DE20005738U1 - Device for examining the molecular interactions of soluble or suspendable active substances with solid-phase-bound peptide or peptoid target molecules through capillary plates with a large inner surface - Google Patents

Description

Device for investigating the molecular interactions of soluble or suspendable active substances with solid-phase-bound peptide or peptoid target molecules through capillary plates with a large inner surface

State of the art

In contrast to solid-phase immobilized miniaturized nucleic acid libraries, about which a large number of publications in specialist journals and patents exist (SPA Fodor et al. (1991), Light-directed, spatially addressable parallel chemical synthesis. Science 251:767-773; EM Southern et al. (1992), Analyzing and comparing nucleic acid sequences by hybridization to arrays of oligonucleotides: evaluation using experimental models. Genomics 13:1008-1017; G. McGaIl. et al. (1996), Light-directed synthesis of high-density oligonucleotide arrays using semiconductor photoresists. Proc. Natl. Acad. Sei. USA 26:13555-13560; M. Chee et al. (1996), Accessing genetic information with high density DNA arrays. Science 274:610-614; S. Singh-Gasson et al. (1999), Maskless fabrication of light-directed oligonucleotide microarrays using a digital micromirror array. Nat. Biotechnol. 17:974-978; SPA Fodor et al. (1995), Arrays of materials attached to a substrate. US 5744305; SPA Fodor et al. (1995), Very large scale immobilized polymer synthesis. US 5424186; GH. McGail et al.

(1995), Spatially-addressable immobilization of oligonucleotides and other biological polymers on surfaces. US 5412087; A:S. Heuermann (1999), Method and device for photolithographic production of DNA, PNA and protein chips. WO 9960156A2; GH McGaIl and NQ. Nam (2000), Synthesis of oligonucleotide arrays using photocleavable protecting groups. US 6022963), solid phase-immobilized, highly miniaturized peptide or peptidomimetic libraries have so far only been described in a few works (SPA Fodor et al. (1991), Light-directed, spatially addressable parallel chemical synthesis. Science 251:767-773; CP . Holmes et al. (1995), The use of light-directed combinatorial peptide synthesis in epitope mapping. MC Pirrung et al. (1992), Large scale photolithographic solid phase synthesis of polypeptides and receptor binding. US 5143854; MC Pirrung et al. (1995), Large scale photolithographic solid phase synthesis of an array of polymers.

One of the main reasons why miniaturized peptide libraries, so-called "peptide chips", have not yet found widespread use is the relatively high cost of

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the production of such arrangements if the usual photolithographic synthesis processes on planar supports used for "DNA chips" are used. In the case of nucleic acids, these processes make use of the fact that there are only 4 different naturally occurring nucleotides (deoxyadenosine, deoxycytidine, deoxyguanosine and thymidine) for the construction of deoxyribonucleic acid oligomers, that the coupling times for the construction of the oligonucleotides are relatively short (less than 30 minutes) and that the yields of the individual coupling steps are very good (more than 99%). All three criteria have a decisive influence on the production time for such chips and thus the economic efficiency of the entire process.

In the photolithographic synthesis of oligonucleotides, a carrier that is completely protected with a photolabile protective group is first deprotected by irradiation with light at the points where, for example, a thymidine phosphate is to be applied, and then the entire carrier is incubated with thymidine phosphoamidite that is photolabile protected at the 5'-OH and suitable coupling reagents. Coupling thus only takes place at the desired points and the entire process must be repeated for the other three nucleotides before the first dinucleotide can be synthesized. For a chip that is coated with 20-mer oligonucleotides, 80 coupling, washing and deprotection cycles are therefore required.

