of arrays directly made from chemical compounds synthesised on beads by combinatorial solid phase synthesis. It solves the complete task from generation of the random ordered bead array (bead plate) with chemical compounds, their transfer onto a microarray (assay plate) via a print process and the performance of a biological assay. The printing step which is generating the assay plate as print from the bead plate could be repeated many times until the beads on the bead plate are depleted from chemical compounds.

It also masters the task, that the transfer process is completely independent from the number of beads or chemical compounds. All chemical compounds will be transferred within one step onto the array. Whilst the transfer process the spatial order is preserved, so that after a successful assay a positive "dot" could be allocated to its progenitor bead. The whole procedure is shown in figure 0.

The invention combines the advantages of a bead based array for the generation of an array, but is than transferring the chemical compounds onto a surface, so that the advantages of a standard mircoarray could be applied whilst performing a biological assay.

The task is now solved by the first time with the described method for production and application of random ordered arrays of chemical compounds, as described hereafter and for example in claim 1. Specific embodiments of the invention are also described in claim 2 to 35. Applications of this method for the performance of drug-screening, biomedical assays and diagnosis are also described in Claims 36 to 41. In one embodiment, the method of the invention comprises the following combination of features to allow the generation of random ordered bead arrays, the transfer from each bead its chemical compound onto a microarrays (one bead generates one dot) and the performance of a biochemical assay. Therefore it comprises the following points

• The synthesis and chemical attributes of the chemical compounds on solid phase synthesis beads. These attributes allow the release of the chemical compounds from bead, the transfer from bead to array surface and the anchoring of them onto the array surface

• The immobilisation of the beads to a monolayer as source for the chemical compounds • The assay plate with the according chemical properties to allow the anchoring and a one bead to on dot transfer of the chemical compounds

• A method to perform a biochemical assay and to detect active substances within a dot

• A routine to allocate each active dot on the assay plate to its progenitor bead on the bead plate.

The whole process is shown in figure 0. The method comprises chemical compounds that are bound to the beads. The chemical compounds are synthesised to have the following principles.

• They contain a chemical protection group which could be orthogonal cleaved to all synthesis steps (principle 1)

• A residue which comprises the transfer of the chemical compounds from bead to the assay plate, as well as for anchoring the chemical compounds onto the assay plate (principle 2)

• A biocompatible spacer, which is separating the different chemical residues from each other so that they don't interfere with each other (principle 3)

Principle 1 to 3 could be introduced before, whilst or after the synthesis. The preferred introduction of this principles are made whilst the solid phase synthesis.

The preferred synthesis of the chemical compounds may be made with classical combinatorial solid phase synthesis the starting molecules are bound onto pre-treated, functionalised beads and sequentially build up to the final chemical compounds. As starting molecule a residue preferably comprises already principle 1. With the use of the combinatorial chemistry and a good planning of the synthesis steps and chemical reactions complex chemical compound libraries with a large number of different chemical compounds could be generated within a week.

After the synthesis the beads from different libraries could be mixed to generate a larger library of chemical compounds. Also beads bearing other compounds could be used as markers for bioassays or to allocate them within the bead matrix on the bead plate. Within this mix of beads all chemical compounds are randomly distributed.

All mixed synthesis beads bear chemical compounds, each comprising principle 1 to 3 but each bearing different chemical parts. This means they behave the same way and comprise all three principles whilst the transfer process, but they have distinct different chemical residues which are important for their activity whilst the biochemical assay.

The beads with the chemical compounds were immobilised onto a bead plate (preferably on a planar plate). The immobilisation procedure allows the formation of a bead monolayer. This could be done (but not only) by a biocompatible non-toxic adhesive layer on the surface of the bead plate. Whilst the transfer process the beads are immobilised in a random ordered manner and form like such a bead array. The procedure to immobilize the beads on the bead plate may include powder coating, dip coating or centrifugal coating. To remove multilayer beads a process may be performed which is called combing and removes simply of the multilayer beads by moving an edge close to the surface of the bead plate.

Finally a bead array of random ordered beads is formed on the bead plate. Each bead bearing a different chemical compound, which have the same chemical residues except the part of which belongs to the chemical library.

For simplification of the deconvolution, the revealing of the structure of the chemical compound, it is preferred to make a mass spectroscopic analysis but other methods for the structural analysis of chemical compounds could also be used.

To apply different sublibraries a compartmentation of the bead plate could be performed. For this purpose a specific mixture or sublibrary could be filled in cavities, which than are used for coating of the bead plate. A microtiter plate could be used as cavity for storage of this sublibraries and for the coating. The transfer again could be made like on the non- compartmented bead plate, but will lead to a compartmented bead plate. This allows sub- compartments and therefore subarrays.

The transfer process from bead plate to assay surface can be performed easily by the skilled person. For example, a planar bead plate is brought in close physical contact to the assay plate, which is assisted or mediated with a medium which is ensuring or enforcing a single contact zone from each bead to the assay plate. Within this zone, later on, the compounds are deposited on the surface of the assay plate and is generating a "dot" which is coated with the chemical compound from its progenitor bead. In a preferred method, water or cell media is used as transfer media.

