EP1833608A1

EP1833608A1 - Microreactor

Info

Publication number: EP1833608A1
Application number: EP05850267A
Authority: EP
Inventors: Matthias Franzreb; Tilmann Rogge
Original assignee: Forschungszentrum Karlsruhe GmbH
Current assignee: Karlsruher Institut fuer Technologie KIT
Priority date: 2004-12-24
Filing date: 2005-12-14
Publication date: 2007-09-19
Also published as: DE102004062534B4; WO2006069627A1; DE102004062534A1

Abstract

The aim of the invention is to create a device that provides a microfluidic system in which particle fractions (beads) can be guided in a serial and directed manner through ducts and reactions chambers without creating a net fluid movement. Said aim is achieved by combining a small-scale fluid movement that causes the particles to move with a switchable force (blocking force) which fixes the particles. The inventive device can be used in bioanalysis or chemical synthesis.

Description

microreactor

The invention relates to a device for transporting at least one magnetic particle fraction through a microfluidic system according to the preamble of the first claim.

Microfluidic systems are central handling systems for fluids, such as liquids or gases, with or without solids in micro- and nanotechnology, and are found particularly in the field of life sciences or biomedicine, where nano-objects in the form of large biomolecules, e.g. Peptides or proteins, must be handled [I]. Since direct handling of such small objects is seldom possible, so-called beads are often used in the field of life sciences. Beads are polymer bodies, usually spheres, on the functionalized surface of which e.g. DNA or proteins bound and so become manageable for synthesis or analysis. In this growing market, commercial devices are already being offered today, with which analyzes are carried out with the aid of individual beads [2]. In addition, there are several bead-based analytical methods or devices that have a high degree of parallelization and operate with liquid volumes as low as 10 microliters. Frequently, electrical fields [3] or so-called laser tweezers [4] [5] are used for the targeted handling of such beads. Magnetic forces are rarely used in microtechnology, as they are difficult to produce microtechnically, with magnetic forces being particularly suitable due to the low interaction with biological materials and processes [6].

Magnetic beads are commonly used in biochemistry today and are distributed by several commercial vendors (eg http://www.magneticmicrosphere.com/supply.htm). Such beads are usually superparamagnetic and monodisperse with diameters from 1 micron to 10 microns available and are used for analysis and synthesis purposes. The handling of magnetic microbeads can be done on a larger scale with the help of so-called high-gradient magnetic separators [7]. In the case of smaller volumes, a separation or fixation of magnetic microbeads usually takes place by simple permanent magnets based on rare earths. However, this approach is quite inflexible and requires to release the fixation always moving components that allow a spatial separation between the reaction vessel with Magnetbeads and permanent magnet. By contrast, a procedure in which the magnetic beads are brought into the area of influence of soft magnetic structures is considerably more flexible. To fix the magnetic beads, the structures are magnetized via an external magnetic field. To release only the external magnetic field has to be switched off, ie no moving component is necessary. A corresponding structure was developed and patented for the separation of magnetic beads from so-called microtiter plates [DE 10 057 396].

Critical point in the work with biochemical substances in biopharmaceutical and pharmaceutical research are the high costs of the substances produced in part by elaborate synthesis methods. The actual investigations require only small quantities of material, as new analysis methods show (for example Genechip ©, the company Affymetrix, www.affymetrix.com), but an economical handling of these substances is difficult. Microfluidic systems are ideal for working with such materials due to their low dead volume. However, this advantage diminishes when it is necessary to flush the system completely to introduce a new substance into the microfluidic system.

The object of the invention is therefore to provide a microfluidic system in which particle fractions (beads) are guided serially and directionally through channels and reaction chambers without a net movement of the fluid taking place.

The object is achieved by a device having the features of the first patent claim. The dependent claims give advantageous embodiments again.

Mass transfer in fluidic systems usually takes place via the movement of the fluid, with which the substances contained therein are moved to different locations.

