PATTERNED, DENDRIMERIC SUBSTRATE SURFACES AND PRODUCTION AND USE THEREOF

Abstract

The present invention relates to a patterned substrate comprising first regions having first dendrimer structures and second regions having second dendrimer structures on a surface of the substrate, as well as a to method for manufacturing a patterned substrate and the use of a patterned substrate for the chemical synthesis of a chemical synthesis product, as a characterizing platform and/or as a platform for cell treatment and/or cell cultivation.

Claims

1. A patterned substrate comprising first regions and second regions on a surface of the substrate, wherein the first regions have first dendrimer structures and the second regions have second dendrimer structures; the dendrimer structures are each covalently connected with the substrate surface; the second regions enclose the first regions; and the second dendrimer structures have at least one structural element different from the structural elements of the first dendrimer structures.

2. The patterned substrate according to claim 1, wherein the dendrimer structures have at least two successive repeating units and the repeating units each have at least two branching points independently from each other.

3. The patterned substrate according to claim 1, wherein the dendrimer structures each have an alkylene silyl group, and the dendrimer structures are connected with the substrate surface via the respective alkylene silyl group.

4. The patterned substrate according to claim 3, wherein the alkylene silyl group has a structure having the following general formula (1) ##STR00016## wherein R.sup.1 and R.sup.2 are independently from each other selected from the group consisting of an alkyl group, a cycloalkyl group, an aryl group, a heteroaryl group, -OA, wherein A is independently from each other selected from the group consisting of an alkyl group, a cycloalkyl group, an aryl group, and a heteroaryl group; and n is an integer from 0 to 6.

5. The patterned substrate according to claim 2, wherein the first dendrimer structures and the second dendrimer structures each have repeating units having the following general formula (2) or (3) ##STR00017## wherein X and Z are independently from each other NR.sup.3 or O, wherein each R.sup.3 is independently from each other selected from the group consisting of hydrogen, an alkyl group, a cycloalkyl group, an aryl group, and a heteroaryl group; and m is an integer of at least 1.

6. The patterned substrate according to claim 1, wherein the first dendrimer structures have terminal groups having the following general formula (4) or (5) ##STR00018## wherein X and Z are independently from each other NR.sup.3 or O, wherein each R.sup.3 is independently from each other selected from the group consisting of hydrogen, an alkyl group, a cycloalkyl group, an aryl group, and a heteroaryl group; ##STR00019## wherein R.sup.4 is selected from the group consisting of -NG.sup.1G.sup.2, —NO.sub.2, —CN, -OG.sup.3, —C(O)G.sup.4, —C(O)NG.sup.5G.sup.6, -COOG.sup.7 and —SO.sub.3G.sup.8, wherein G.sup.1 to G.sup.8 are independently from each other selected from the group consisting of hydrogen, an alkyl group, a cycloalkyl group, an aryl group, and a heteroaryl group; and o is an integer from 1 to 6.

7. The patterned substrate according to claim 1, wherein the second dendrimer structures have terminal groups having the following general formula (6) ##STR00020## wherein p is an integer from 0 to 10 and q is an integer from 3 to 15.

8. The patterned substrate according to claim 1, wherein the surface of the substrate having the first and second regions has an electrically conductive material.

9. A method for manufacturing a patterned substrate according to claim 1, comprising applying second dendrimer structures in second regions and first dendrimer structures in first regions on a surface of the substrate, wherein the dendrimer structures are each covalently connected with the substrate surface; the second regions enclose the first regions; and the second dendrimer structures have at least one structural element different from the structural elements of the first dendrimer structures.