After the first contact between bead plate and assay plate the chemical compounds are released, preferably by physical, chemical, biological or enzymatic ways like photo cleavage, pH-change or likewise, according to principle 1. With this the chemical compounds are released from the bead and will diffuse within the transfer media to the contact zone onto the assay plate. Principle 2 allows this diffusion, cause it makes the molecules soluble for the transfer, whilst principle 3 allows the anchoring of the compound on the assay plat. Thus after the release the chemical compounds leave the bead and will anchor onto the assay plate forming one single dot from each bead on the assay plate. Such a chemical print is generated from the bead array on the bead plate. Due to the printing process an one-to-one allocation could be made between each bead on the bead plate and each dot on the assay plate.

The contact zone between the bead of the bead plate and the assay plate depends on the materials of the beads, the surface of the assay plate, the size of the bead, the amount and chemical properties of the transfer medium and applied pressure. All this variables have to be optimized to ensure a homogenous transfer from each chemical compound of each bead to each dot on the assay plate. Principle 2 is optimized to allow this transfer. The anchoring of the chemical compounds onto the assay surface could be made by any physico-chemical interaction like covalent, ionic, electrostatic, magnetic, adhesive, adsorptive or ligand-receptor- interactions.

The assay plate generated as print of the bead array is mirror symmetrical in respect to the bead array (figure 1).

Each bead is generating one dot on the surface of the assay plate. Due to the immobilisation of the beads on the bead array the pattern of beads is printed in a one-to-one allocation onto the assay plate. Principle 1 allows the release of the compounds if the print could be performed. Principle 2 allows the transfer and anchors the compound in a manner so that the compounds of a bead are only generating a dot on the assay plate directly underneath the bead (figure 0). The chemical compounds form a dense monolayer in this dot due to the structure of principle 2. Whilst Principle 3 allows now a screening, due to the fact that the biocompatible spacer is optimized to prevent non-specific interactions.

Due to the excessive amount of chemical compounds on each bead the transfer process could be repeated until all chemical compounds are transferred and the beads are empty. The amount of transferred chemical compounds could be controlled by contact time between bead plate and assay plate and by releasing time according to principle 1. This could be used to perform dilutions and will lead to arrays with different compound densities and concentration rows.

Like on any microarray any test that is performable as biochemical assay could be performed on the so generated assay plate.

After a performance of an biomedical or diagnostic assay a positive or negative identified dot could be allocated to its progenitor bead on the bead plate. The allocation is made due to a doping of the beads with beads that are generating a colour on the assay surface, which allows to allocate this pattern to the pattern of the bead plate. This could be made due to pattern recognition and is based on the theorems pronounced by Riemann 1849 about manifolds in geometry.

This marker principle is used to make the allocation. The coloured beads contain coloured chemical compounds bearing principle 1 to 3 like all other beads, but with coloured or fluorescent residues. The coloured beads are mixed to all other beads so that each transfer will generate a fluorescent pattern on each assay plate (like the dark dots in figure 1). According to the allocation of 3 dots to their progenitor beads all beads and dots could be allocated to each other by using mathematical algorithms like, torsion, shrinking and rotation.

After the identification of a positive dot, the according progenitor bead could be allocated. The allocated bead than could be analysed for its chemical compound preferably by MALDI or mass spectroscopy. Than the analysed chemical compound could be determined as positive and therefore active compound.

The invention covers all applications to this method of transfer. The main applications are used whilst biochemical or biological probing, in which large amounts of chemical compounds are tested for binding against peptides, proteins or other molecules. In this screening process the chemical compounds could be any chemical molecules, but are preferably peptides. Especially the analysis of biological activity of the chemical compounds in assays with cell biological samples, ligand-receptor measurements, antibody-binding tests, epitope mapping or likewise tests in field of diagnostics.

The invention is predestined as method in the field of pharmacy for the screening of structure- effect-relationship for drug screening, where the interaction of one single molecule is tested against a large number of binding partners. This binding partners are the chemical compounds which could be transferred by the method comprised by the patent.

The present invention therefore solves for the first time the spatial resolved transfer of a random ordered bead array to a random ordered array of chemical compounds without any pipetting steps and a low technical effort. It also solve the problem of increasing time amount with increasing number of chemical compounds that have to be transferred. It solves as well the problem of screening of bead arrays and of manufacturing of chemical arrays by combining both advantages without the disadvantages. The beads are used to form fast a random ordered bead array, than the printing step allows to generate a normal array and this could be used for normal screening. An one-to-one allocation allows than to identify the active dot and the active bead. So a chemical analysis of the chemical compound from the bead could be made according to the positive dot on the microarray, without any pipetting steps for the transfer of the compounds or the array generation.

The invention interconnects the bead arrays to the surface arrays by a simple printing step which transfers the chemical compounds from each bead to a according single dot on the microarray. Each dot consists of the chemical compounds according to its progenitor bead. The assays could be performed later on the assay plate as on any commercially available microarray. With this printing step any pipetting step for the generation of the microarray is circumvent. This printing step solves the problem that the time amount doesn't increase if the number of chemical compounds is increased. In all other methods a dramatic increase of time consumption is observed. Therefore the time consumption of the transfer step to generate the microarray is independent of the number of transferred chemical compounds.

