WO2003099440A1 - Method for transferring molecules from a chemically reacting first flow into an adjacent chemically second reacting flow - Google Patents

Abstract

The invention relates to a method for transferring a molecule, molecule complex or microparticle located in a first flow into a second flow, which is flowing adjacent to the first flow and is in contact therewith at least in areas along an interface. The at least two laminar flows have different chemical compositions and, in particular, provoke reactions (e.g. catalysts, buffer solutions) that are incompatible with one another on the molecule, molecule complex or microparticle to the transferred. Due to the laminar flow, the chemical composition in both flows are preserved despite the existence of contact surfaces. In order to transfer the molecule, molecule complex or microparticle, an electric field is applied at least in the contact area of the interface of the at least two flows. This electric field is oriented essentially perpendicular to the direction of flow or to the directions of flow of the at least two flows.

Description

 Method for transferring molecules from a chemically reacting first stream to an adjacent chemically reacting second stream

The invention relates to a method for transferring molecules from a chemically reacting first stream into an adjacent chemically reacting second stream, that is to say a method in which molecules are selectively transferred between at least two chemically reacting flows.

Technical fields of application

The processing of biopolymers using chemical and enzymatic reagents often requires a large number of steps under different conditions in biotechnology. This requires an increasing number of steps, variety, parallelism and integration with increasingly programmable reaction management. Such biopolymer processing operations include synthesis, amplification, separation, selection, modification, cutting and assembly, labeling, detection, sorting, nano assembly and reaction control. Especially in "lab-on-a-chip" applications, a complete automated and self-sufficient sample processing is sought. The invention contributes to this broad technical field of application, in which computer-controlled and parallel transfer of biopolymers in small volumes between a large number of different reaction solutions.

State of the art

Many biotechnological processes are based on the combination of several reaction steps, which are often based on very different and incompatible re- action conditions are instructed. Working with restriction enzymes, ligases and polymerases are characteristic examples of this, which requires the change of reaction buffers. In other cases, it is necessary to process principally compatible substeps in a certain order. Changing the reaction conditions for biopolymers is complex and requires specific separation and concentration steps, for example the desalting of nucleic acids. It forms a significant technical barrier to integration if samples are not to be immobilized and processed identically. Regardless of whether these transfer steps are carried out automatically or manually, they increase both the time required and the risk of contamination and cause material losses on samples.

Highly parallel sample handling and laboratory automation in the field of biotechnology show two different approaches: open chip technology (DNA chips, antibody chips etc.) and closed microfluidic systems (lab-on-a-chip). Successful approaches to date to implement reaction systems in microstructures above 1000 samples are based on the miniaturization of open reaction chambers (ie the first category). Substantially serial pipetting robots are then required to transfer samples from one chamber to the next and limit the sample throughput when molecular transfer steps are required. This is especially the case when biomolecules in solution have to be switched from one reaction condition to another, e.g. to conduct reactions under a different buffer condition. Electroelution and electro-desalination are common but complex methods to process larger amounts of samples between reaction steps. They are used between liquids at rest.

Different reaction conditions can be brought into close proximity in closed microsystems with the help of a laminar flow technique. Here, two or more flows are run parallel or antiparallel side by side in a reaction chamber. The low Reynolds number and the small diffusion coefficients (compared to contact times) lead to a negligible mixing of the two solutions at the interface. Such a technique has been proposed previously. Biomolecules can then (e.g. on magnetic beads) be transferred from one solution to the next. However, this transfer is relatively difficult to integrate and control; a parallel individual reaction with magnets is complex.

The combination of microreactor systems with electrical fields is generally limited to arrangements that generate fields in the longitudinal direction of the flow and thus only use the electrophoretic effect for particle separation or serve to transport the particles. The fields created can be pulsed or harmonically modulated fields that are actually capable of avoiding electrolysis within certain parameter limits (concentration, current density, potential differences). An adaptation to the particle types, however, is hardly possible. The use of protected electrodes by gels or polymers prevents the rigid attachment of ions and the redox conversion of the particles to be transported. The static biochips currently mostly used only allow a few process steps per chip. This makes further process integration very difficult.

task

A large number of individually programmable reaction steps in parallel biopolymer samples are to be carried out in small volumes under different reaction conditions. This should be done without complex separation or pipetting steps or mechanical or hydrodynamic switching, which prevent integration for highly parallel sample throughput. solution

To achieve this object, the invention proposes a method according to claim 1; individual embodiments of the invention are specified in the subclaims.

The invention therefore describes a method for converting a molecule, molecular complex or microparticle in a first flow into a second flow flowing adjacent to the first flow and contacting it at least in regions along a boundary layer, the at least two laminar flows having different chemical compositions, which contain, in particular, mutually incompatible reaction agents (for example catalysts, buffers) in order to cause reactions on the molecule, molecular complex or microparticles to be transferred, it also being provided that

that the chemical compositions in the two flows are maintained despite the contact areas by the laminar flows and - that at least in the contact area of the boundary layer of the at least two flows for the transfer of the molecule, molecular complex or microparticle an electrical field is applied that is essentially transverse to the direction of flow or is directed to the flow directions of the at least two flows.