10. The method according to claim 9, comprising the steps of (a) providing a substrate, which comprises a surface having hydroxyl groups or silanol groups; (b) reacting the hydroxyl groups or silanol groups of this surface with an alkenyl silane having the following general formula (12) to form alkenyl silyl groups on the surface ##STR00021## wherein R.sup.1 and R.sup.2 are independently from each other selected from the group consisting of an alkyl group, a cycloalkyl group, an aryl group, a heteroaryl group, -OA, wherein A is independently from each other selected from the group consisting of an alkyl group, a cycloalkyl group, an aryl group, and a heteroaryl group; R.sup.5 is a halogen or -OQ, wherein Q is selected from the group consisting of an alkyl group, a cycloalkyl group, an aryl group, and a heteroaryl group; and n is an integer from 0 to 6; (c) reacting with a thiol compound having the following general formula (7) ##STR00022## wherein X and Z are independently from each other NR.sup.3 or 0, wherein each R.sup.3 is independently from each other selected from the group consisting of hydrogen, an alkyl group, a cycloalkyl group, an aryl group, and a heteroaryl group; (d) reacting with a carboxylic acid having the following general formula (8) or (9) ##STR00023## wherein m is an integer of at least 1; (e) at least 1 time repeating the steps (c) and (d) in combination; (f) selective reacting of a part of the alkenyl or alkynyl groups, which were obtained from the last conducting of step (d), either with (i) a thiol compound having the following general formula (10) to obtain second dendrimer structures ##STR00024## wherein p is an integer from 0 to 10 and q is an integer from 3 to 15; or with (ii) a thiol compound having the general formula (7) or a thiol compound having the following general formula (11) to obtain first dendrimer structures ##STR00025## wherein R.sup.4 is selected from the group consisting of -NG.sup.1G.sup.2, —NO.sub.2, —CN, OG.sup.3, —C(O)G.sup.4, —C(O)NG.sup.5G.sup.6, -COOG.sup.7 and —SO.sub.3G.sup.8, wherein G.sup.1 to G.sup.8 are independently from each other selected from the group consisting of hydrogen, an alkyl group, a cycloalkyl group, an aryl group, and a heteroaryl group; and o is an integer from 1 to 6; (g) reacting the remaining alkenyl or alkynyl groups either with (i) a thiol compound having the general formula (7) or a thiol compound having the general formula (11) to obtain first dendrimer structures in case second dendrimer structures were formed in step (f); or with (ii) a thiol compound having the general formula (10) to obtain second dendrimer structures in case first dendrimer structures were formed in step (f).

11. The method according to claim 10, wherein step (f) comprises the following steps (f1) to (f4): (f1) applying the corresponding thiol compound on the surface having alkenyl or alkynyl groups; (f2) covering the surface having the thiol compound with a photomask; (f3) irradiating the surface having the thiol compound and the photomask with UV light; and (f4) removing the photomask.

12. The method according to claim 10, wherein step (g) comprises the following steps (g1) and (g2): (g1) applying the corresponding thiol compound on the surface having the remaining alkenyl or alkynyl groups; and (g2) irradiating the surface having the thiol compound with UV light.

13. A method for the chemical synthesis of a chemical synthesis product, comprising using a patterned substrate according to claim 1 in the method as a characterizing platform and/or as a platform for cell treatment and/or cell cultivation.

14. The method according to claim 13, wherein the patterned substrate is at first used for the chemical synthesis of a chemical synthesis product and then as a characterizing platform for characterizing the chemical synthesis product and/or for treating at least one cell with the chemical synthesis product on the patterned substrate.

Description

[0124] The Figures show:

[0125] FIG. 1: Schematic illustration of the chemical structure of a poly(thioether) dendrimer of the third generation starting from a core (G0).

[0126] FIG. 2: Manufacturing process of patterned dendrimeric surfaces. First, a glass surface (or a glass-like surface, such as indium-tin oxide (ITO)) is activated and modified with chloro(dimethyl)vinylsilane. After that the dendrimeric structure is synthesized in a repeating photochemical thiol-ene-click reaction with thioglycerol, followed by an esterification with 4-pentenoic acid. The patterning of the surface is carried out with 1H,1H,2H,2H-perfluorodecanethiol (PFDT) and thioglycerol to produce hydrophilic/omniphilic spots, which are delimited from each other by hydrophobic/omniphobic barriers.

[0127] FIG. 3: Reaction cycle for synthesizing dendrimeric structures at a surface. The cycle consists of a photochemical thiol-ene-click reaction with thioglycerol (compound 2), followed by an esterification with 4-pentenoic acid (compound 4).

[0128] FIG. 4: Measurement of the static water contact angle on PFDT- and cysteamine-modified regions on dendrimer slides of generation G0 (corresponds to conventional LSTL slides) to generation G4.