However, in the case of peptides, the number of components that can be used for coupling is significantly higher than in the case of nucleic acids. There are 20 proteinogenic amino acids, some naturally occurring non-proteinogenic amino acids (e.g. ornithine), the same number of corresponding D-amino acids and a continuously increasing number of artificial amino acids, such as cyclohexylalanine, aminoisobutyric acid, norvaline, etc., also in D and L forms. Taken together, it can be assumed that around 100 different amino acids are currently available for the chemical synthesis of peptides and peptidomimetics. If - conservatively speaking - only half of these reagents are used to synthesize a substance library, 1,000 coupling and deprotection cycles result in the synthesis of a 20-mer peptide or peptidomimetic library. In addition, solid phase peptide synthesis usually yields coupling yields of 85-90% with reaction times of about 30 min, so that usually at least one repetition of the coupling step with fresh reagents is necessary to achieve the required synthesis yields. This

means that extremely long synthesis times of weeks to several months must be taken into account for the production of a peptide or peptidomimetic library immobilized on a two-dimensional support when photolithographic processes are used. Consequently, only photolithographic syntheses of short peptides with low sequence variability have been described to date (SPA Fodor et al. (1991), Light-directed, spatially addressable parallel chemical synthesis. Science 251:767-773; CP. Holmes et al. (1995), The use of lightdirected combinatorial peptide synthesis in epitope mapping. Biopolymers 37:199-211; MC Pirrung et al. (1992), Large scale photolithographic solid phase synthesis of polypeptides and receptor binding thereof. US 5143854).

An alternative method that significantly reduces the number of steps is based on the simultaneous deprotection of all carrier-bound reaction partners followed by the parallel or sequential site-directed application of the different amino acid reaction mixtures into defined sample areas. This also allows libraries of longer and complex peptides or peptidomimetics to be synthesized within an acceptable time frame. The main problem here, however, is the expected cross-contamination of the reactants if the sample areas are close to one another. A dense packing of the sample areas is desirable in order to sufficiently miniaturize the substance library.

One possible solution to the problem is to increase the interfacial tension in the areas between the sample areas of the planar support so that the reaction mixtures remain as small droplets in the area of the sample areas. To achieve this goal, the support surface between the sample areas must be fluoroalkylated, as described in TM. Brennan (1995), Method and apparatus for conducting an array of chemical reactions on α support surface, US 5474796; TM. Brennan (1997), Method and apparatus for conducting an array of chemical reactions on α support surface, EP 703825B1 and TM. Brennan (1999), Method and apparatus for conducting an array of chemical reactions on α support surface, US 5985551. However, the method has the disadvantage that the drops are highly exposed to evaporation without a protective coating, which can be a significant problem , especially with very small sample volumes.
An alternative solution to the cross-contamination problem is to use a porous membrane that collects the amino acid reaction mixture at the site of application

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immediately absorbed (R. Frank (1992), Spot synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on α membrane support. Tetrahedron 48:9217-9232; R. Frank and S. Güler (1992), Process for the rapid synthesis of support-bound or free peptides or oligonucleotides, flat material produced thereby, use and device for carrying out the process. WO 92/04366; J. Schneider-Mergener (1994), Process for the synthesis and selection of sequences from covalently linked building blocks. WO 94/20521). However, the disordered capillaries in the porous membrane lead to cross-contamination with increasing miniaturization of the sample areas and the relatively large surface area of the membrane also leads to evaporation of the solvent in this process and thus under certain circumstances also to an impairment of the coupling reaction. Another problem that occurs when using the cellulose membranes usually used here is the sometimes very strong heterogeneity of the synthesized product. In a mass spectroscopic examination of the entire material detached from a sample area, both amino-terminal and carboxy-terminal shortened peptides are found. These are caused by the fact that the synthesis stops too early within the membrane for steric reasons and that chain restarts can still take place in later synthesis cycles through esterification of amino acids directly on the cellulose substrate. In particular, peptides that are carboxy-terminally shortened by 1-4 amino acids are detected (D. Goehmann and A. Frey, unpublished results). Complete blocking of the hydroxyl groups of the cellulose by acetylation or similar measures to prevent chain restarts is not permitted, as this increases the solubility of the carrier in the solvents usually used for solid-phase peptide synthesis and significantly reduces mechanical stability (D. Goehmann and A. Frey, unpublished results). Another disadvantage of using membranes is their optical properties. Cellulose membranes in particular exhibit strong intrinsic fluorescence in the emission range of many commercially available fluorophores, so that the sensitivity of the detection of active substances that bind to the target molecules is greatly reduced (J. Helfmann, unpublished results).