The mass transfer within the device according to the invention is not carried out as usual by the flowing fluid, but by the Transport of the beads on the principle of a "fluidic ratchet".

By generating a blocking force during the movement of the fluid, the beads can be fixed.

Ratchet is the name for a device z. B. tool in which a locking device allows only one direction of movement (freewheel), in the opposite direction it locks and moves an object z. B. screw or belt. The directions of movement can be reversible. The device according to the invention involves the provision of a microfluidic system in which particle fractions (beads) can be guided serially and directionally through channels and reaction chambers without a larger-scale fluid movement taking place. For this purpose, a small-scale fluid movement, which causes a movement of the particles, combined with a switchable force (blocking force), which at least fixed but at least significantly reduces the speed of movement of the particles. The fluid movement may be generated mechanically or electrically (e.g., electro-osmosis). The blocking force can be generated by magnetic fields acting on magnetic beads, by electrochemical induced electro fields induced by electrostatic fields due to dielectric constant differences of fluid and particles (dielectrophoresis, electrostatics) by optical fields generating forces due to refractive effects according to laser tweezers Surface forces that attach to the bead surfaces.

An actuator generates a periodic, small-scale forward and backward movement (freewheeling) of the fluid in the channel system. By generating an inhomogeneous magnetic field (blocking device) during the return movement of the fluid, the beads can be fixed during the return movement. Due to the fixation during the return movement of the fluid and the solution of the fixation during the forward movement, a directed movement of the beads through the channel system results without a net movement of the fluid. The directions of movement can be reversed.

Superparamagnetic particles are introduced into a fluidic channel system. As long as no other forces act on these particles, These particles are carried along with every movement of the fluid in the channel system. If a movement direction of the particles is blocked during a periodic fluid movement, the particles are transported in one direction. The surrounding fluid is moved by this periodic movement only by the volume amount of the particles in the reverse direction. The periodic movement of the fluid otherwise leads to no significant mixing, since in the smallest channel systems, a turbulent mixture is extremely difficult to achieve. The volume of the reaction chambers is extremely low, whereby the required amount of reactants is very low. Channel dimensions of a few micrometers and volumes of the reaction chambers in the nanoliter range are achieved.

Magnetic forces

In order to create a bead movement on the principle of a fluidic ratchet sufficiently large magnetic forces must be generated and suitable magnetic beads are available. The magnetic blocking force on the superparamagnetic particles should preferably be in the range of 10-100 pN. The magnetic force on the particles results from the volume and the susceptibility of the particles as well as from the product of field strength times the gradient of the magnetic field. While the achievable field strengths are limited to a few teslas, very soft field micrographs can generate very high field gradients over short distances.

The magnetic holding force is achieved by soft magnetic microstructures immediately adjacent to the fluid region, which distort an externally generated magnetic field. The smallest, lateral dimensions of these structures should correspond approximately to the diameter of the beads used, while the vertical dimensions should be three to ten times. The production takes place after resist structuring with mask technique by electroplating. Subsequently, the structures are cast with plastic. The plastic fulfills two functions. On the one hand, this creates a flat surface, which does not affect the bead movement. adversely. On the other hand, the plastic serves as a bonding partner for the

Housing part with the fluidic channel structures.

Exemplary production of a soft magnetic microstructure

1. Deposit on substrate (silicon or glass) electroplating start layer

2. Spin up the resist and structure it

3. NiFe electroplating

4. Spin on the sealing layer

Essential for the use of magnetic forces in micrometer dimensions is the generation of highly inhomogeneous magnetic fields, it is shown that even without soft magnetic microstructures the> 10pN for 4μm particles can be achieved [9]. By using soft magnetic microstructures, the particles can be much smaller or the background magnetic field weaker. Suitable are u. a. soft magnetic structures made of permalloy (80% Ni and 20% Fe). For example, Permalloy columns with a diameter of 5 μm and a height of 90 μm can be produced by X-ray lithography and electroplating with a saturation magnetization of 0.93 T [10].