The transfer process could generate in an ideal way a monomolecular layer of the chemical compounds and therefore a performance of an assay would lead to a significant signal yield, which could also be counted as advantage of the invention.

The preparation of the bead plate could be seen as master, which could be copied multiple times onto an assay plate. Therefore multiple copies of each master could be made. This multiple copying is another advantage of the invention. The transfer process itself is instantaneously and could be done within 20 seconds and as said is completely independent from the number of transferred chemical compounds, used beads or size of assay or bead plate.

In the shown experiments synthesis beads with 20 micron are used to build a bead plate. The transfer of this random ordered bead array onto a standard microscope slide can lead to a density of at least 250,000 Beads per cmΛ2 and at least 250,000 dots per cmΛ2 on the microarray. This is comparable at least to commercially available microarrays.

The spatial resolution of dots after performance of a biochemical assay was about 10% of the bead diameter which means 2 microns or less.

The random order of the chemical compounds on the bead plate and the assay plate, the extremely fast production and transfer time, together with the possibility of multiple copies allow it to make multiple and reproducible tests with the generated microarrays. This allows to remove and recalculate random artefacts or variability whilst the assay performance and ensures a robust and stable assay. This is another advantage of this invention.

Due to the fast and easy control of the release condition whilst the transfer the density, dot size and concentration of the chemical compounds on the assay plate could be immediately controlled. This invention comprises therefore the fast production of a variety of arrays which isn't possible with all the other methods. No other method allows this easy control.

More details and special preferred applications of this invention together with more attributes and advantages of this invention are shown in the following description of the application examples in combination with the claims, subclaims, invention attributes and any combination of them. The application examples are only to illustrate the invention and the invention is not limited to these examples. The following figures show:

Figure O Schematic view of the transfer process a) formation of a bead monolayer on the master plate b) physical contact between the master and the assay plate c) release of chemical compounds from bead (principle 1 ) d) transfer from bead and anchoring on the assay plate surfaces of the molecules (principle 2) e) removal of master plate and repeated printing f) performance of biochemical assay with less background due to spacing between anchoring and screening part of the chemical compounds (principle 3) g) allocation of positive dots to their progenitor beads

Figure 1 Schematic view of the random pattern on the bead plate (right) and the corresponding mirror symmetrical pattern on the assay plate after performance of an biological assay. Positive dots are shown in black.

Figure 2 Synthesis scheme for the synthesis of the photo cleavable compound A-

Bromomethyl-3-nitrobenzoic acid on a synthesis bead followed by methylamylation.

Figure 3 Synthesis scheme for photolytic cleavage of synthesised chemical compounds from synthesis bead with 4-Bromomethyl-3-nitrobenzoic acid.

Figure 4 Chemical structure of Pam3Cys-OH.

Figure 5 Chemical structure of immobilised chemical compound bearing all 3 principles

Figure 6 Chemical structure of a intermediate after coupling of Pam3Cys-OH and before further coupling of biological active substances after spacing with β-Alanine.

Figure 7 Synthesis scheme for coupling of photo cleavable linker (step 1 ).

Figure 8 Synthesis scheme for methylamidation of photo cleavable linker (step 2).

Figure 9 Synthesis scheme for coupling of Fmoc-aminoacid (Step 3).

Figure 10 Synthesis scheme for deprotection of Fmoc-aminoacid (Step 4).

Figure 11 Synthesis scheme for photolytic cleavage of conjugates (Step 7).

Figure 12 ESI-MS OfPBm3CyS-SK3K(PA-PA-ACa-ACa-BiOtIn)-NCH3.

Figure 13 ESI-MS of Pam3Cys-SK3K(βA-βA-Dipeptide)-NCH3-library with 400 different chemical compounds (aan=proteinogenic amino acid). Figure 14 Enlargement of Figure 13 with assignment of some compounds of the peptide collection

Figure 15 Microscopy picture of bead array generated by powder coating and combing with synthesis beads (020 μm, image size 780x600 μm).

Figure 16 Microscopy picture of bead array generated by dip coating and combing with synthesis beads (020 μm, image size 2600x2000 μm).

Figure 17 Microscopy picture of bead array generated by centrifugal coating and combing with synthesis beads (020 μm, image size 780 x600 μm).

Figure 18 Scheme about the three principles of the chemical compound bound to the synthesis bead

Figure 19 Scheme about immobilised bead on the bead plate. The immobilisation is shown on the example of a adhesive thin film.

Figure 20 Scheme of the transfer step of chemical compounds. The transfer medium comprises a contact zone which determines the dot which is generated after the transfer. The release of the compounds is made in this example by radiation.

Figure 21 Scheme of the build dot after the transfer step. Each bead is transferred to one single dot. So that each beads bearing only on chemical compound is generating only one dot with exactly one chemical compound

Figure 22 Chemical structure of Pam3Cys-SK3K(Carboxyfluorescein)-NCH3. Figure 23 Chemical structure of Pam3Cys-SK3K(Aca-Tetramethylrhodamin)-NCH3.

Figure 24 Microscopic picture of the generated dot pattern of fluorescent beads (upper left picture), the first copy as array (lower left) and a second copy as array (lower right).