Another approach to solving the cross-contamination problem when loading a planar support with a compound library is to use a microreactor system in which the planar support forms a vessel wall for a variety of

reaction cavities and at the same time the carrier medium for the target molecules. The reaction cavities thus enclosed on all sides are supplied with the desired reagents and washing solutions, for example by repositioning the microreactor block or with the help of microchannels, electroosmotic pumps and microvalves in the reactor block. Waste products are removed in an analogous manner (JL Winkler et al. (1995), Very large scale immobilized polymer synthesis using mechanically directed flow paths. US 5384261; PJ. Zanzucchi et al. (1997), Method of synthesis of plurality of compounds in parallel using a partitioned solid support. US 5643738; PJ. Zanzucchi et al. (1998), Partitioned microelectronic device array. US 5755942; SC Cherukuri et al. (1999), Method and system for inhibiting crosscontamination in fluids of combinatorial chemistry device. US 5980704). A crucial disadvantage of such arrangements, however, is their susceptibility to particulate contamination in the reaction solutions, particularly when the channels are only a few micrometers in diameter and the flow direction of the reagents and waste products in the microreactor block is changed several times. The risk of blockage of the microreactor system is particularly high in coupling reactions with carbodiimides, since the organic urea derivatives produced in this process have a strong tendency to crystallize. In the worst case, the entire microreactor block must then be replaced.

The existing methods or those described in the literature or patents all have significant disadvantages. Either they do not offer a large surface area and therefore no high detection sensitivity with small external dimensions, or the synthesis times are too long overall. The optical detection is greatly impaired by self-fluorescence and chain terminations lead to an inhomogeneous sample. If the substance library is densely packed, cross-contamination between different sample areas can be observed. With small volumes, evaporation influences the results. Handling is cumbersome or very sensitive to particles.

Inventive solution

The detection sensitivity for the interaction (e.g. binding) of active substances in the liquid phase with solid-phase-bound target molecules is essentially determined by the number of solid-phase-bound target molecules. With an ideally planar carrier, the solid phase available for binding is limited to the outer surface of the carrier. However, an optical detection system (e.g. fluorescence detection) is always able to additionally detect a certain volume above and below the outer surface.

The invention relates to a structure which can consist of glass, quartz, ceramic, plastic, semi-metal or metal and which, due to a high density of small capillaries [1] (diameter 5-100 μm), has a large inner surface which is several times the outer surface (see Figure 1). The entire thickness of this capillary plate [4] (thickness up to 10 mm) and thus the entire length of the capillaries [1] and their surface is recorded by a detection apparatus. Since the maximum occupancy (number of molecules) of the capillary plate [4] is determined by the size of the inner surface, and this is many times (100 to 1000 times) larger than the outer surface, the detection sensitivity increases to the same extent when using the capillary plate [4] compared to a planar sample arrangement.

Surprisingly, it has been shown that high-quality peptide and peptoid molecules can be synthesized on a silanized surface and the number of chain restarts can be almost completely eliminated. If an extended anchor molecule, which serves as a spacer, is additionally coupled between the surface and the target molecule, the chain terminations are greatly reduced and the accessibility of the active ingredients is significantly improved.

In order to produce a library of target molecules, each of which can be synthesized or bound to a specific area of the capillary plate [4], the inner surface of the capillary plate [4] must first be made capable of covalently binding the target molecules. This is done by applying an organosilane layer that has functional groups for anchoring peptide or peptoid target molecules. Preferably, a γ-aminopropyltrialkoxysilane is used for this purpose, but other organofunctional silanes, such as γ-mercaptopropyltrialkoxysilane, are also suitable for this purpose. When using metal or semi-metal capillary plates,

An oxide layer must first be created to bind the silane by surface oxidation of the capillary plate.

A sample area [3] is then created on this organofunctionalized capillary plate [4] by applying a substance at a specific location (e.g. by pipetting).