Fluidic system

The fluid transport of particles through channels and along surfaces has been studied for many decades and is described in detail [11].

In addition to the geometric parameters and the surface forces, the particle motion depends on the flow velocity of the fluid, and can be well realized with spheres [12] as well as biological entities such as cells [13]. If no turbulences are formed during the periodic fluid movement, it is expected that the mass transfer within the fluid will not be significantly greater than the diffusion rate. As the extensive literature in the field of micromixers shows [14], [8] the intended induction of turbulence in microfluidic systems is difficult to achieve. The device according to the invention fulfills various requirements for this purpose. The fluid movement must be large enough to move particles through the fluid. These are in the channel system flow rates of about 1 - 10 mm / s necessary. The speed of the

Particles in the fluid channels from the ratio of channel size to particle size, the fluid velocity, the adhesion of the particles to the channel walls and the shape of the particles from.

The fluid channels must be designed so that a periodic fluid movement within the fluidic structures can spread well. It is important that the system is sufficiently incompressible and does not store the fluid movement elastically. Since the flow rate and thus also the movement of the beads depends on the channel cross-section, the flow rate can also be varied within the system. Thus broadening of the channel cross-section in the region of the reaction chambers can extend the residence time. The filling of the reaction chambers and the continuous supply of reactants are ensured by a slow flow through the reaction chambers perpendicular to the direction of movement of the beads. This also allows the complete replacement of the ingredients of individual reaction chambers.

The fluidic channels should have a cross-section that approximately corresponds to the bead size. For example, with a bead size of 4 μm, the channel width and height should not exceed 10 μm. Structures with these dimensions can be produced both by photolithography and X-ray lithography. Which method is most suitable depends on the required structural quality and the suitable plastics.

The production of microstructures is carried out in many ways: with optical lithography (SU8, polyimide), by hot stamping (mold insert production by LIGA method or machining technique) or by X-ray deep lithography. As a result, it is possible to meet even the highest demands on structural dimensions, down to the submicrometer range, with optical quality of sidewall roughnesses and aspect ratios of 20 or more.

bonding

A bead movement within the fluidic fluid generated by the fluid

Systems requires good propagation of fluid movement within of the fluid area. Air pockets or deformations of the microstructures would interfere and must be avoided. Furthermore, variations in the channel geometry lead to changes in the flow velocity. Therefore, it is important to make a pressure-resistant bond with little variation in bond area thickness. For plastic structures, sealing processes are suitable in which thin sealing layers are produced by photodegradation (see above) or spin-coating and then connected by pressure and heat in a corresponding bonding device.

With the help of bonding methods, it is also possible to produce much smaller fluidic structures than before with typical channel cross-sections of 50 μm x 50 μm.

actuator

For the construction of the device according to the invention microfluidic actuators is necessary at least at one point. Firstly, a periodic fluid movements must be generated, and / or requires the work with minimal amounts of material, for example, a metering devices with fast switching times. Piezo actuators are suitable for both tasks, for example. Thus, a piezo-operated microvalve with switching times of less than 2 ms, whose design principle [DE 199 49 912] is likewise suitable for generating a periodic stroke, is available.

Particular advantages of these actuators lies in the short switching times (typically one millisecond) and the large force generated thereby. The coupling of the mechanical movement into the system can take place either directly or via a translation system. Alternatively, actuators can be represented via pressure spring systems or by shafts to electrically operated motors.

Connection concept of fluid supply / Produktentnähme

The functional principle of the device according to the invention requires a periodic fluid movement, which can only be used efficiently if the system is incompressible and the fluid has only a freely movable interface at the exit (eg gas bubble). This requires Rigid fluid supply or high flow resistance in the fluid supply area. Furthermore, a simple Bead-removal is possible at any time. For this purpose, the beads are collected in at least one chamber and flushed out as needed.

