DE102005048260A1 - A method and apparatus for handling a liquid sample using rotation with a time-varying rotary vector - Google Patents

Abstract

A method for handling a liquid sample comprises introducing the sample into a chamber, in which a detection structure is arranged, which enables detection of a property of the sample, and rotating the chamber with a time-varying rotation vector, which is a multiple acceleration and a multiple Braking has to generate convection currents of the sample in the chamber by hydrodynamic inertial effects in order to lead the sample past the detection structure. Such a method is particularly suitable for carrying out microarray experiments.

Description

The The present application relates to a method and an apparatus to handle a liquid Sample using a rotation with a time-varying Rotation vector suitable for mounting the sample on a detection structure, For example, a microarray to pass.

One Microarray is a parallel arrangement of a plurality of different, but defined "catcher structures", which are also called reaction points can be in a miniaturized grid (for example, 500 microns) on a mostly flat substrate. The catcher structures can For example, catcher molecules (in English "sample molecules" or "capture probes "), like DNA, cDNA, proteins, antibodies, biological cells or the like, be. A "microarray-based Test "allows detection the molecules complementary to the capture molecules. Microarray-based tests are of great economic interest, for example in gene expression analysis, SNP analysis (SNP = Single nucleotide polymorphism) or for personalized medicine.

A "microarray-based Test "consists of one Procedure with several substeps. First, a defined microarray, So the substrate with the immobilized capture molecules, in contact with the examining liquid brought so that the complementary molecules react with the catcher molecules can. The reaction can take many hours and is of the characteristic times dependent, it takes until the complementary molecules have reached the catcher molecules and reacts there have hybridized, for example, a DNA.

By continuous "stirring" or a suitable fluid guide can the complementary molecules very much catcher molecules be brought very close so that the diffusion lengths are strong shortened and thus the durations for the reaction, for example, the hybridization, greatly reduced can be. After the reaction of the molecules with the microarray further process steps, such as washing the substrate and thus the microarray, d. H. the removal of unbound or non-specifically bound molecules Exchange of the fluid to be examined by a defined Buffer, or feeding of reagents, for example fluorescently labeled detection antibody the microarray.

According to the state In the art, for example, microarrays are processed by the sample and the relevant reagents are pipetted manually. Due to the planar surface of the substrate, typically in the form of a microscope slide here the consumption of sample and reagents high. Besides that is the overflow of the Microarray reaction points with the sample to be examined are not controlled. Due to the non-continuous closed substrate it can very quickly lead to drying effects at individual reaction points come that negatively affect the outcome of the experiment.

Out the prior art, it is also known, microarrays in one process a closed fluidic structure. Here are it technical solutions For example, by a targeted mixing of the sample in reaction chambers using surface acoustic waves (Engl. SAW = surface acoustic wave). Such techniques have been used, for example, by the Company Advalytix AG (www.advalytix.de) discloses.

A alternative technique was a targeted mixing of the Sample in reaction chambers by a coupled rotational movement to perform two axes. Such a technique is available at M.A. Bynum et al., "Hybridization Enhancement using Microfluidic Planetary Centrifugal Mixing ", Analytical Chemistry, Vol. 76, No. 23, December 1, 2004, pages 7,039-7,044. According to this technique Samples are applied to seal slides and with a Microarray covered to form a chamber. The chamber will then used in a planetary centrifuge. The centrifuge Turn the chamber around one outside the chamber axis of rotation at a rotational speed of 1,200 revolutions per minute. In addition, the chamber still at a rate of 10 revolutions per minute about its own axis turned.

systems, using a pressure-driven or electrically-driven convection, in Rinie van Beuningen et al., "Fast and specific hybridization using flowthrough microarrays on porous metal oxide ", Clinical Chemistry, 47 (10), p.1931-1933, 2001, and Vincent Benoit et al., "Evaluation of three-dimensional microchannel glass biochips for multiplexed nucleic acid fluorescence hybridization assays ", Analytical Chemistry, 73 (11), 5.2412-2420, 2001, described.

A Chaotic advection is described in Mark K. McQuain et al microarray hybridization ", Analytical Biochemistry, 325 (2), 5.215-226, 2004.

Sample oscillation using microfluidic hybridization and mäan Such array channel is described by Yingjie Liu and Cory B. Rauch, "DNA Probe Attachment on Plastic Surfaces and Microfluidic Hybridization Array Channel Devices with Sample Oscillation", Analytical Biochemistry, 317 (1), pp. 76-84, 2003.