[0129] FIG. 5: Comparison of the advancing angle (θ.sub.adv) and the receding angle (θ.sub.rec) of water droplets on PFDT-modified and cysteamine-modified regions between conventional slides (LSTL slides and G0 slides, respectively) with dendrimer slides of generation G3.

[0130] FIG. 6: Droplets of different solvents (low surface tension (ethanol) to high surface tension (water) from left to right) on a patterned dendrimer slide.

[0131] FIG. 7: Comparison of the water contact angle of differently modified dendrimer slides, which were manufactured by various methods (thiol-ene and thiol-yne chemistry).

[0132] FIG. 8: Measurement of the electrical current at four different points (each at a distance of 1 cm) on the surface of dendrimer slides.

[0133] FIG. 9a-d: Results of the cell viability screening. (a) On spots of the dendrimer slides, all three tested cell lines (HeLa, HEK293T, and Jurkat) have shown an excellent cell viability of more than 90%. (b) Microscopy image of HeLa cells (viability: 92.5±3.3%) on spots of dendrimer slides. (c) Microscopy image of HEK293T cells (viability: 98.2±0.2%) on spots of dendrimer slides. (d) Microscopy image of Jurkat cells (viability: 99.2±0.1%) on spots of dendrimer slides. Scale indicator: 50 μm.

[0134] FIG. 10: Droplet array and fluorescence microscopic image in the propidiumiodide channel of purified dendrimer slides after cell experiments. For an easier visualisation a negative image has been produced from the fluorescence microscopic image in a subsequent image processing. After mechanical cleaning by a toothbrush, a sponge and soap no cell debris could be found on the surface. Droplet arrays may still be produced afterwards.

[0135] FIG. 11: 3 component reaction for synthesis of lipid-like molecules (lipidoids) and the precursor molecules used for the same. For synthesis, in each case 1.5 μL amine component and 1.5 μL thiolactone-pyridyldisulfide solution were combined on the spots of the array (round, diameter of 2.83 mm) by a dispenser.

[0136] FIG. 12: Evaluation of the toxicity screening. The cell viability in [%] after 24 h of incubation of HEK293T cells, which have been seeded on a lipidoid library synthesized beforehand, is shown.

[0137] The present invention is discussed in more detail by the following non-limiting examples.

[0138] General

[0139] General Standard Operation Procedures:

[0140] Activation and Silanization of Slides (Holders):

[0141] In order to activate glass holders (for example, regular silicon oxide glass holders (Schott Glas) and indium-tin oxide-coated glass holders (ITO slides, Bruker)) the same were cleaned by plasma treatment for 10 minutes at room temperature. The silanization of the surface of the glass holder was carried out in a gas phase including 100 μL chloro(dimethyl)vinylsilane (Sigma-Aldrich) for 10 hours at 80° C. under vacuum (50 mbar). First, the surface was rinsed with acetone and then with ethanol and dried with compressed air.

[0142] General Standard Operation Procedure of a Cycle to Generate a Branch of the Dendrimer Structure (One Generation) Formed at a Smooth Surface with 4-Pentenoic Acid:

[0143] On the surface of the slides, which were silanized beforehand, 250 μL of a 10 vol % 1-thioglycerol solution (in ethanol/H.sub.2O (1:1), 10 mg.Math.mL.sup.−1 2,2-dimethoxy-2-phenylacetophenone (DMPAP, Sigma-Aldrich); Sigma-Aldrich) were applied. After that the solution was covered with a quartz glass and the slide was exposed with UV light having a wavelength of 260 nm (OAI model 30) at an intensity of 3 mW.Math.cm.sup.−2 for 2 minutes at room temperature. The surface was rinsed with ethanol and dried with compressed air. The slide was immersed in an esterifying solution (45 mL acetone (Merck), 56 mg 4-(dimethylamino)pyridine (DMAP, Novabiochem), 125 μL 4-pentenoic acid (Sigma-Aldrich) and 180 μL N,N′-diisopropylcarbodiimine (DIC, Alfa Aesar)) cooled to −20° C. for 4 hours with stirring and then the temperature of the reaction solution was slowly raised to room temperature. First, the surface was washed with acetone and then with ethanol and dried with compressed air.