Overlay of array on assay plate and beads on bead plate. (Image size

520 x400 μm).

In the overlay rhodamine-beads are bright grey, fluorescein beads are white and non labelled beads are dark grey, whilst the arrays on the assay plates only show the fluorescein in green and the rhodamine in red. This is due to the non-specific fluorescence of the beads which normally is a great hindrance for the assays.

Figure 25 Microscope images of sub-compartments within the bead arrays of the master plate. The master plate was manufactured with centrifugal coating.

Figure 26 Master plate with 65 sub-compartments on the storage plate. Each compartment contains another chemical sub library and doping with different amounts of fluorophores, wielding in different colours.

Figure 27 ESI-MS spectra of a biotinylated compound Figure 28 Master plate with massive doping of rhodamine (red) or fluorescein (green) bearing beads. Cross contaminations after printing processes lead to yellow colour. The arrows show missing beads on the master plate. The circles mark special positions of beads which are allocated to the assay plate shown in figure 29. Especially the larger bead (lower right circle) generates a larger dot in figure 29.

Figure 29 Assay plate generated from master plate in figure 28. Clearly visible that there is no cross contamination between the dots and beads (white arrows) could stick to the surface, but could be removed by washing steps. A larger dot was generated (lower right circle) by a larger bead (see figure 28)

Figure 30 Performance of a bioassay by binding of streptavidine (green) onto the dots of an assay plate. Before the incubation (left) of streptavidine only the pattern of the red dots (doping with red labelled beads on the master plate) is visible and a contamination (green). After the incubation (right) the dots which bound streptavidine are clearly visible.

Figure 31 Left side is before, right after binding assay. All assay plates bear a red and a green pattern as 100% control and for establishment of an internal coordinate system to find back the same position within the assay plate. From top to bottom different ratios of red and green labelled streptavidine has been used, (green to red ratio: top 3:1 ; 1 :1 ; 1 :2 and bottom 1 :4).

Figure 32 Example for an allocation. The bead array on the master plate (upper left, transmission light micrography) was printed onto an assay plate (upper right, fluorescence microscopic image). An overlay of these two pictures showed a perfect fit (lower left). Also a fluorescence image for green beads of the master plate showed that the beads which are illuminate are over green dots on the assay plate and in any case not on any red dot (lower right).

Figure 33 Another example of allocation. The master plate (upper left) was printed several times. Due to different process parameters the generated arrays on the assay plates show differences. But in total the pattern of the red dots could be allocated to each other easily by eye. In cases of larger images a software is needed to allocate to each dot its progenitor bead. But this image is only a example to show that the allocation works fine.

Figure 34 Regaining of a bead by laser microdissection. First (upper left) a bead is marked and than cut out by a laser. A final laser impulse is shooting the bead out of the area of the plate (upper right) into a eppendorf cup (lower left). The regained bead was a fluorescent one. Its fluorescence was directly proven in the cup (lower right). Application examples

Example A Applications of the three principles

Pertaining to the invention a realisation of a synthesis strategy was shown as example to allow the transfer as described before. Whilst the synthesis a multitude of chemical functions could also be implemented.

Principle 1 - orthogonal cleavable linker

Basic principle is that this linker is cleavable and reacts orthogonal to all other deprotection steps and synthesis conditions. The NH2-funktionality of the photo cleavable 4-Bromomethyl- 3-nitrobenzoic acid bound to the synthesis bead is substituted via methylamine to an amid group and allows than the coupling or synthesis of any chemical compound. Therefore the classical solid phase synthesis with Fmoc-, Boc- and Aloe- together with tBu- protection groups could be applied. After finished synthesis and dispersing the beads in a random ordered array onto the bead plate the synthesised chemical compounds could be released by a well directed radiation with UV-light. The applied dose (product of intensity x time) determines the amount of cleaved and therefore released chemical compound. The photo cleavable linker is stable during the whole synthesis process. Due to the UV light the covalent binding breaks as such that the released chemical compound bears only an additional methyl group. Due to the difference in size of whole molecule and methyl group it could be suggested that it wont interfere with biomedical assays. The synthesis scheme is shown in figure 2, the cleavage reaction in figure 3.

principle 2 - transfer principle

This principle bears the transfer and the anchoring. The transfer parts have to be adapted onto the used transfer media and the anchor part has to be adapted onto the surface the compound should be anchored.

In this application example a hydrophilic transfer media (water) is used and the anchoring is made by a lipophilic (hydrophobic biomembrane like surface). Therefore the used compound was Pam3Cys-OH (Figure 4) of the company EMC microcollections GmbH was elongated with serine followed by 4 lysines. This molecule provides a lipophilic part (the fatty acids) which will anchor the molecule on the hydrophobic surface and a hydrophilic part (the lysines, K4) which allow the compound be transferred from the bead onto the surface via the hydrophilic solution. The hydrophilicity of the liquid and the hydrophobicity of the surface determine the contact angle of the liquid and therefore the size of the contact area. The assay plate was functionalised with trimethoxypalmitoylsilan to gain a lipophilic surface for the Pam3 anchor of the chemical compound. Contact angles have been between 100° and 115° against pure water

The Pam3Cys-compound anchors instantaneously onto this hydrophobic surface by hydrophilic interactions and build up a molecular monolayer. A density of 0.8 nmΛ2 per molecule could be observed. The adhesion of this molecules to this surface was high enough that assays could be performed in hydrous solutions. Only with a mixture of organic solvents together with heat and mechanical shear forces the compound could be removed.

