DE19642777A1 - Reactor for microchemical or microbiological syntheses - Google Patents

Abstract

The invention relates to a pipette which can be used in laboratory automation for a large number of processes for the miniaturization, parallelization and simplification of filtering processes. The aim of the invention is to create a pipette which filters absorbed liquid, which acts as a reactor for microchemical, microbiological or physicochemical reactions and which is particularly suitable for laboratory automation and the processing of large numbers of samples in the ml - sub ml range. Said aim is achieved by providing the pipette (1) in the area of its tip with an integrated means of obturation (3,4) and a filtering element (5).

Description

The invention relates to a reactor for microchemical or microbiological syntheses according to the preamble of the claims. she is particularly for use in the field of highly parallel chemical synthesis determined.

There are relatively large-lumen reaction vessels to carry out known microchemical reactions from a container with at least one opening, the for solid phase reactions Openings can be covered with filters or frits. These serve the combination of several substances in solid, dissolved or liquid form. To carry out reactions with liquid components, the filled liquid substances into the reaction vessel using a pipette. In recent years, there has been a development to simplify the Laboratory enforced; it consists in immobilizing one Reaction partner. Elaborate precipitation or extraction work are therefore superfluous. Carrying out the solid phase reactions only requires the addition of solutions and subsequent Filtration or suction steps to remove those in solution Remove the reaction partner again. The procedure is relatively easy too automate and is suitable for handling large numbers of samples in the ml volume range. It is used regularly at today biochemical or chemical reactions such as peptide synthesis, oligonucleotide synthesis, combinatorial chemistry, bioassays such as ELISA or RIA and chromatography in a "batch" process, such as ion exchange during DNA purification.

The liquids are usually dosed using pipettes. The solid phases are in a vessel with or without a filter base. From the AMS 422 product information from Abimed (PO Box 11 11, 40736 Langenfeld) is a one-way flow reactor with inserted Frit known. However, this system is relatively large and not for parallelization and automation are suitable. Is the Solid phase in the vessel with filter bottom, centrifugal forces or  Vacuum applied to separate the solutions from the solid phase. In addition to the filters, devices for collecting the Liquids needed. The solid phase is in the vessel without Filter bottom, the solutions are removed by pipetting, whereby care must be taken to ensure that the solid phases do not enter the pipettes reach. This problem led to the development, for example of pearls with a magnetizable core, which are created by the application of a magnetic field can be drawn to the vessel wall and thus less are at risk of being detected by the pipetting step.

As is well known, highly parallel chemical syntheses allow the representation of substance libraries in a comparatively very short time. Such Syntheses are usually carried out on a solid support material carried out. This facilitates the processing of the samples and that Shift the reaction equilibrium. The carrier material for the Synthesis is generally chemically modified Polymer resin balls, polymer resin blocks or layers, or glass beads or plates.

There are two types of highly parallel synthetic processes: the synthesis in the mixture and the synthesis of individual samples. Mixing methods have the disadvantage that the information about the Identity of the substances or their synthesis protocols is lost and through resynthesis and / or complex biological test procedures must be procured (deconvolution process).

One solution is the "mix-and-split" synthesis. Here are Substance libraries created with the goal, per polymer resin ball only to represent a substance. The identity of the substance can be derived if enough material is available for analysis. Since the However, the amount of substance that can be obtained from a pearl is often very small are, or are required for the bioassay Coding method developed; in a parallel synthesis Reaction history of the pearl chemically on the polymer resin captured.

This contrasts with methods in which all components of the Substance pools in areas that are spatially separated from the outset be synthesized. The solid phase carrier material is in a  given predetermined two-dimensional arrangement ("array"); the is the respective reaction protocol or the corresponding target structure usually defined by an xy coordinate. A very illustrative Example was developed by Fodor et al in the article "Light-Directed, Spatially Addressable Parallel Chemical Synthesis "Science 251 (1991) pp. 767-773 published. In it are photosensitive Protective groups coated glass supports by photolithographic Processes broken down into a thousand microscopic substance fields.

Other methods also use glass plates or pearl-filled ones micro-compartmentalized silicon wafers, "chips".

Synthesis on rods coated with polymer resin (pin Technology, see "Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid "H.M. Geysen et al, Proc. Natl. Acad. Sci. USA 81 (1984) pp. 3998-4002) has the advantage with that widely used 96-well microtiter plate format compatible be. The ones that have already been tried and tested in solid phase synthesis Backing materials can be used with the commercial variant of the pin Technology cannot be processed. However, pins are already there have been described, which are filled with glass or polymer beads can ("Diversomers: An approach to nonpeptide, nonoligomeric chemical diversity "S. Hobbs De Witt et al, Proc. Natl. Acad. Sci. USA 90 (1993) pp. 6909-6913). In all of these cases it affects desired parallelization and automation of the work steps unfavorable that the pins, chips or tubes are only carriers for the fixed Represent phase; the fluid supply is not integrated in the system and must be done separately by immersion or rinsing, see the Review article "Multiple Peptide Synthesis Methods and Their Applications "G. Jung et al, Angew. Chem. Int. Ed. Engl. 31 (1992) pp 367-383, also in Angew. Chem. 104 (1992) p. 375 ff.