[0144] General Standard Operation Procedure of a Cycle to Generate a Branch of the Dendrimer Structure (One Generation) Formed at a Smooth Surface with 4-Pentynoic Acid:

[0145] On the surface of the slides, which were silanized beforehand, 250 μL of a 10 vol % 1-thioglycerol solution (in ethanol/H.sub.2O (1:1), 10 mg.Math.mL.sup.−1 2,2-dimethoxy-2-phenylacetophenone (DMPAP, Sigma-Aldrich); Sigma-Aldrich) were applied. After that the solution was covered with a quartz glass and the slide was exposed with UV light having a wavelength of 260 nm (OAI model 30) at an intensity of 3 mW.Math.cm.sup.−2 for 2 minutes at room temperature. The surface was rinsed with ethanol and dried with compressed air. The slide was immersed in an esterifying solution (45 mL acetone (Merck), 56 mg 4-(dimethylamino)pyridine (DMAP, Novabiochem), 111.6 mg 4-pentynoic acid (Sigma-Aldrich) and 180 μL N,N′-diisopropylcarbodiimine (DIC, Alfa Aesar)) cooled to −20° C. for 4 hours with stirring and then the temperature of the reaction solution was slowly raised to room temperature. First, the surface was washed with acetone and then with ethanol and dried with compressed air.

[0146] Patterning of the Surface with Perfluorodecanethiol and 1-Thioglycerol:

[0147] 300 μL of a 20 vol % 1H,1H,2H,2H-perfluorodecanethiol solution (10 mg.Math.mL.sup.−1 2,2-dimethoxy-2-phenylacetophenone (DMPAP, Sigma-Aldrich); Sigma-Aldrich) in acetone (Merck) were applied on a surface, which was modified beforehand, and exposed through a photomask (Rose Fotomasken) for 1.5 minutes at room temperature with UV light having a wavelength of 260 nm (OAI model 30) at an intensity of 3 mW.Math.cm.sup.−2. The surface was rinsed with acetone and dried with compressed air. 300 μL of a 10 vol % 1-thioglycerol solution (in ethanol/H.sub.2O (1:1), 10 mg.Math.mL.sup.−1 2,2-dimethoxy-2-phenylacetophenone (DMPAP, Sigma-Aldrich); Sigma Aldrich) were applied on the surface and exposed through a quartz glass with UV light having a wavelength of 260 nm (OAI model 30) at an intensity of 3 mW.Math.cm.sup.−2 for 1.5 minutes at room temperature. The surface was rinsed with ethanol and dried with compressed air.

[0148] Patterning of the Surface with Perfluorodecanethiol and Cysteamine Hydrochloride:

[0149] 300 μL of a 20 vol % 1H,1H,2H,2H-perfluorodecanethiol solution (10 mg.Math.mL.sup.−1 2,2-dimethoxy-2-phenylacetophenone (DMPAP, Sigma-Aldrich); Sigma-Aldrich) in acetone (Merck) were applied on a surface, which was modified beforehand, and exposed through a photomask (Rose Fotomasken) for 1.5 minutes at room temperature with UV light having a wavelength of 260 nm (OAI model 30) at an intensity of 3 mW.Math.cm.sup.−2. The surface was rinsed with acetone and dried with compressed air. 300 μL of a 10 wt % cysteamine hydrochloride solution (in ethanol/H.sub.2O (1:1), 10 mg.Math.mL.sup.−1 2,2-dimethoxy-2-phenylacetophenone (DMPAP, Sigma-Aldrich); Alfa Aesar) were applied on the surface and exposed through a quartz glass with UV light having a wavelength of 260 nm (OAI model 30) at an intensity of 3 mW.Math.cm.sup.−2 for 1.5 minutes at room temperature. The surface was rinsed with ethanol and dried with compressed air.

[0150] Water Contact Angle Measurements:

[0151] The surfaces of the glass holders were characterized by water contact angle measurements using the Drop Shape Analyzer DSA25 (Krüss). The advancing angle (θ.sub.ac), the receding angle (θ.sub.rec) and the static angle (θ.sub.stat) were measured. To this end, 40 μL deionized water were applied on the respective surface at a rate of 0.3 μL.Math.s.sup.−1 and withdrawn by suction again and in the course thereof the corresponding angles were measured.

Example 1: Preparation of Dendrimer Slides with 2-Pentenoic Acid

[0152] The preparation of a dendrimer slide is schematically shown in FIG. 2.