The synthesis of proteins, peptides and the like a. has become increasingly important in recent years. It is not only the cost-effective production of large amounts of material technically interesting, but also methods of flexible production of small amounts of material that get along with very small amounts usually extremely expensive precursors. The required amounts of substance are only a few nanograms, so that even for a simple prototype of the biosynthetic reactor with the production of sufficient amounts of substance is to be expected. This makes it possible to qualify and quantify the synthesis reaction when the process parameters are varied. In a Merrifield solid phase synthesis (AMS) adapted to magnetic beads, targeted peptides are generated. To beads with specifically cleavable spacers (spacer) which carry at their end the starting molecules for the AMS is carried out by means of the device according to the invention, the AMS to the desired peptide length. For this purpose, the beads are guided through the individual reaction areas of the device. The device according to the invention allows for purposes in which only small amounts of material are needed, a fast and material-saving synthesis of complex molecules, for example peptides, proteins, oligonucleotides, DNA, oligosaccharides or RNA, whose synthesis is carried out by successive individual reactions. Small quantities of substances, but with a wide range of variations, are needed, for example, in the context of drug discovery and development in pharmaceutics and biomedicine.

Using the device according to the invention, the quantities of substances and times required for sequencing proteins or DNA sections can be further reduced. For this purpose, the proteins or DNA sections are bound to beads and analyzed stepwise during the passage of various reaction chambers. The device according to the invention can by additional components be extended for detection, such as magnetoelectric [16], by (integrated) optical systems [2] or electrochemical [17]. A combination of synthesis, reaction and analysis can also be carried out with the device according to the invention. For example, molecules can be synthesized in a first area, exposed to various substances in a subsequent area, and then directly analyzed. Furthermore, sensors can be introduced into the reaction chambers or the fluid channels to more precisely control the reactions.

The invention and its details are explained in more detail below by way of example with reference to embodiments with reference to figures. Show it

Fig. 1 System elements and principle for magnetic ratchet Fig. 2 Exemplary microstructure for generating an inhomogeneous magnetic field

Fig. 3 Exemplary production of soft magnetic microstructures

FIG. 4 Exemplary Production of a Fluid Structure FIG. 5 Exemplary Production of a Bond Connection FIG. 6 Exemplary Construction of a Device According to the Invention FIG

Fig. 1 shows a schematic sectional view of the essential elements and the principle of a fluidic ratchet. With an actuator 1 for generating a fluid flow 8 in the fluid channels 6. The fluid flow 8 moves the beads 4. Equipped with a mixing chamber volume 3 and a microstructured soft iron magnetic core 2 for generating a magnetic blocking force. The device is completed with a housing 5. Depending on the direction of movement of the actuator 1, the fluid moves 8 and with him the beads 7. If the blocking force is turned on, the beads are fixed in the direction of magnetic structure. 9

FIG. 2 shows a schematic sectional view with the field lines 10 of an inhomogeneous magnetic field which has been switched on, produced by an NEN soft iron magnetic core 2 in microstructure is embedded in plastic 11. The magnetic beads 4 are fixed from the fluid-filled channel 6 direction 9 magnetic core 12.

3 shows, by way of example, the schematic illustration of the production of the soft-magnetic microstructure in which an electroplating start layer 16 is deposited on substrate 13 (for example silicon or glass), then resist 15 is applied by spin coating and patterned, followed by electroplating with, for example Permalloy (NiFe in the ratio (80/20) and the spin coating of the sealing layer 14.

4 shows, by way of example, the schematic production of a microfluidic channel structure ^" .8 Openings 19, which serve to supply fluid, are introduced into the substrate 13. These holes can be introduced mechanically (for example drilling, lasing), wet-chemically or by reactive ion etching. The trench structures are formed by structuring (stripping) the resist spun onto the substrate (for example SU8, PMMA, polyimide).

FIG. 5 shows the bonding of the structures produced in FIG. 3 and FIG. 4 by pressure 20 and heat 20, thereby creating the microfluidic channel structures 21.