The invention is therefore based on the object, a simple assembled reactor for microchemical or microbiological To create syntheses that are significant in laboratory operations Simplification and acceleration of the synthetic steps as well as a Reduction of the instrumental effort in the synthesis enables  and also for laboratory automation and Processing large numbers of samples in the ml range is particularly suitable.

According to the invention, this object is achieved by the characterizing Features of the first claim solved. The present invention thus includes a reactor that combines a pipette and represents a reaction vessel. This type of reactor and pipette has or in the lower area, near the pipette tip multiple outlets and / or one covered with filters or frits Opening for the inlet and / or outlet of the liquids and one at the top Approach to connecting a dosing aid. Can be advantageous within the Pipette an openable closure means, preferably in the form of a magnetizable ball can be provided with the peripheral wall the pipette or by storing it on an inserted ring Ball valve forms.

It was found that the pipette and the reaction vessel were combined in one element surprisingly essential for simplification and Acceleration of the synthesis process steps contributes. Centrifuges, Collecting vessels, filters and suction or precipitation devices and the invention makes them unnecessary to operate.

Another unexpected benefit is the shorter response time through better mixing of the reactants. When unit of The liquid phase can be pipette and reaction vessel as often as required the dissolved reagents are taken up and released again are in a particularly efficient way on the solid phase passed by. The reactions are much faster Bring reaction balance.

Conventional filtration or suction processes do not allow this and either have to put up with much longer incubation times or take action to get through the solid phase in the reaction vessel To move gas flows, mechanical shaking or ultrasound, or in an oven or bath or block.

The invention encompasses any combination of chemical or biological modified surfaces and a pipette tip. The modified Surface can be part of the pipette tip inner surface,  or the pipette tip can be used as a container for polymer resin beads or Serve glass particles. Furthermore, the pipette tip can be used as a holder for Glass capillaries or glass vessels act, responding to Solid phase surfaces takes place in the glass containers. The invention also includes pipette tips for homogeneous chemical and enzymatic synthesis, as well as for immunoassays. Here are presented reagents in covalently bound, adsorbed or adherent form in or on the pipette tip.

The commercially available disposable pipette tips for volumes are preferred from 0.01 to 5 ml, which with reactive solid phases in bulk or structured form or filled as a block and have a filter, to allow the exchange of liquids while doing the Hold back solid phases.

A pipette tip made of polypropylene with a Volume from 0.01 to 1 ml, which is filled with synthetic resin and their lower opening with a magnetizable steel ball or glass ball is locked; its side wall can be drilled with 0.1 mm Diameter must be perforated. The perforation allows the exchange of Liquid. If necessary, the ball can be magnetically lifted from the outside to remove the synthetic resin from the tip or the Allow entry of synthetic pearls into the tip.

The reactor according to the invention can be used in this way, for example be that the pipette tips with chemically or biologically active Molecules or groups are loaded. This modified Pipette tips are then - preferably by automatic Manipulation - filled with solvents or washing reagents. Here find chemical syntheses or biological ones in the pipette tips Interactions instead.

A manual synthesis of a dipeptide is carried out as an example. For this purpose, a capillary (2 ml volume, EM) with 80 beads is used an amino acid and an acid-labile linker Polystyrene resin (Fmoc-Ala-HMPB, Novabiochem) filled and into one Pipette tip inserted. The capillary is made using a automatic pipette (Eppendorf) five times with 20% piperidine / DMF and  five times with a solution of Fmoc-Phe-OH (0.25 M), HBTU (0.25 M), HOBt (0.25 M), DIEA (0.5 M) in DMF and rinsed three times with DMF. The beads are transferred to 0.25 ml 5% TFA / DCM and after 15 min filtered off. The filtrate is evaporated and in 0.1 ml acetonitrile / 0.1% TFA added. 20 ml are linearized by means of HPLC (RP 18 column, Gradient from 5 to 95% acetonitrile / water in 35 min, UV detection at 220 nm) analyzed. Peak identification is done by comparison with Reference substances. The HPL chromatogram shows the product Fmoc- Phe-Ma-OH (retention time tR 23,088 min) in high purity and good Yield.