As other anchoring and transfer methods ionic, polar, ligand-receptor and even magnetic techniques are thinkable. Other techniques could also be applied.

Principle 3 - biocompatible spacer

This principle works closely together with principle 2. And like in this example both principles could be combined within one chemical residue. Principle 3 is providing chemical inertness in biochemical tests and assays. This means it separates the two other principles from the later on performed assay as "simple" spacer. It prevents the toxicity of the Pam3Cys-conjugates by elongate it with 4 lysines. In other cases this part could be elongated to reduce again toxicity or other side effects of the principle 1 or 2. In our example we additionally added 2 molecules of β-Alanine.

The whole example conjugate is shown in Figure 5. The biological active part of the chemical compound could be synthesised via traditional solid phase synthesis. All chemical compounds are identical according to principle 1 to 3. Therefore they reacted in the same way for the transfer steps. Mainly not influenced by the different biological active parts of the molecules. This decouples the transfer from the screening compound in terms of transferability.

Example B - Solid phase synthesis of the basic compound

TentaGel S-NH2 was used as synthesis beads. First the photo cleavable linker 4-Bromomethyl- 3-nitrobenzoic acid was coupled (Step 1) and than substituted with methylamine (Step 2) and finalized principle 1. Than the principle 2 and parts of principle 3 were synthesised by stepwise coupling of Fmoc-Lys(Aloc)-OH, 3 molecules of Fmoc-Lys(Boc)-OH and a Fmoc-Ser(tBu)-OH. The Fmoc protection group was cleaved of after each coupling step respectively. (Step 3 and 4). After final cleavage of Fmoc-protection group Pam3Cys-OH was coupled as lipophilic anchor and finished principle 2. this basic conjugate is shown in Figure 6. After cleavage of the Aloe- protection group (Step 5) of the first lysine 2 molecules of Fmoc-β-Alanine-OH (finalising principle 2) and several biological active conjugates were coupled to the sidegroup of this lysine. Figure 5 shows the final basis conjugate after the cleavage of the Boc- all other protection groups (Step 6).

After photo cleavage (Step 7) and separation from the beads the resulting chemical compound showed hydrophobic attributes but was soluble in water with > 1 mg/ml.

Each synthesis step was monitored with HPLC-ESI-MS. Therefore the protection groups were cleaved (Step 6) and than a photo cleavage was performed (Step 7). The single synthesis steps are explained in detail as followed. All resin used amounts are calculated to 1 gram of starting resin.

Step 1 - Coupling of photo cleavable linker (Figure 2 and 7)

1 g TentaGel S-NH2-resin (Rapp Polymer GmbH; loading capacity 0,25 mmol/g; bead diameter 20 μm) was washed 4 times with 5 ml DMF each.

4-Bromomethyl-3-nitrobenzoic acid (3 Equiv.; 0,195 g; 0,75 mmol) and HOBt-H2O (3 Equiv.;

0,115 g; 0,75 mmol) was dissolved in 12,5 ml DMF, DIC (3 Equiv.; 1 ,16 ml; 7,5 mmol) were added and the solution was stirred for 30 min at 2O0C

The washed resin was incubated with this solution and shook for 16 h at 200C.

The resin was filtered, washed (each 6 x 5 ml DMF, DCM, MeOH) and dried under vacuum.

Accomplished reaction was proven with Kaiser-Ninhydrin-Test.

Step 2 - Methylamidation of photo cleavable linker (Figure 2 and 8)

To the dry resin was reconstituted inδ ml water free DMSO and 8 M Methyl-amine in EtOH

(20 Equiv.; 0,575 ml; 4,6 mmol) was added.

The mixture was shaken at 200C for 3-3,5 h.

The resin was filtered and washed (3 x 8 ml DMSO, 6 x 8 ml DMF, 6 x 8 ml DCM, 6 x 8 ml

MeOH, 3x8 ml DMF).

The accomplished reaction was proven by a positive Chloranil-Test.

Step 3 - Coupling of Fmoc-amino acids (Figure 9)

3 Equiv. of any Fmoc-aminoacid or any chemical compound bearing a carboxy-group like Pam3Cys-OH, was dissolved together with HOBt H2O (3 Equiv.; 0,115g; 0,75 mmol) in 8 ml DMF. To this mixture DIC (3 Equiv.; 1 ,16 ml; 7,5mmol) was added. In case of Pam3Cys-OH a mixture of DCM and DMF (1 :1 v:v) was used as solvent to enhance the solubility of this compound.

The final mixture was added to the washed resin and incubated under shaking for at least 45 min. In cases of larger or low soluble molecules or fatty acids the incubation time could be elongated for several hours. The resin was filtered and washed with 8 x 8 ml DMF.

The completeness of the reaction was proven by a negative Chloranil-test.