[0153] Regular silicon oxide glass holders (Schott Glas) and indium-tin oxide-coated glass holders (ITO slides, Bruker) were used for the preparation of dendrimer slides. For activation, the glass holders were cleaned by a plasma treatment and silanized, as indicated in the general standard operation procedure. The surfaces of the holders were silanized in order to render them more reactive by introducing double bonds (see structure 1 in FIG. 3).

[0154] After that the surfaces were further functionalized in order to form dendrimeric structures thereon. FIG. 3 shows the two iteratively running synthesis steps which constitute one cycle and one degree of branching and thus one generation of the surface structure. After reacting the reactive surfaces, which have been functionalized with double bonds (see structure 1 in FIG. 3) with thioglycerol (see compound 2 in FIG. 3) in a first step of the cycle, the surfaces have numerous hydroxy groups (see structure 3 in FIG. 3), which are reacted in the second step of the cycle in an esterification with 4-pentenoic acid (see compound 4 in FIG. 3). Thus, the hydroxy terminal groups of the surfaces are converted into terminal groups having double bonds (see structure 5 in FIG. 3). These double bonds may again be reacted in a further cycle in a thiol-ene-reaction with thioglycerol (see compound 2 in FIG. 3; right side), whereby again functional hydroxy terminal groups are produced, and after that may be reacted with 4-pentenoic acid. The two steps of the cycle may be further repeated, wherein in each passing of the cycle the degree of branching raises by 2.sup.n (n: number of cycles). The produced dendrimers may be chemically denoted as poly(thioether) dendrimers.

[0155] The above-described processes were applied on the holders by carrying out the general standard operation procedure with regard to one cycle (one generation) for generating a branch of the dendrimer structure formed at a smooth surface with 4-pentenoic acid. In total, the cycle was carried out three times, as shown, for example, in FIG. 2. After the third passing of the cycle the number of originally one immobilized double bond could be raised to eight double bonds (see FIG. 1).

[0156] After that those terminal double bonds were site-specifically reacted in further photochemical thiol-ene-click reactions with strongly hydrophobic (1H,1H,2H,2H-perfluorodecanethiol (PFDT)) and hydrophilic (thioglycerol) molecules by a photomask or by a laser beam. Thus, the surfaces could be patterned such that thioglycerol-functionalized spots were obtained, which are separated from each other by boundaries of PFDT-funktionalized barriers.

[0157] The above-described terminal functionalizations were applied on the holders by carrying out the general standard operation procedure concerning the patterning of the surface with periluorodecanethiol and 1-thioglycerol.

[0158] In this case, spots having different diameters, e.g. between 500 μm and 2.83 mm, and an arbitrary geometrie could be produced.

Example 2: Preparation of Dendrimer Slides with 2-Pentynoic Acid

[0159] In analogy to example 1, a dendrimeric surface modification was also carried out with 4-pentynoic acid instead of 4-pentenoic acid. In this case, the dendrimeric structure is formed by a thiol-yne-reaction and finally patterned. The repeating units were applied on the holder using the general standard operation procedure with respect to one cycle (one generation) to generate a branch of the dendrimer structure formed on a smooth surface with 4-pentynoic acid.

Example 3: Investigation of the Influence of the Degree of Branching of Different Dendrimer Generations on the Static Water Contact Angle

[0160] The influence of the degree of branching of different dendrimer generations on the static water contact angle was investigated. To this end, dendrimer slides were prepared, which were esterified with 4-pentenoic acid. In the last step, respective modifications of the surfaces were carried out with PFDT in order to characterize the properties of the hydrophobic/omniphobic boundaries (i.e., the second regions), and with cysteamine in order to characterize the properties of the hydrophilic/omniphilic spots (i.e., the first regions). The results are shown in FIG. 4.

[0161] Already after the second generation of the dendrimer slides a raise of the static contact angles on the PFDT-modified regions and a clear decline of the static contact angle on the cysteamine-modified regions can be noted. After that the contact angle changes only slightly. Substrates having a third dendrimer generation had, for example, static contact angles on the PFDT-modified regions of θ.sub.stat(H.sub.2O)=111.5±2.9° and on the cysteamine-modified regions of θ.sub.stat (H.sub.2O)=33.0±3.0°.