An automated synthesis is carried out, for example, using the dodecapeptide H-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln-OH. Ten perforated pipette tips with ball valves were filled with 2 mg synthetic resin Fmoc-Gln (Trt) - SASRIN (Bachern, Heidelberg). The tips are placed on a pipetting robot (Tomtec Quadra, Zinsser Analytic, Frankfurt) and subjected to eleven synthesis cycles by sucking up, incubating and dispensing reagents placed in microtiter plate vessels. A synthesis cycle consists of the following steps:

• 1 × incubation for 2.3 min with 0.05 ml dichloromethane,
• 2 × incubation for 2.3 min each with 0.05 ml dimethylformamide (DMF),
• 4 × 2 min incubation with 0.05 ml 20% piperidine / (DMF) from one Vial with a total of 0.15 ml reagent solution,
• 1 × 2 min incubation with 0.05 ml dichloromethane,
• 3 × 2 min incubation with 0.05 ml DMF,
• 6 × 2.25 min incubation with 0.05 ml 0.5 M Fmoc-amino acid OP- Ester, 1 M hydroxybeniotriazole (HOBt)) 1 M diisopropylethylamine (DIEA) from a tube with a total of 0.17 ml reagent solution,
• 1 × incubation for 2.3 min with 0.05 ml DMF,
• 2 × incubation for 2.3 min each with 0.05 ml acetic anhydride / DIEA / HOBt,
• 1 × incubation for 2.3 min with 0.05 ml dichloromethane.

The products obtained from the pipette tips are included Trifluoroacetic acid / water split off, deprotected and synthetic resin isolated by ether precipitation. The analysis of these products with High pressure liquid chromatography (HPLC) shows purities of over 95%; their analysis using mass spectrometry confirms the  Correctness of structures. Differences between the products from the different pipette tips were not found. The quality of the Substances are of the same quality as that of a commercially available, HPLC-purified reference substance (hirudin 54-65 desulfated, Bachem, Heidelberg).

The invention is described below with the aid of the schematic drawing of four embodiments explained in more detail. Show it:

Fig. 1 is a pipette tip with a magnetic ball,

Fig. 2 is a closed bottom pipette tip,

Fig. 3 shows a pipette tip, which is closed by frit or filter with a reactive surface and

Fig. 4 is a sealed through frit or filter without reactive surface pipette.

In Fig. 1, a reactor according to the invention is shown in which a conventional in itself pipette 1 having a neck 2 for a not shown dosing, slot-like in the lower, narrowed peripheral portion at the inlet or outlet-side end 3 with perforated or Openings 4 is provided. In the end 3 on the inlet or outlet side, a ball 5 forming a ball valve is also provided, which is preferably made of a magnetizable material. A reactive carrier 6 in pearl shape is introduced above the ball 5 , the fill level of these pearls preferably being dimensioned such that all openings 4 are detected. The openings 4 serve for the exchange of liquid, while the ball valve formed by the ball 5 and the inlet or outlet-side end 3 serves to receive or remove the beads (particles) 6 from the tip of the pipette 1 .

Fig. 2 represented a second embodiment illustrates the pipette 1, but which, is carried out of the pipette sealed exit the lower, narrowed end 3. In this embodiment there are again hole-like or slot-like openings 4 and a reactive carrier 6 in the form of beads, but no ball valve is provided.

In Fig. 3, a pipette 1 is provided at the inlet or outlet end 3 with a frit or a filter 7 , which is given a correspondingly reactive surface, so that the separate reactive carrier in pearl form can be omitted. The frit or filter 7 contains the openings for the liquid exchange.

Fig. 4 illustrates a further embodiment in which the outlet side end on or 3, although sealed with a frit or filter 7, the reactive carrier is 6, however, in turn, introduced in the form of beads in the pipette. 1 This has the advantage that the reactive support 6 is easier to replace if necessary.

Claims (11)

1. Reactor for microchemical and / or microbiological reactions, which comprises a pipette with a dosing approach, characterized in that a reactive carrier with at least one immobilized reaction partner is provided in the pipette near its lower, narrowed end. 2. Reactor according to claim 1, characterized in that the reactive Carrier is formed in the form of particles. 3. Reactor according to claim 1, characterized in that the reactive Carrier is designed as a frit or filter. 4. Reactor according to claim 2, characterized in that the pipette in their lower peripheral area with hole or slit-shaped Openings are provided. 5. Reactor according to claims 2 and 4, characterized in that the particles the peripheral area with the hole or slit-shaped Remove openings. 6. Reactor according to at least one of the preceding claims, characterized in that the pipette is narrowed at its lower End is closed. 7. Reactor according to claim 6, characterized in that the pipette its lower, narrowed end can be closed with a ball valve is designed. 8. Reactor according to claim 1, characterized in that the pipette its lower, narrowed end with a frit or a filter is provided.   9. Reactor according to claim 7, characterized in that the ball of Ball valve is designed magnetizable. 10. Reactor according to claim 1, characterized in that the pipette Polypropylene. 11. Reactor according to claim 1 or 10, characterized in that the Volume of the pipette comprises 0.01 to 1 ml.