In the simplest case, the substance can be a reagent that temporarily protects the functional groups of the organosilane layer intended for binding. In a preferred embodiment, however, an anchor molecule is applied to the sample areas [3], with which the target molecules can be raised between 0.5 and 20 nm above the inner surface of the capillary plate [4] in order to enable better binding of the active substance. Fluorenylmethoxycarbonyl (FMOC)-protected a-amino-poly(ethylene glycol)-copropionic acid-N-hydroxybenzotriazole ester is used as an anchor reagent.
used.

Subsequently, all areas of the capillary plate [4] that are not derivatized with protective or anchoring reagents are chemically saturated in such a way that neither synthesis or coupling of target molecules nor nonspecific binding of active substances can occur there at a later point in time.

By knowing the substance that is bound or synthesized at each location, a parallel analysis of the interactions of different target molecules with applied active substances is possible. The prerequisite for this is that no mutual contamination occurs during the location-dependent application or synthesis of the target molecules.

In a continuation of the inventive concept, this is achieved by the parallel arrangement of the capillaries [1] running perpendicular to the plate surface, which are only open to the outer plate surfaces. The resulting absence of any cross-connections between different sample areas [3] successfully prevents cross-contamination even when the sample areas [3] are very dense. A sample area [3] comprises several (e.g. up to 4,000) capillaries [I]. Pipetted substances are drawn into the capillaries [1], which, depending on the liquid, is also supported by generating a negative pressure on one side of the capillary plate. Flowing out of the capillaries [1] across several capillaries [1] into another sample area [3] is impossible, since the liquid is held in the capillary by capillary force. Solutions dripping out of the capillaries [1] due to very low viscosity is prevented with these liquids by

A plate [12] (Figure 2) with a hydro- and oleophobic surface positioned close to the underside of the capillary plate [4] effectively suppresses this.

In continuation of the inventive concept, a simple automation of a solid-phase peptide synthesis carried out in the capillary plate [4] is also possible due to the capillaries [1] being open to both outer plate surfaces. An apparatus for the synchronous application of washing and reagent solutions to all areas of the capillary plate [4] is shown in Figure 2.

The incubation of the substance library with active substance solutions or suspensions as well as rinsing processes before and after are carried out in a preferred embodiment with a simple setup as shown in Figure 3.

In a preferred embodiment, the analysis of the active ingredient binding is carried out optically using fluorescent labels by binding a fluorophore to the active ingredients. In an alternative embodiment, the fluorophores are bound to the target molecules. Figure 4 shows a detection apparatus for reading the fluorescence. For reading, the individual sample volume, which is made up of the thickness of the capillary plate and the sample area [3], is both completely penetrated by excitation light and completely captured by the detection optics. The large number of sample locations is captured by temporally sequential scanning, whereby either the capillary plate is moved two-dimensionally under the stationary optical arrangement or the beam path is moved over the capillary plate. The use of quartz or glass as the material for the capillary plate minimizes the influence of the disturbing external fluorescence according to the invention.

Detailed description of the inventive solution in a preferred embodiment

It is described how the entire capillary plate is manufactured. The sample areas are then created and the substances are synthesized on them. An automated device is described for all processes to which the capillary plate is fully exposed, such as washing. This device is combined with a pipetting robot so that the capillary plate can be washed and deprotected in the same device and the individual synthesis steps can be carried out. To investigate the binding of an active substance to the substance library, another device is described into which the capillary plate

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and brought into contact with the active ingredient. At the same time, optical detection of the binding is carried out.

Derivatization of the capillary plate to accommodate the substance library

The capillary plate is manufactured by etching from homogeneous glass, or in an alternative process from heterogeneous glass. The oxidation of silicon and metal surfaces and the silanization of glass, quartz, silicon, ceramic and oxidized silicon or metal surfaces is state of the art and has already been described many times in the specialist and patent literature (e.g. for the surface oxidation of a silicon carrier: AW Flounders et al. (1997), Patterning of immobilized antibody layers via photolithography and oxygen plasma exposure. Biosensors Bioelectronics 12:447-456; e.g. for silanization: M. Lynn (1975), Inorganic support intermediates: covalent coupling of enzymes on inorganic supports. In: "Immobilized Enzymes, Antigens, Antibodies and Peptides", HH Weetall, ed., Marcel Dekker, New York, NY, USA, pp. 1-48; HM. Weetall (1976), Covalent coupling methods for inorganic support materials. Methods Enzymol. 44:134-148).