Example 4: Comparison of the Advancing Angle (θ.SUB.ad.v) and the Receding Angle (θ.SUB.rec.) of Water Droplets on PFDT-Modified and Cysteamine-Modified Regions Between Conventional Slides (LSTL Slides and G0 Slides, Respectively) with Dendrimer Slides of the Generation G3

[0162] Advancing angles (θ.sub.adv) and receding angles (θ.sub.rec) of water droplets on PFDT-modified and cysteamine-modified regions of conventional slides (LSTL slides and G0 slides, respectively) and dendrimer slides of the generation G3 were measured. The results are shown in FIG. 5. Both angles were clearly increased by the dendrimeric surface on PDFT-modified regions and clearly decreased on cysteamine-modified regions. Due to the large difference between the receding angle on PFDT-modified regions and cysteamine-modified regions, droplet arrays on dendrimer slides may be generated both with solvents with low as well as with high surface tension. However, on conventional LSTL slides without dendrimeric surface no droplet arrays can be formed. The difference between the receding angle on PFDT-modified regions (θ.sub.rec(H.sub.2O)=83.3°) and the receding angle on cysteamine-modified regions (θ.sub.rec(H.sub.2O)=33.7°) is clearly smaller in conventional LSTL slides than in dendrimer slides according to the invention (see FIG. 5).

[0163] The third dendrimer-generation shows a clearly larger difference of the receding contact angle (PFDT: θ.sub.rec(H.sub.2O)=111.2°; cysteamine: θ.sub.rec(H.sub.2O)=6,7°). Due to the large difference of the receding angle on the two differently modified regions also droplet arrays with aqueous solvents having a high surface tension may be generated on patterned dendrimeric surfaces. Since the modification of the surface is a direct covalent modification, also solvents having a low surface tension may be applied without diffusion out of the spots and cross-contamination resulting therefrom (see FIG. 6).

Example 5: Comparison of the Water Contact Angle of Differently Modified Dendrimer Slides, which were Prepared by Different Methods (Thiol-Ene and Thiol-Yne Chemistry)

[0164] Dendrimeric structures were generated by photochemical thiol-ene-click reactions using 4-pentenoic acid and by photochemical thiol-yne-click reactions using 4-pentynoic acid on different flat substrates. Since the degree of branching in a thiol-yne reaction is once more doubled compared with a thiol-ene reaction, the water contact angles of the second generation of thiol-yne generated dendrimeric surfaces were compared with the third generation of thiol-ene generated dendrimeric surfaces (see FIG. 7).

[0165] While the advancing angles of the dendrimer surfaces on PFDT-functionalized regions prepared with 4-pentynoic acid (θ.sub.adv(H.sub.2O)=124.2±0.1°) and cysteamine-functionalized regions (θ.sub.adv(H.sub.2O)=57,3±5.2°) are almost identical with dendrimer surfaces prepared with 4-pentenoic acid, the receding angle of the dendrimer surfaces on the PFDT-functionalized regions prepared with 4-pentynoic acid (θ.sub.rec(H.sub.2O)=91.0±1.1°) is slightly lower and on the cysteamine-functionalized regions (θ.sub.rec(H.sub.2O)=13.2±1.1°) is slightly higher than on the corresponding regions with 4-pentenoic acid dendrimers.

[0166] Besides the investigations of different acid components for the synthesis of the dendrimer, different reagents for the final surface patterning were investigated, too (see FIG. 7). A functionalization with thioglycerol results in a further clear improvement of the hydrophilic/omniphilic properties of the surface. Dendrimeric surfaces (4-pentenoic acid) functionalized with thioglycerol of the third generation have an advancing angle of θ.sub.adv (H.sub.2O)=32.6±2.2° and a receding angle of θ.sub.rec(H.sub.2O)=1.2±0.6°, corresponding to a halving of the corresponding angles compared to the functionalisation with cysteamine (see FIG. 7).

Example 6: Modification of Conductive Surfaces with Dendrimer Structures

[0167] ITO-coated glass slides were modified with dendrimer structures according to the invention. On the corresponding substrates, droplet arrays having small and large surface tension could be generated, too.

[0168] The electric current was measured at respective four positions (at a respective distance of 1 cm) on the surface of the modified glass slides in order to check the provision of the conductivity (see FIG. 8). A 9 V battery having a residual current of 1.1 A and a residual voltage of 8.5 V was used for the measurement.