Step 4 - Cleavage of the Fmoc -protection group (Figure 10)

A solution of piperidin in DMF (8 ml; 30% v:v) was added to the resin. The resin was incubated for 20 min under shaking.

The resin was filtered, washed once with 8 ml DMF and again a piperidin solution in DMF

(8 ml; 30% v:v) was added and incubated for 15 min under shaking.

The resin was filtered and washed with 6 x 8 ml DMF.

The complete reaction was monitored with a Kaiser-Ninhydrin-test. In case of negative test result the cleavage was repeated and the incubation times were doubled.

Step 5 - Cleavage of Aloe-protection group

Pd(PPh3)4 (1 ,1 Equiv.) was dissolved in 10 ml DCM together with 5% acidic acid and 2,5%

Morpholin.

This solution was added to resin, which was first washed 5 times with DCM and the mixture was incubated under shaking for at least 6 h.

The resin was filtered and washed 5 times each with DCM added with 5% acidic acid, pure

DCM, DMF and DCM again.

The completeness of the reaction was proven by a Kaiser-Ninhydrin-Test.

Step 6 - Cleavage of Boc and other side-chain protection groups

8 ml of a 95% TFA and 5% H2O (v:v) mixture was added to the resin and incubated for 40 to

60 min under shaking.

The resin was filtered and washed with 8 x 8 ml DMF.

The completeness of the reaction was proven by a Kaiser-Ninhydrin-Test.

Step 7 - Photolytic cleavage of chemical compounds from bead (Figure 11)

For monitoring of the chemical reaction and as proof that the chemical compound was synthesised 5 mg resin was taken and solved in 1 ml 80% tert. Butylalcohol / water. The mixture was shaken for 10 min and filtered.

The resin was resolved in 1 ml 80% tert. Butylalcohol /water again. This mixture than is radiated for 90 min with UV-light at a wavelength of 365 nm.

The resin was filtered and the filtrate was kept for storage. Again the resin was solved in 1 ml

80% tert. Butyialcohol /water and radiated again for 30 min with UV-light.

Again the resin was filtered and the filtrate was stored away again.

The resin was than incubated with 400 μl ethanol, incubated for 3 min and than filtrated again.

Again the filtrate was stored. All filtrates are now combined together, shock frozen and lyophilised.

The obtained colourless powder was used for further analysis.

Example C - Coupling of biotin to the basic chemical structure

According to example B the synthesised basic chemical compound was elongated with biotin. Biotin was used representative for any potential biochemical active compound and also representative for any chemical residue that could be coupled onto the basic chemical compound.

Coupling reaction

For the synthesis 50 mg resin bearing the basic chemical compound (Figure 5) was used. As spacer (principle 3) 2 molecules β-Alanine and 2 molecules of ε-Aminocaproic acid were coupled onto the Aloe-protected lysine of the basic chemical compound. Finally a biotin was coupled to this conjugate.

After finalised synthesis all side chain protection were cleaved off (Steps 3 to 6 in example B). 10 mg of the resin with the final product were used for photolytic cleavage. To regain a maximum of product the procedure described in example B step 7 was repeated 4 times. Finally 3.2 mg of the product Pam3Cys-SK3K(βA-βA-Aca-Aca-Biotin)-NCH3 could be retained from the beads.

Analysis of reaction product

A small amount if the Pam3Cys-SK3K(βA-βA-Aca-Aca-Biotin)-NCH3was solved in water and analysed via HPLC-ESI-MS.

The according mass spectra is shown in figure 11 (calculated mass of this test compound was 2118 g/mol). Representative peaks are shown and their masses are assigned to the masses and the structures

707,1 [MHs]3+ main product

1059,7 [MH2]2+ main product

712,5 [MH3J3+ oxidised sulphur atom with one oxygen

1067,8 [MH2]2+ oxidised sulphur atom with one oxygen

717,6 [MH3]3+ oxidised sulphur atom with two oxygen

1075,5 [MH2I2+ oxidised sulphur atom with two oxygen

All peaks belong to the expected main product. The oxidised products are generated due to the excessive use of UV-light and are a known side product. Thus it is proven that the product was generated from the basic compound. Example D - Synthesis of a combinatorial library on the basic compound

Based on combinatorial chemistry a standard split-and-mix-synthesis was made with the beads bearing the basic compound (figure 5). First 2 molecules of β-Alanine were added to the side- chain of the lysine to elongate the molecule and ensure principle 3. Than with 2 split-and-mix steps a dipeptide library was generated with the 20 proteinogenic amino acids each. This is leading to 400 different chemical compounds.

8 mg resin from this library was photolytic cleaved of and regained as lyophilisate. The according mass spectra of the analyis (Figure 12 and 13) show clearly the library. The peaks with the highes and lowest masses have been assigned to the compounds and the according dipeptide. Also the most abundant peaks have also been assigned to the according dipeptide. But these peaks also show that several isobar compounds (peptides with the same masses) have been generated. The results are expected according to split-and-mix synthesis.