6 shows an exemplary embodiment of a device according to the invention consists of a microstructured magnet, a microfluidic channel structure, an actuator and fluidic connections.

The supervision of this system shows the fluidic structures. The time required for bead transport 8 periodic fluid movement 7 is generated by an actuator 1, which is located at the beginning of the fluid system. Through an opening 28, the beads are introduced into the system and after the fluidic ratchet principle [Fig. 1] moves through the microfluidic channel. A compensation chamber 24 at the end of the fluid structure with a free liquid level allows the periodic movement. In the mixing chamber volume 25, the residence time of the beads 4 can be regulated by the geometric shape, where column structures lead the beads 4 there. In the last mixing chamber volume 23 of the system, the beads are collected and rinsed out if necessary. Into the mixing chamber volume 25, the reaction substances are fed perpendicularly to the direction of movement via the microfluidic fluid guide. Through introduction 26 and branch 22, the filling of the chambers is facilitated and also allows a continuous regulation of the substance concentration.

bibliography

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[2] Sherry A. Dunbar, Coe A. Vander Zee, Kerry G. Oliver, Kevin L. Karem, and James W. Jacobson, Quantitative, Multiplexed Detection of Bacterial Pathogens: DNA and protein applications of the Luminex LabMAP ™ system, Journal of Microbiological Methods, Volume 53, Issue 2, May 2003, Pages 245-252.

[3] Michael Pycraft Hughes, Nanoelectromechanics in Engineering and Biology, 2003, Boca Raton, London, New York, Washington, D.C.-CRC Press LLC.

[4] G. Romano, L. Sacconi, M. Capitanio and FS Pavone, Force and Torque Measurements Using Magnetic Microbeads for Single Molecule Biophysics, Optics Communications, Volume 215, Issues 4-6, 15 January 2003, Pages 323-331 ,

[5] Sibani L. Biswal and Alice P. Gast, Mechanics of semiflexible chains formed by polyethylene glycol-linked paramagnetic particles, Physical Review E 68, 021402 (2003).

[6] D. Niarchos, Magnetic MEMS: key issues and some applications, Sensors and Actuators A: Physical, Volume 106, Issues, 1-3, 15 September 2003, pages 255-262.

[7] C. Hoffmann, M. Franzreb, W.H. Hell, "A novel high-gra- ted magnetic separator (HGMS) design for biotech applications", IEEE Trans, on Appl. Superconductivity, 12, No.1, 2002, Pages 963-966.

[8] H. Suzuki and CM. Ho, A Magnetic Force Driven Chaotic Micro Mixer, Proc. 15th IEEE Int. Conf. MEMS ¹ 02, Las Vegas, (2002), Pages 40-43.

[9] Jin-Woo Choi, Chong H. Ahn, Shekhar Bhansali and H. Thurman Henderson, A new magnetic bead-based, filterless bio-separator with planar electromagnet surfaces for Tegrated Bio-detection Systems, Sensors and Actuators B:

Chemical, Volume 68, Issues 1-3, 25 August 2000, Pages 34-39.

[10] A. Thommes, W. Stark, W.Bacher, Galvanic Deposition of Iron-Nickel in LIGA Microstructures, FZKA 5586, Scientific Reports, Forschungszentrum Karlsruhe GmbH, Karlsruhe, 1995.

[11] Ronald F. Probstein, Physicochemical Hydrodynamics, John Wiley & Sons Inc., New York Chichester Brisbane Toronto Singapore, 19952.

[12] Q. Han and J.D. Hunt, Particle pushing: critical flow rate required to put into motion, Journal of Crystal Growth, Volume 152, Issue 3, 1 June 1995, pages 221-227.

[13] Cheng Dong and Xiao X. Lei, Biomechanics of cell rolling: shear flow, cell-surface adhesion, and cell deformability, Journal of Biomechanics, Volume 33, Issue 1, January 2000, Pages 35-43.