The Use of shear flows in K. Pappaert et al., "Enhancement of DNA micro-array analysis using shear-driven micro-channel flow system ", Journal of Chromatography A, 1014 (1-2), Pp. 1-9, 2003; Johan Vanderhoeven et al., "Exploiting the benefits of miniaturization for the enhancement of DNA microarrays ", 25 (21-22), p.3677-3686, 2004, and Johan Vanderhoeven et al., "DNA microarray enhancement using a continuously and discontinuously rotating microchamber, "Analytical Chemistry, 77 (14), 5.4474-4480, 2005, described.

Cavity-induced micro trends Robin Hui Liu et al., "Hybridization enhancement using cavitation microstreaming ", Analytical Chemistry, 75 (8), 5.1911-1917, 2003, described.

Finally, acoustically-induced convection is described by Andreas Toegl et al., "Enhancing results of microarray hybridizations through microagitation", Journal of Biomolecular Techniques, 14 (3), pp. 1957-204, Achim Wixforth, "Flat fluidics: Acoustically driven planar microfluidic devices for biological and chemical applications ", in Proceedings of the 13 ^th International Conference on Solid-State sensors, Actuators and Microsystems (Transducers'05), June 5-9, Seoul, Korea, pp 143-146. IEEE, 2005.

M. Grumann u. a., "Batch Mode Mixing on Centrifugal Microfluidic Platforms ", Lab Chip, 2005, 5, pp. 560-565 a mixing of liquids in a mixing chamber in which the mixing chamber is in rotation with changing periodically Is subjected to rotation.

adversely in the known state of the art for processing microarrays are the necessarily active and complex systems for excitation and control to a targeted mixing of the sample in reaction chambers to reach. Furthermore require some of the alternative systems, for example, pumped Systems, inflows and outflows, their additional Dead volumes negatively impact the sensitivity of the arrays.

The minimal lateral dimensions of the detection structure are through the lateral dimensions of the 2-dimensional microarray (typically in the range of one square centimeter) through the manufacturing process specified. At the same time, the dead volume of the chamber should be minimized shall be. From a technical point of view, this is mainly about the litigation the problem, in a wide and as shallow chamber (im Case of the present invention aspect ratios of well below 1:10) with volumes in the microliter range a liquid sample on a detection structure to pass by.

The The object underlying the present invention is that an apparatus and method for handling a liquid sample create, which allow it with a simple structure, an increased proportion a sample arranged in a sample chamber at a detection structure to pass by.

These The object is achieved by a method according to claim 1 and a device solved according to claim 13.

The present invention provides a method for handling a liquid sample comprising the steps of:
Introducing the sample into a chamber in which a detection structure enabling detection of a property of the sample is arranged, and
Rotating the chamber with a time-varying rotational vector, the multiple acceleration and a multiple deceleration, to generate convection currents of the sample in the chamber by hydrodynamic inertial effects, to lead the sample past the detection structure.

The present invention further provides an apparatus for handling a liquid sample, comprising:
a chamber in which a detection structure enabling detection of a property of the sample is arranged;
a drive device configured to subject the chamber to rotation; and
a controller configured to control the drive means to rotate the chamber with a time-varying rotary vector having multiple accelerations and multiple decelerations to generate convection currents of a sample within the chamber by hydrodynamic inertial forces Pass the sample past the detection structure.

The The present invention is based on the recognition that by a Rotation with a time-varying Rotation vector produced convection currents are advantageously exploited can, a big one Proportion of a sample arranged in a sample chamber at a detection structure to pass by or around the parts of a sample exposed to the detection structure is to change in an easy and fast way.

at embodiments of the invention the acceleration phases through several intervals with dormant or constantly rotating chamber are interrupted, which is advantageous for to be effected Reactions can be.

at preferred embodiments According to the invention, the detection structure is represented by a microarray, i. H. a parallel arrangement of a plurality of different, but defined catcher structures or catcher molecules that on a carrier are immobilized, formed.

In this regard is suitable the present invention in particular for the processing of Microarrays, with a quasi-instantaneous active introduction of Sample and reagents to the reactants in the microarray points by rotating the chamber with a time-varying Rotary vector, which as a shaking mode can be designated takes place. Furthermore, this is done by a quickly removing unbound reactants from the microarray points, i.e. the individual catcher structures or catcher molecules.

at preferred embodiments of the present invention form a microarray body and a lid together a reaction chamber, whereby by alternating Rotational movements in the reaction chamber convection in the chamber is brought about, which the mixing (between sample and microarray) in one Microarray experiment accelerated. Thus, due to a forced convection of a in a reaction chamber arranged sample the time, for example for one DNA hybridization be reduced.

at preferred embodiments can the reaction chamber completely with liquid filled be.

at embodiments According to the present invention, the chamber may be formed in a body, being the body a substrate and a cover chip has. One or more such Body can in a rotor can be used, which via a rotary motor in rotation is displaceable. The detection structure, for example the microarray, may be immobilized on the substrate while in the lid chip has a channel structure structured with inlet area, reaction cavity and outlet area is. Alternatively, the detection structure on the part of the body, for example the lid, be immobilized, in which the channel structures formed are while the lid can be a planar member. The channel structure can one or more inlet regions, one or more sample chambers, For example, reaction chambers, and one or more outlet areas exhibit.

at embodiments According to the present invention, the body itself as a body of revolution, for example a disc, be formed, which is rotatable about an axis of rotation is. Such a rotary body can also be formed of substrate and cover chip and a Channel structure required to implement the present invention or for parallelization a plurality of corresponding channel structures, the star-shaped are arranged on the disc.