Example E - Manufacturing of a bead layer on the master plate

One main part of the invention is to generate a primary array of beads. To remain the spatial order of this random ordered array the beads on the master plate have to be immobilised. A monolayer would be preferred finally. As master plate a plastic polymer on which an adhesive thin film has been coated was used. The thin film is thinner than the radius of the beads. The adhesive layer is UV- permeable and biological inert (in means that it doesn't produce nonspecific binding in a biochemical assay). For the generation of an immobilised bead monolayer the following 4 steps have been performed.

Specification of the synthesis beads

The synthesis beads bearing the chemical compounds show in a dry and non-swollen state a uniform size and shape of a 20 micron diameter sphere. 1 gram of this resin consists of roughly 240 million beads with a loading capacity of 1 picomol molecules of each chemical compound. This means that each bead could carry 6-10'11 molecules. All beads are uniform in means of shape, size, chemical functionalisation, loading capacity and surface coating. For the purpose of peptide chemistry a NH2-functionalisation was used in this case.

Singularisation of the beads

To transfer the beads onto the master plate and into a bead monolayer firstly the beads have to be "singularised". This means that each bead has to be a single bead and does not stick together with others. This is a prerequisite condition for the monolayer formation. For the singularisation the beads have been solved in 600C warm water free tert. Butylalcohol and were sonicated for 20 min in an ultrasonic bath. Thereafter the suspension was shaken for another 20 min, at a temperature above 4O0C. In case that no homogenous suspension could be generated the beads were filtered and resuspended in n-Methyl-pyrrolidon and shaken for 1 h, washed than 3 times in diethyl ether. Than again treated with the 600C warm tert. Butylalcohol.

As soon as a homogenous suspension was generated the bead solution was shock frosted in liquid nitrogen and than lyophilised at -800C and 0,5 mbar until a fine powder was gained, with an amount of less than 0,5 %o non-singled beads.

Transfer of the beads onto a master plate For this step three different coating methods have proven as successful.

Powder coating is a widely used technique and allows coating of the master plate very easy. The master plate is put in the upper part of the coating device and the powder is blown up from the bottom of the device. Beads which are fast enough to penetrate the adhesive layer penetrate it and will stick into it. Whilst beads which are to slow will only touch it and fall back into the containment to the other beads and will get blown up again.

Dip coating is another widely used technique. It could be used to coat the master plate but it is less efficient like the powder coating and dorms more multilayers than monolayers. In case of dip coating the master plate is simply dipped into an excess of beads and shaken. The movement together with the dipping ensured that the beads would stick onto the master plate. Due to electrostatic charging multilayers could also be generated. In that case the multilayers have been removed with a brush.

Centrifugal coating is a coating principle developed for this invention. It is not know to the author if there had been a similar development elsewhere. Even if this coating principle is not of importance for the invention, it allows making sub compartments within the bead layer on the master plate, preferably for each compartment a sub library from the synthesis is used. The working principle is shown in figure 30.

The singularised beads were put into a compartment and the compartment is sealed with the master plate. The adhesive thin film will seal the compartment. Within a first centrifugal step the beads are centrifuged into the adhesive layer. Than the compartment is taken out and put back into the centrifuge in a reversed position. With the next centrifugation step the beads are forced back into the compartment. Only the beads which are stick into the adhesive layer will stay on the master plate. At the end the master plate is removed. The penetration depth of the beads into the adhesive layer could be controlled by the revolutions per minute of the centrifuge and the centrifugal time. Combing 1 : Final immobilisation of the beads on the master plate

Microscopy images showed that not all beads have been brought into a nice order in means of penetration depth into the adhesive thin film or multilayers. To ensure a bead monolayer a technique called combing was used. To bring all beads into the same penetration depth a metal edge was carried in a defined distance over the master plate surface. The angle (in direction of the edge movement) between the edge and the master plate was less than 30°, so that the beads have been pressed into the adhesive layer. This step was closing non filled structures with multilayer beads and ensured that all beads stuck into the adhesive.

Combing 2: Removal of non-immobilised beads

Now the metal edge was moved again over the master plate in an angle larger than 135°. In this case now like a snowplough all beads which are higher than the given distance between master plate surface and the metal edge are carried away by the metal edge.

Finally the master plate was treated with an gas blow to remove dust and non-adhered beads. The generated master plate than was a bead monolayer array with nearly no multilayer beads.

Comparison of the coating techniques

All 3 techniques are sufficient to generate bead monolayers. Powder coating and dip coating are generated high density bead monolayers (Figure 14 and 15). Centrifugal coating also allowed a monolayer with a less high density (figure 16), but with the possibility to generate compartments of different sub libraries, which hasn't been possible with the other techniques. The size and shape of the master plate is for all 3 coating techniques not limited.

Example F -Transfer of the chemical compounds from the beads of the master plate into a array onto the assay plate

Exemplary for the screening compounds a chemical structure was designed and synthesised bearing the photo cleavable linker 4-Bromomethyl-3-nitrobenzoic acid which was coupled to the amino group of the synthesis beads (principle 1). As bioinert spacer the pentapeptide Serin- Lysin-Lysin-Lysin-Lysin with a side chain elongation of β-Alanin-β-Alanin was made (principle 3). For the anchoring on the assay plate the liphophilic group Pam3Cys-OH was used (principle 2). The pentapeptide Serin-Lysin-Lysin-Lysin-Lysin also enhanced the solubility of the molecule in water and counts therefore also to principle 2. As assay plate a microscope slide was used after silanisation with trimethoxypalmitoylsilan performing a hydrophobic surface.