In the preferred embodiment, an untreated capillary plate [4] with surface oxide, ceramic or glass coating is freed of adsorbed organic compounds either in an oxidizing acid, such as chromic sulfuric acid or hot concentrated nitric acid, for one hour, washed five to ten times by suction through highly pure water (on a suction filter, glass frit or a rinsing device specially developed for the capillary plate [4] (Figure 2)) and dried at 250 ^° C.

Device for fully automatic washing, saturation and deprotection
Fully automated washing, saturation and deprotection is the preferred embodiment for the preparation of the substance library (Figure 2).
For assembly, the capillary plate [4] is placed on the lower part of a synthesis table [6], which is firmly connected to a pipetting robot. The edge areas [5] of the capillary plate are placed on a soft, solvent-resistant seal [7]. Spacers [8] are then placed around the

Capillary plate [4] is pushed close to the edge areas [5] and secured against displacement by threaded bolts [9] so that the capillary plate [4] is fixed in place on the sample table. Then the upper part of the synthesis table [10] is put on the threaded bolts [9] and pulled against the capillary plate [4] and the spacer [8] or the lower part of the synthesis table [6] using a nut [11] so that the seals [7] in the upper and lower parts of the synthesis table [10 and 6] are pressed together and seal the capillary plate [4] to the side.
To feed the sample areas [3], the hydrophobically and oleophobically coated piston [12] is pushed to the bottom of the capillary plate [4] so that no reaction solution can escape downwards from the capillary plate [4] during the coupling reaction.

For washing after completion of the coupling reaction, the entire capillary plate [4] is covered with washing solution from the washing solution inflows [13], the piston [12] is moved downwards and a vacuum is applied to the outlet [14] to remove the solutions sucked through the capillary plate [4]. The piston [12] is then pushed back to the underside of the capillary plate [4], the capillary plate [4] is again covered with washing solution and the washing process is repeated as desired (Figure 2A). After completion of the washing process, air is sucked through the capillary plate [4] until all solvent residues have evaporated. The piston is then pushed back to the capillary plate [4] and another pipetting cycle is carried out, the plate is saturated or deprotected.

To saturate unreacted amino functions and to deprotect amino functions, the piston [12] is pushed downwards and the cavity, which is created by pulling back the piston [12] below the capillary plate [4], is filled with the desired reaction solution through the reagent inflow [15]. With the outlet [14] and reagent inflow [15] closed, the piston is slowly moved up and down for the duration of the reaction so that the reaction solution flows in a uniform flow through the capillaries [1] and the reaction can take place (Figure 2B).

The capillary plate [4] is then washed as described above.

Device for binding active substances to the immobilized on the capillary plate
Substance library

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To investigate the binding of drugs to the substance library located on the inner surface of the capillary plate [4], a device is necessary with which a drug solution or suspension can be flushed evenly through all capillaries [1] so that each drug component can come into contact with each target molecule species (Figure 3).

In the preferred embodiment, the capillary plate [4] is placed on the lower part of the incubation table [16] for assembly. The edge areas [5] of the capillary plate come to rest on a soft seal [17]. Spacers [18] are then placed around the capillary plate [4], the upper part of the incubation table [19] is placed on top and pressed by a clamp [20] using pressure springs [21] onto the capillary plate [4] and the spacer [18] or the lower part of the incubation table [16], so that the seals [17] in the upper and lower parts of the incubation table [19 and 16] are pressed together and seal the capillary plate [4] to the side. An ultrasound transmitter [22] is then pushed into a recess in the spacer [18] until it comes into contact with the edge area [5] of the capillary plate [4].