[0169] In this manner, after each step of preparing the dendrimeric surface the electric current was measured (see Table 1). Furthermore, the influence of the solvent in the esterification steps was investigated.

TABLE-US-00001 TABLE 1 Measurement of the electrical current at four positions (P1-4; see FIG. 8) on surfaces after various steps in the production of dendrimeric slides. Time after start Step P1 [A] P2 [A] P3 [A] P4 [A] Solvent [d] Non-treated 0.048 0.034 0.032 0.024 0 (positive control) Plasma 0.042 0.035 0.031 0.023 0 activation Silanization 0.025 0.021 0.020 0.015 1 1. Esterification 0.024 0.021 0.015 0.011 Acetone 1.5 1. Esterification 0.024 0.021 0.020 0.014 DCM 1.5 2. Esterification 0.026 0.022 0.019 0.017 Acetone 2 2. Esterification 0.025 0.022 0.019 0.017 DCM 2 3. Esterification 0.028 0.026 0.025 0.021 Acetone 2.5 3. Esterification 0.028 0.021 0.020 0.020 DCM 2.5 Patterned 0.032 0.028 0.025 0.022 Acetone 2.5 Patterned 0.027 0.024 0.021 0.018 DCM 2.5 Regular glass 0 0 0 0 Acetone 0 slide (negative control) Patterned glass 0 0 0 0 Acetone 2.5 slide (G3; negative control)

[0170] No difference between the use of acetone and dichloromethane (DCM) could be noted. The electric current halves after the silanization, then, however, remains constant up to the final patterning (see Table 1). Thus, no conductive platforms could be obtained. Thus, after the completed on-chip synthesis of a substance, also a direct on-chip characterization by MALDI-TOF MS or other analysis methods requiring an electrically conductive platform may be carried out.

Example 7: Cell Viability Screening on Dendrimer Slides

[0171] Cell viability screenings with various cell lines (adherend cells: HeLa, HEK293T; suspension cells: Jurkat) were carried out in order to evaluate the compatibility of the surface of the patterned substrates according to the invention with (cell) biological experiments.

[0172] To this end, HeLa and HEK293T cells were cultivated in Dulbecco's Modified Eagle Medium (DMEM, Life Technologies) having 15% v/v fetal bovine serum (FBS; PAA Laboratories) and 1% v/v penicillin-streptomycin (Life Technologies). Jurkat cells were cultivated in RPMI 1640 Medium (Gibco) having 15% v/v FBS (PAA Laboratories) and 1% v/v penicillin-streptomycin (Life Technologies).

[0173] 3 μL of a HeLa and HEK293T cell suspension each having a concentration of 50,000 cells/mL and 3 μL of a Jurkat-cell suspension having a concentration of 80,000 cells/mL were applied by a dispenser (I-DOT, Dispendix) per spot of the substrate. The diameter of the round spots was 2.83 mm and the distance between the spots was 1.67 mm. After 24 h of cultivation (at 37° C. and 5% CO.sub.2 concentration) the cells were stained with Hoechst 33342 (1:900; 10 mg.Math.mL.sup.−1; Invitrogen) to determine the total cell number (staining of the cell nucleus) and propidiumiodide (PI; 1:1350; 1.00 mg mL.sup.−1; Invitrogen) to determine the number of dead cells (staining of dead cells). The evaluation was carried out by fluorescence microscopy by a Keyence BZ9000 (Keyence) and the software ImageJ (Funktion: Analyze Particles). The number of viable cells was calculated from the difference of the total cell number and the number of dead cells. The cell viability was indicated as a quotient of the number of viable cells and the total number of cells in percent [%] (see FIG. 9).

[0174] Any one of the tested cell lines demonstrated a very good cell viability (>90%) on the spots of the dendrimer slides (see FIG. 9a). Furthermore, all three tested cell lines have shown the respective typical morphology (see FIG. 9b-d). Thus, the patterned substrates according to the invention are excellently suitable for biological cell screenings.