[14] St. Ehlers, K. Elgeti, T. Menzel and G. Wießmeier, Mixing in the off-stream of a microchannel system * 1, Chemical Engineering and Processing, Volume 39, Issue 4, July 2000, Pages 291-298 ,

[15] T. Rogge, Z. Rummler, W.K. Schomburg, development of a piezo-driven microvalve - from the idea to pre-series production, FZKA 6671, Scientific Reports, Forschungszentrum Karlsruhe GmbH, Karlsruhe, 2001.

[16] MM Miller, PE Sheehan, RL Gemstone, CR Tamahaaha, L. Zhong, S. Bounnak, LJ Whitman and RJ Colton, A DNA array sensor utilizing magnetic microbeads and magnetoelectronic detection, Journal of Magnetism and Magnetic Materials, Volume 225, Issues 1-2, 2001, Pages 138-144.

[17] Joseph Wang, Nanoparticle-based electrochemical DNA detection, Analytica Chimica Acta, In Press, Corrected Proof, Available online 15 June 2003.

LIST OF REFERENCE NUMBERS

1 actuator

2 soft iron magnetic core in microstructure

3 reaction chamber

4 magnetic bead

5 housing

6 Microfluidic channel filled with a fluid

7 Movement direction of the magnetic beads, depending on the fluid flow and the magnetic blocking force

8 Generated, directed fluid flow

9 Fixing direction of the magnetic beads

10 field lines

11 Kunstcff or other suitable polymers

12 Fixation in the direction of the soft iron magnetic core

13 substrate

14 sealing layer

15 resist

16 electroplated layer

17 Microstructured soft iron magnet

18 Microfluidic channel

19 openings

20 pressure and / or heat

21 Microfluidic channel

22 diversion

23 mixing chamber volume for removing the beads

24 Opening as compensation chamber

25 mixing chamber volume

26 Introduction

27 Transport direction of the reactants in the microfluidic fluid guide

28 openings for introducing the beads into the microfluidic system

Claims

claims

1. A device for transporting at least one magnetic particle fraction through a microfluidic system comprising, a) at least one microfluidic channel with a fluid comprising the magnetic particle fraction, b) means for generating a fluid flow axially to the microfluidic channel with two switching positions corresponding to the two Flow directions, c) can be added external magnetic field in the channel for temporary fixation of the magnetic particle fractions, wherein in an operating condition, a cyclic change of the two switching positions takes place and wherein the magnetic field is connected only in a switching position.

2. Apparatus according to claim 1, characterized in that the microfluidic channel adjacent to its full length of soft magnetic material.

3. Device according to one of claims 1 or 2, characterized in that the microfluidic system is wholly or partially incorporated by optical lithography, hot stamping, injection molding or by X-ray lithography in a substrate.

4. Device according to one of claims 1 to 3, characterized in that the microfluidic system is positively closed with a cover plate.

5. Apparatus according to claim 4, characterized in that the cover plate has at least two interconnected by the microfluidic channel openings.

6. Device according to one of claims 1 to 5, characterized in that the means acting directly on the fluid Actuator includes.

7. Apparatus according to claim 6, characterized in that the actuator comprises a piezo-bending actuator or pressure-spring system.

8. Device according to one of claims 1 to 7, characterized in that the device comprises at least one further microfluidic fluid guide, which crosses the microfluidic channel to form a mixing chamber volume.

9. Device according to one of claims 1 to 8, characterized in that the microfluidic channel has at least one introduction or branch for another fluid.

10. Use of the device according to one of claims 1 to 9 for carrying out a solid phase synthesis of magnetic particle fractions.

11. Use of the device according to one of claims 1 to 9 for bioanalytics with fixed on at least one magnetic particle fraction biomolecules.

12. Use according to claim 11, characterized in that the biomolecules comprise proteins, peptides, DNA, RNA or cells, prokaryotic or eukaryotic.

13. Use of the device according to one of claims 1 to 9 for chemical analysis or production with fixed on at least one magnetic particle fraction chemical reactants or catalysts.