To load the device, the piston [23] is pushed downwards and the cavity created by pulling back the piston [23] below the capillary plate [4] is filled with the desired active substance solution or suspension through the filling nozzle [24]. The volume of the active substance solution or suspension should be at least 1.5 times the internal volume of the capillary plate [4] plus the volume of the cavity [25] located above the capillary plate [4].
For incubation, the piston [23] is slowly moved upwards with the outlet [26] and filler neck [24] closed so that the air from the capillaries [1] can escape into the cavity [25]. Air still trapped in the capillaries [1] can be removed by sonication with the ultrasound generator [22]. When the piston [23] is pushed up to the capillary plate [4], the cavity [25] is completely filled with the active ingredient solution or suspension and both the air and the excess active ingredient solution or suspension escape through side channels [27] into the compensation vessels [28]. By repeatedly raising and lowering the piston [23] with a short stroke, the solution or suspension in the capillaries [1] is mixed with the solution or suspension in the cavity [25] and any air bubbles still in the cavity [25] are gradually expelled into the compensation vessels [28].

To empty the device, the piston [23] is pushed back until the outlet [26] is exposed, through which the active substance solution or suspension is then removed by applying negative pressure.
The device is washed in an analogous manner using suitable washing solutions.

The spatially resolved detection of the drug reaction with the target molecules can also be carried out in the device, since the cavity [25] located above the capillary plate [4] is limited at the top by a transparent window [29] and thus, for example, the binding or the cleavage of fluorophores in the device can be detected or followed with the aid of the measuring device described in Figure 4.

Device for detecting the binding of active substances to target molecules

Optical methods that allow penetration of the bridges between the capillaries are used to detect bonds in the capillary plate. The most diverse optical methods are based on fluorescence labels (e.g. MV Rogers (1997), Light on high-throughput screening: fluorescence-based assay technologies, Drug discovery Today 2:156-160). The fluorescence label is produced by coupling a fluorophore either to the target molecules or to the active substances. For reading, in the preferred embodiment, the individual sample volume, which corresponds to the sample area [3] over the entire thickness of the capillary plate, is both completely penetrated by excitation light and completely detected by the detection optics. To prevent crosstalk from neighboring sample areas, only one sample location is illuminated at a time and detection is limited to just one sample location. The large number of sample locations is detected by sequential scanning. The measurement at a sample location can be carried out either continuously or with repetitive pulses. In the first case, irradiation is carried out using a continuously emitting bandpass-filtered light source or a laser, whereby the narrow-band excitation light is adapted to the absorption of the fluorescent marker. Detection is carried out narrow-band in the range of the fluorescence emission of the fluorescent marker using a suitable photodetector. In the pulsed fluorescence excitation shown in Figure 4, a laser [30] is used for illumination, the pulse duration of which is at least one third of the lifetime of the marking fluorophore. The collimated beam of the laser [31] is

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a wavelength-selective beam splitter [32] in the direction of the capillary plate [4]. The beam is focused on a sample area [3] of the capillary plate [4] by an objective [33] so that the entire sample volume of a sample area [3] is irradiated. The isotropically emitted fluorescent light [34] of the fluorescence marking is collimated by the same objective [33] and passes through the selective beam splitter [32] due to the longer wavelength of the fluorescence. Scattered light from the excitation laser is strongly suppressed by a narrow-band filter [35], but the fluorescent light is allowed to pass with the highest possible transmission. The fluorescent light is focused on the photodetector [37] using another objective [36]. Through time-resolved detection with a rise time of less than one third of the fluorescence lifetime of the fluorophore, the fluorescence signal can be detected particularly advantageously in a time-gate integration technique with a boxcar integrator [38] after both the elastic scattered light and Raman-scattered light have decayed. The time gate is opened approximately after twice the laser pulse duration and remains open for approximately twice the fluorophore lifetime. A trigger signal from the laser [39] provides the time base for the time gate. With the help of a signal from the laser [40] that is proportional to the laser pulse energy, the fluorescence light can be normalized via a second channel of the boxcar integrator [38], thus correcting for fluctuations in the laser pulse energy. All data are collected in a computer [41] and a plot of the fluorescence intensity over the respective sample location can be displayed and the detected signals can be assigned to the target molecules.