Example 8: Reusability of the Dendrimer Slides

[0175] The dendrimer slides from example 7, which have been used for the biological cell viability screenings, were cleaned and tested as to whether they may be reused. To this end, the slides were first rinsed with acetone and ethanol, which resulted in a lysis of the cells remaining thereon. In this context, a cloudy staining of the spots indicated a precipitation of the proteins present in the cells. For sterilisation the slides were immersed in an aqueous 70 vol % ethanol solution for two weeks. After that the cloudiness of the spots was still visible.

[0176] Microscopic images have shown still existing cell debris on the surface of the spots. The staining of the dead cells by propidiumiodide was still visible by fluorescence microscopy, too. After that parts of the slide surface were mechanically cleaned by means of a commercially available toothbrush and soap and after that with a sponge. The slide was again rinsed with ethanol and acetone and dried with compressed air. FIG. 10 shows the result of the cleaning process.

[0177] A clear line between the mechanically cleaned surface and the non-cleaned surface can be observed in the propidiumiodide channel by fluorescence microscopy (see FIG. 10). Also by transmitted-light microscopy in visible light no cell debris on the mechanically cleaned spots of the dendrimer surface could be observed anymore. Nevertheless, droplet arrays could also be generated on the mechanically cleaned spots. No difference between the shape and volume could be observed between cleaned and non-cleaned spots (see FIG. 10). Thus, the surfaces of the used dendrimer slides are mechanically stable and reusable. In contrast, the surface of HSTL slides known from the prior art would be destroyed by this process.

Example 9: Toxicity Screening with Dendrimer Slides

[0178] The possibility of a chemical synthesis with a biological screening on a single patterned substrate according to the invention was investigated. To this end, first a library of 25 different lipid-like molecules (lipidoids) was synthesized in a combinatorial manner in a liquid phase on a dendrimer slide (round spots, diameter of 2.83 mm, barrier diameter of 1.67 mm). The synthesis is based on a three components reaction, wherein a primary amine (A1-A5) initiates the reaction by ring opening of a thiolactone (T10/T12/T14), and which terminates by a disulfide exchange with a pyridyldisulfide (PY10/PY12/PY14). The precursor molecules which were used are shown in FIG. 11.

[0179] For the combinatorial synthesis of the lipidoid library in each case 1.5 μL of the amine component (dissolved in DMSO; 1:24 v/v) (A1-A5) and 1.5 μL of a mixture (dissolved in DMSO) of thiolactone derivative (1.67 mg.Math.mL.sup.−1) and pyridyldisulfide derivative (1.75 mg.Math.mL.sup.−1) (mixtures: T10_PY10/T12_PY12/T14_PY14/T14_PY12/T14_PY10) were combined. The solutions were applied by dispensers (I-DOT, Dispendix) in a combinatorial format. After 2 hours reaction time at room temperature the solvent was removed under reduced pressure. After that 3 μL of a HEK293T cell suspension (50,000 cells/mL) were printed by a dispenser onto each spot of the array in order to determine the toxicity of the synthesis products. The slide was incubated for 24 hours at 37° C. and 5% CO.sub.2 concentration and after that the cells were stained with Hoechst 33342 (1:900; 10 mg.Math.mL.sup.−1; Invitrogen) to determine the total cell number (staining of the cell nucleus) and propidiumiodide (PI; 1:1350; 1.00 mg.Math.mL.sup.−1; Invitrogen) to determine the number of dead cells (staining of dead cells). The evaluation was carried out by fluorescence microscopy (Keyence BZ9000 (Keyence)) and the software ImageJ (function: Analyze Particles). The number of viable cells was calculated from the difference of the total cell number and the number of dead cells. The cell viability was indicated as the quotient of the number of viable cells and the total number of cells in percent [%] (see FIG. 12).

[0180] As it can be seen in FIG. 12, all substances were found to be very toxic in the tested concentration. Having a cell viability of 33.0±4.4%, the lipidoid A1_T14_PY10 was found to be the least toxic. For the negative control with non-treated cells again an excellent cell viability of 97.2±1.3% could be observed. This clearly shows that the toxic effect was not caused by the surface and confirms the preceding cell viability screenings of example 7. In the following, the finding from this primary screening may serve to provide a lead structure for chemical structure-optimizing experiments.

[0181] Thus, it could be successfully shown that on patterned substrates according to the invention chemical synthesis in an aqueous phase may be directly combined with biological screenings on a single platform in a droplet array format.