The invention thus proposes (bio) molecules or (bio) molecule complexes induced by electrical fields from a first flowing medium with which the molecule can initiate a (bio) chemical reaction into a second flowing medium to which the molecule can also react (bio-) chemically. The transfer of the reactant in the form of the (bio) molecule or complex thus takes place in the flow. With the method according to the invention, it is possible to selectively address molecules, molecular complexes or particles in the flows by applying electrical fields which are directed essentially across the flow. The peculiarity of the invention consists precisely in this selective addressing, which takes place through the parameters of the electric field. Inhomogeneous electric fields have proven to be particularly advantageous for transverse transport. The transverse transport according to the invention preferably utilizes the dipole moment of a molecule, a molecular complex or a particle, which follows this in particular in an inhomogeneous electric field.

According to the invention, the type of application of the electric fields is selected such that the flowing media are not exposed to any electrochemical reactions.

Outlines of the solutions

1. Different miscible solutions are carried out under parallel or anti-parallel laminar flow conditions in direct contact over a certain distance without mixing overall. This can be done in closed microreactors in channels. Under the flow velocities used (<< 1 m / s) and dimensions (channels with widths and depths on the scale of lμm - 10mm), hydrodynamic turbulence is not present and thermal convection is negligible. In the absence of electrical fields, the exchange of molecules between neighboring rivers only takes place via diffusion. Diffusion causes the liquids to mix only within a boundary layer (mixture layer). The width of the mixture layer increases with the contact length and depends on the flow rate. Typical diffusion coefficients of biomolecules e.g. B. are in the range 10 ^"u m ² / s. 2. Since parallel / anti-parallel laminar flows do not mix, very different reaction conditions can be brought into close proximity. This is of particular interest if components of the individual solutions mutually interfere or consume each other through inhibition or unwanted reaction. Instead of allowing the individual non-compatible reaction steps to be spatially separated and run one after the other, only the biomolecule of interest is transported from one reaction solution to another across the flow direction of the rivers. This eliminates additional intermediate steps such as desalination or cleaning.

3. Due to the direct contact of the different solutions, charged molecules can be transferred from one solution to the next by means of an electric field without being hindered by a barrier. Electrodes are inserted into the channel in a sequence that runs transversely to the direction of flow so that they do not disturb the flow conditions. The electrode spacing ranges between lμm and lOOμm depending on the channel width and task. If the induced transport path is longer than the width of the mixture layer, molecules can be affected by a reaction condition, i.e. Reaction solution to the other (and returned if desired). The molecules can be the biomolecules themselves or other reagents (see Fig. 1).

4. The electrodes are addressed individually and pulsed with digital voltages or by means of analog voltages in such a way that particle transport starts due to electrophoresis or electromigration across the flow direction. By using digital voltages, with which the pulse duration and duty cycle can be set, electrolytic phenomena, especially in the high salt area, can be avoided by undermining the electrode kinetics. The well-known use of protected

Electrodes with gels or polymers prevent the rigid attachment of ions and the redox conversion of the particles to be transported. 5. The individual control of the electrodes also makes it possible to adapt the electrical parameters to the types of particles to be moved, so that neighboring types of particles are influenced less or not at all.

6. The solutions are put together in such a way that the mobility of the ions and the degree of dissociation of the auxiliary substances (conductive salt, buffer) have little effect on the movement of the actual particles due to shielding effects on the electrodes or mixing of the buffers or reaction solutions, especially at the interfaces ,

7. By introducing a partition with short connecting channels at regular intervals, after short transfer periods under constant flow conditions, time for mixing processes and reactions in the separate solutions can be left without the two solutions mixing during this time (see Fig. 2).

Improvements and advantages over the prior art

The use of the neighboring 'laminar flow technology allows a high degree of integration and prevents contamination. The electrode-controlled transfer of biomolecules between solutions is quick and reversible and can even cause the samples to concentrate with minimal losses.

The use of the digital fields enables individual control of a large number of electrodes, so that the particles can be specifically addressed and disruptive effects such as electrolytic phenomena can be avoided. Very small field strengths can be achieved by using small rectangular electrodes with small distances between them. The fields can be made inhomogeneous, so that particles with a stronger dipole than ion character can also be influenced effectively.

By using laminar flows, reactions with very different reaction environments can be combined in close proximity. There is no inhibition or unwanted reaction between reaction components of different process steps. No additional cleaning steps to remove unnecessary or disruptive reaction components, by-products or degradation products are required. Among other things, the response time is reduced. In addition, the risk of contamination from manual steps is reduced. There is no need to switch between different reaction environments. This eliminates problems such as caused by a change in the surface condition (adsorption). No complex technology (valves, switches, temperature gradients) is necessary.