Description of the characters

Figure 1 contains a schematic representation of a capillary plate [4] in plan view and
as a section. The individual capillaries [1] are separated from each other by web areas [2].
separated. The sample areas [3] include a large number of capillary openings. The
The edge area of the plate [5] contains no capillaries.

Figure 2 shows a device for fully automatic washing, saturation and deprotection
the capillary plate [4] during the synthesis of the substance library.
the capillary plate [4] between the upper part [10] and the lower part [6] of the synthesis table
and secured with the help of appropriate seals [7] and spacers [8] over

a threaded bolt [9] with a nut [11] fixes it in position. The upper part of the synthesis table [10] contains the washing solution inlets [13], the lower part [6] contains the reagent inlet [15] and outlet [14]. A piston [12] can be moved in the lower part of the synthesis table [6] and pushed to the underside of the capillary plate [4].

Figure 3 shows a device for binding active substances to the substance library immobilized on the capillary plate. The capillary plate [4] is inserted between the upper part [19] and lower part [16] of the incubation table and is fixed in position with the help of appropriate seals [17] and spacers [18] by a clamp [20] with a pressure spring [21]. An ultrasound transmitter [22] is attached to the side of the capillary plate [4]. In the lower part of the incubation table there are filler necks [24] and drains [26] for the active substance solutions, which can be pressed through the capillary plate [4] into the upper cavity [25] using a piston [23]. Side channels [27] lead from the cavity [25] into an equalizing vessel, which is used to collect displaced air and excess active substance solution. The cavity [25] is closed at the top with a transparent window [29], so that direct optical detection of reactions in the device is possible.

Figure 4 contains a schematic representation of a device for detecting the binding of active substances to the substance library using optical methods. Fluorophore-labeled target molecules on the capillary plate [4] are excited to fluorescence by a laser beam [31] generated by a pulsed diode laser [30], which is directed onto the sample area by a wavelength-selective beam splitter [32] and an objective [33]. The emitted fluorescent light [34] passes through the beam splitter [32], a bandpass filter [35] and another objective [36] before it hits the photodetector [37]. To integrate the fluorescence signal within a time gate, a boxcar integrator [38] receives a temporal trigger signal [39] from the laser and, to normalize the fluorescence signal, a signal proportional to the laser energy [40]. A computer [41] controls a movable sample table [42] so that the sample areas can be measured one after the other. The standardized fluorescence signal is displayed above the sample location.

Legend

1 capillary

2 Bridge area between capillaries

3 Sample area

4 Capillary plate

5 Edge area of the capillary plate

6 Lower part of the synthesis table

7 Solvent resistant seal

8 Spacers 9 Threaded bolts

10 Top of the synthesis table

11 Mother

12 pistons

13 Washing solution inlet 14 Outlet

15 Reagent inflow

16 Lower part of the incubation table

17 Seal

18 spacers

19 Upper part of the incubation table

20 bracket

21 Pressure spring

22 ultrasonic transducers

23 pistons

24 Filler neck

25 Cavity

26 Procedure

27 Channel

28 Expansion vessel

29 Transparent window

30 Pulsed diode laser

31 Laser beam

32 beam splitters

33 Lens

34 Emitted fluorescent light

35 Bandpass filters

36 Lens

37 Photodetector

38 Boxcar Integrator

39 Trigger signal from laser

40 Signal from the laser proportional to the laser pulse energy

41 calculator

42 Movable sample table 10

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Claims (31)