Bead arrays generated like described in example E were used as master plates with a size of a microscope slide. The coated area was 24 x 70 mm with roughly 4,000,000 beads per master plate. The beads were wetted with water. An excess of water was removed with a gas blow. Than the master plate was brought in a close physical contact with the hydrophobic assay plate. The hydrophobic coating of the assay plate ensures that a small singular contact area is generated. Each bead is generating exactly one contact area in the shape of a circular dot.

By radiation with UV-light (365 nm) the chemical compounds were released from the bead by photolytic cleavage of the linker (principle 1). The chemical compounds diffuse within the liquid layer and anchor instantaneously in an aligned order on the hydrophobic assay plate (principle 2). The spacer within the molecule (principle 3) now ensures that the anchor group sticks to the surface whilst the screening compound part is directed away from the surface and therefore will be exposed in a binding assay.

After removal of the bead carrying master plate an array of dots bearing the chemical compounds remain on the assay plate. The pattern of the dots could be allocated to the bead pattern due to a doping with beads bearing a fluorescent labelled compound.

Due to the fact that the beads have a chemical loading capacity in the range of a picomol and a dot only consumes 200 to 300 attomol there is enough material to generate (mathematically) more than 1000 copies from each master plate. Several copies (up to 20) have been made manually. By changes in the illumination time for the cleavage the amount of released chemical compounds could be determined. In a efficient printing step only the amount of molecules which could be anchored on a dot will be released.

Example G - Evaluation of transfer step

Visual control by transfer of fluorescent chemical compounds

As well for internal control of a successful transfer as for the use as a internal pattern and therefore coordinate system a mixture of beads bearing a dipeptide library was doped with beads bearing either Carboxyfluorescein (figure 22) or a rhodamine (figure 23) or a biotin (figure 28 and 29). With this bead set master plates have been generated like described in example E and like in example F described transferred as chemical copy onto several assay plates. The transfer was made manually, but even than the copies generated showed a similar pattern. An allocation between each pattern of the arrays on each assay plate could be made with the bead array on the master plate (figure 28 and 29 as well as 32 and 33).

Performance of a biochemical assay on the array of the assay plate

To show a biochemical assay one of the best understood binding pairs in biochemistry was used, biotin and streptavidine. Biotin is a small molecule which could easily coupled to a chemical compound (figure 27), whilst streptavidine is used widely in many biochemical assays and staining steps. Therefore a master plate has been generated with different beads (non-biotinylated, biotinylated and red fluorescent). The red fluorescent beads generate a pattern of red fluorescent dots on the surface of the assay plate (figure 30 left). This pattern allows the recovery of this position after performance of assay. Before the assay only the red fluorescence is visible (figure 30 left). After the assay the red pattern has slightly changed due to washing effects but could be still allocated to the original pattern. But now a green fluorescence occurred (figure 30 right) as a significant sign for binding of green labelled streptavidine. Thus it could be proven that it is possible to perform a biochemical binding assay on the assay plate surfaces.

Performance of a ratiometric assay

For DNA-diagnostics a ratiometric assay is a commonly used technique. Within this test two different samples were used. One sample is marked with a green, the other with a red fluorophore and than mixed. The mixture is poured onto a microarray and both samples than compete with each other for binding on the array. In cases that both samples bind equally strong to the spot 50% is green and 50% is red, which is resulting in a yellow colour. In cases that one sample binds stronger the colour will shift to that of the strongest binder. Such an assay could be simulated by using a known pair of molecular binders. In this case as chemical compound a biotin was used. Streptavidin is binding biotin. Therefore a amount of streptavidine was labelled green and another red. A mixture of both streptavidine derivatives was made in different ratios.

Assay plates have been generated containing a biontinylated compound and a doping with red and green labelled compounds. Before the binding only this pattern of red and green dots is visible (figure 31 left). After the incubation with the streptavidine mixture the before non- visible dots are now fluorescent, according to the ratio of the streptavidines to each other, in a different colour (figure 31 right). Thus is proving that this assay could be used as ratiometric assay as well.

One-to-one allocation between dots on the assay plate to beads on the carrier plate

To generate an internal pattern all master plates were doped with a small amount of rhodamine labelled beads. This beads bearing a chemical compound which shows a bright right fluorescence. This compound is transferred like the other chemical compounds from all other beads. The pattern of the red fluorescence on the assay plate could therefore be compared to each other. If it is possible to find 3 dots and their according beads all other beads could be allocated to each other. A sample of such an allocation is shown in figure 32 and 33. Regaining of a single bead

For screening purposes it is of interest to regain the bead from the master plate which is allocated to a positive dot on the assay plate. This could be made with laser microdissection. Like from a cell tissue the polymer of the master plate could be cut out with the laser and the bead is catapulted out into a compartment like an eppendorf cup. The sequence of marking, cutting and the regained bead in the cup is shown in figure 34 The bead could be used like any other for analysis.

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Referenziert von
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