Application example of the invention

Chemical amplification of nucleic acids:

The polymerase chain reaction (PCR) is a standard method for amplifying DNA. It is based on the alternating denaturation of template DNA, subsequent hybridization of specific primers and their elongation. The classic denaturing takes place thermally, but can also be triggered chemically. A pH change can be used for chemical denaturation (50-100mM NaOH), but also substances such as urea (8.3M) or formamide (50%). If the laminar flows from Fig. 1 are an enzyme solution and a denaturing solution, DNA can be alternately transported into the enzyme / amplification and denaturing solution using the electrodes. This fulfills the prerequisite for amplification. A transition from the enzyme / amplification solution to the denaturing solution and back corresponds to a classic PCR cycle. Alternatives are

1. Molecular diagnostics:

The clinical diagnosis of pathogenic organisms usually consists of several reaction steps, the reaction conditions of which are incompatible. The most important steps are the disruption of the cells and the extraction and detection of the nucleic acid. As a rule, it is possible to disrupt cells by adding lysis buffer in order to release genomic or plasmid DNA. After digestion, unnecessary cell components, such as cell wall pieces or proteins, have to be removed. This DNA preparation can be solved by adjacent laminar flows if the cells are placed in cell lysis buffer in one channel and a transport buffer in the adjacent channel. According to the invention, the DNA will be transferred from the cell lysis buffer into a transport buffer using electrical fields which are directed transversely to the direction of flow and can be used directly for further manipulations, such as restriction, or for detection, e.g. by means of hybridization.

2. Restriction / ligation:

A problem with the multiple restriction digestion of DNA is the sometimes very different buffer conditions of the individual restriction enzymes. Adjacent laminar flows can solve this problem if the various buffers with the corresponding enzymes run side by side and only the DNA is transferred. The cut DNA can be ligated in a further channel with the appropriate enzyme / reaction solution with a target sequence. 3. DNA synthesis:

The synthesis of nucleic acids consists of the repeated sequence of several work steps. A start nucleoside is elongated by coupling 5 'chemically protected nucleosides. The protective group is then removed and the next nucleoside is coupled. At the end of the synthesis, the product is chemically oxidized. The individual steps can be solved by the arrangement of the corresponding laminar flows and the electrode-induced transport of the growing synthesis product according to the invention.

The drawing, to which reference has already been made, shows:

Fig. 1 A microstructured channel system with sudden contact of the laminar flows. The white arrows indicate the direction of the laminar flows (1). (2) marks the contact point of these rivers. The dark rectangles (3) stand for electrode arrays with which components can be transferred from one flow to another. The method is not limited to two parallel channels

(left) but also works with 3 or more rivers (right).

Fig. 2 A microstructured channel system with a switching channel (4). The labeling is analogous to Fig. 1. Here, too, the process is not limited to two channels.

Claims

EXPECTATIONS 1.Method for converting a molecule, molecular complex or microparticle in a first flow into a second flow flowing adjacent to the first flow and contacting it at least in regions along a boundary layer, the at least two laminar flows having different chemical compositions and in particular reactions incompatible with one another ( cause catalysts, buffer solutions) on the molecule, molecular complex or microparticle to be transferred, characterized in that the chemical compositions in the two flows are maintained despite the contact surfaces by the laminar flows and that at least in the contact area of the boundary layer of the at least two flows for transferring the molecule , Molecular complex or microparticle, an electric field is applied which is essentially transverse to the flow direction or to the flow directions n which is directed at least two currents (Fig. 1, 2). 2. The method according to claim 1, characterized in that more than two flows are provided which at least partially make contact along boundary layers and that electric fields are selectively applied essentially transversely to the respective boundary layers between two flows (Fig. 1, right). 3. The method according to claim 1 or 2, characterized in that the flows are guided in an integrated microreactor network (Fig. 1, 2). 4. The method according to any one of claims 1 to 3, characterized in that the contact area extends along the entire boundary layer (Fig. 1). 5. The method according to any one of claims 1 to 3, characterized in that the boundary layer is formed in spaced apart passages forming the contact areas (Fig. 2). 6. The method according to any one of claims 1 to 5, characterized in that each contact area is assigned at least one group of at least two electrodes for applying an electrical voltage for generating an electrical field, the electrodes of each group being substantially transverse to the flow direction or to follow the flow directions one after the other (Fig. 1, 2). 7. The method according to claim 6, characterized in that each group has a plurality of electrodes arranged side by side and that an electric field is generated sequentially between adjacent electrodes (Fig. 1, 2). 8. Use of the method according to one of claims 1 to 7 for automated multi-step reaction control of biomolecules, the individual steps having to be done in a defined order. 9. Use of the method according to one of claims 1 to 7 for programmable reaction control, wherein the sequence and choice of the individual reactions can be controlled by programming the temporal electrode voltages. 10. Use of the method according to one of claims 1 to 7 for molecular diagnostic applications. 11. Use of the method according to one of claims 1 to 7 for molecular computing. 12. Use of the method according to one of claims 1 to 7 for drug screening. 13. Use of the method according to one of claims 1 to 7 for "lab-on-a-chip" laboratory automation.