1. Device for studying the molecular interaction of soluble or suspendable active substances with solid-phase-bound peptide or peptoid target molecules, characterized in that the surface required for this is created within a plate in the form of capillaries. The capillaries run from one side of the plate to the opposite side, but are not connected to one another. 2. Device according to 1, characterized in that a plurality of parallel capillaries are arranged in the plate, which draw liquids into the capillaries by capillary force and hold them therein. 3. Device according to 1, characterized in that the capillary plate is made of one of the materials glass, quartz, silicon, plastic, semi-metal, ceramic, metal or a combination of these materials. 4. Device according to 1, characterized in that the inner surface is capable of covalently binding target molecules or a synthesis of the target molecules takes place. 5. Device according to 4, characterized in that the inner surface is covered by an organosilane layer which has functional groups for anchoring peptide or peptoid target molecules. 6. Device according to 4, characterized in that γ-aminopropyltrialkoxysilane or other organofunctional silanes are used for silanization. 7. Device according to 4, characterized in that when using metal or semi-metal capillary plates, an oxide layer for binding the silane is created by surface oxidation of the capillary plate before silanization. 8. Device according to one of claims 4 to 7, characterized in that locally limited sample areas are created in the capillary plate. 9. Device according to one of claims 4 to 7, characterized in that a plurality of sample areas each with a diameter of up to 2,000 µm or a number of up to 4,000 adjacent capillaries are applied on a capillary plate. 10. Device according to 8 or 9, characterized in that the capillaries have a length and the capillary plate a thickness of 100 to 2,000 µm. 11. Device according to one of the preceding claims, characterized in that the functional group of the organosilane layer is temporarily protected by pipetting a substance onto the capillary plate. The area thus protected represents a sample area. 12. Device according to one of the preceding claims, characterized in that by pipetting a reagent onto the capillary plate, a protected anchor molecule is formed at each binding site of the sample area, the anchor molecule simultaneously serving as a spacer to the inner surface. 13. Device according to one of the preceding claims, characterized in that a protected sample area is created by pipetting on fluorenylmethoxycarbonyl (FMOC)-protected α-aminopoly(ethylene glycol)-ω-propionic acid N-hydroxybenzotriazole ester. 14. Device according to one of claims 11 to 13, characterized in that all non-protected areas of the capillary plate are chemically saturated. 15. Device according to 14, characterized in that the protective groups in the sample areas of the entire capillary plate are removed. 16. Device according to 15, characterized in that peptide or peptoid target molecules are synthesized location-dependently sequentially from amino acids in the sample areas of the capillary plate. 17. Device according to 15, characterized in that the complete peptide or peptoid target molecules are bound location-dependently in the sample areas of the capillary plate. 18. Device according to 16 or 17, characterized in that a substance library is constructed by the location-dependent different sequences of peptide or peptoid target molecules. 19. Device according to 18, characterized in that on at least one side of the capillary plate there is a closed and variable volume provided with a valve into which liquids with soluble or suspendable active substances as well as other liquids and gases can be admitted and sucked off. 20. Device according to 18, characterized in that gas bubbles can be released by coupling ultrasound to the capillary plate. 21. Device according to 18, characterized in that the reaction rates in the capillaries are increased by coupling ultrasound. 22. Device according to one of the preceding claims, characterized in that the binding of the active substances to the target molecules is detected. 23. Device according to one of the preceding claims, characterized in that a change in the target molecules can be caused and detected by the active substances. 24. Device according to 22 or 23, characterized in that the capillary plate is transparent or opaque and is freely accessible for optical measurement. 25. Device according to 24, characterized in that soluble or suspendable active substances are fluorescently labeled and are introduced into the capillaries. 26. Device according to 25, characterized in that after a certain time the unbound active substances are removed and the bound active substances are detected by means of the fluorescent label. 27. Device according to 24, characterized in that the solid phase-bound peptide or peptoid target molecules are fluorescently labeled. 28. Device according to 27, characterized in that soluble or suspendable active substances, acids or other liquids are flushed into the capillaries, flushed out again after a certain time and the presence of the still bound fluorescently labeled target molecules is detected. 29. Device according to 26 or 28, characterized in that the fluorescent label is excited and detected with light in the UV, visible or near infrared. 30. Device according to 29, characterized in that the fluorescence excitation is carried out with a pulsed diode laser with a pulse duration of less than 10 ns. 31. Device according to 30, characterized in that the detection of the fluorescence for suppressing scattered light takes place in a time gate after the excitation pulse has decayed.