According to embodiments According to the present invention, the channel structure may have an outlet channel which is fluidically connected to the chamber and a siphon structure which in a given centrifugal force field to a defined filling level of the liquid sample leads in the chamber. This makes it possible especially in processes where successively different liquids be supplied to a detection structure are intended to avoid drying out the collection structure safely.

Under a siphon structure is generally a gravitational field, in the Case of the present invention, the centrifugal field, exposed Structure to understand which a reservoir and a connecting channel comprising one end connected to the reservoir and the other end has an outlet. The system is with a liquid at least partially filled.

To The system now strives for the principle of communicating tubes a hydrostatic equilibrium state, in which all Menisci take the same position in the direction of the force field. If the exhaust duct is designed so that it follows the principle the communicating tubes Completely with liquid filled, flows, if the position of the outlet below the liquid level in the hydrostatic Balance is as long as liquid from the outlet until the liquid level in the reservoir at the height the outlet is located. In sanitary engineering, the continuous liquid column of the Connection channel as odor stop, which is odor-proof even after rinsing remains.

As soon as the connection channel completely with liquid filled is, the liquid level can also independently of the course of this connecting channel solely by the height of his Outlet be adjusted. By lowering the outlet below of the reservoir bottom, the reservoir is emptied as long as the continuous liquid column not is interrupted. Is the connection of the connection channel for example, at the bottom of the reservoir, the reservoir is completely emptied.

The full filling The siphon can either by the principle of communicating tubes through fill up into the reservoir until the common liquid level in the hydrostatic Equilibrium state above the highest point of the connection channel lies, and / or by the capillary driven filling of Channel.

When Siphon effect is here generally the regulation of the level in a reservoir through the course and the position of the connection channel be understood on the principle of communicating tubes.

In alternative embodiments of the invention, the detection structure may comprise a sensor structure comprising one or more sensor regions responsive to a sample located in the region thereof to enable conclusions to be drawn about properties of the samples. Examples of such sensor structures are oxygen sensors, CO ₂ sensors or biosensors that use biomolecules to detect properties of a sample.

The Channel structures are preferably designed such that the Interplay of centrifugal force and capillary force with synchronized Sample addition and reagent addition, for example, a complete processing of a microarray experiment. This can be a siphon structure in the centrifugal force field one by the time course of the rotation frequency and defined the liquid volumes added at certain times Make the course of the filling height, what about precisely control the reaction conditions in the reaction chamber to let. In particular, when performing a microarray experiment the siphon structure cause the reaction space always at least so far with liquid filled is that the microarray is covered by this, if one after the other the sample, a washing liquid and reagents are fed into the reaction space. Thus, that can Microarray under throughout be processed controllable fluidic conditions, since in the centrifugal force field of the siphon channel determines the filling level in the reaction chamber.

The The present invention is in accordance with preferred embodiments thus in an apparatus and method for processing Microarrays on a preferably planar substrate, wherein the Invention, the integration of the example described below, typical steps of a microarray-based experiment allows. These steps involve adding the sample to a reaction chamber and blocking them, optionally washing several times the reaction chamber and thus the microarray, the detection, d. H. dyeing, of the microarray and a following, again optionally multiple washing of the microarray. Also reading the microarray on each any conventional manner could be done to get integrated.

Consequently allows the present invention provides a nearly complete integration of all necessary Steps to process a microarray-based experiment, whereby the reduction of manual work also helps the reproducibility of microarray-based experiments too increase. The body, in which the fluidic structures are formed, preferably consists of a substrate and a lid, can be produced easily and inexpensively and can therefore be used as a disposable article.

at In a pilot experiment, the reaction chamber had a volume of about 50 μL, the total volume consisting of sample, reagents and buffers at the performed Experiment at only 500 μL was. This generally represents a significant reduction in consumption the sometimes very expensive fluids These volumes are in contrast to experiments that at one free, ie not limited by a chamber microarray surface are performed very small and left through a targeted optimization of the chamber shape even more to reduce.

conditioned through the optimized hydrodynamic conditions created by the Application of rotation achieved with a time-varying rotation vector can be and the alternative or supportive by a meandering reaction chamber can be achieved let himself go reduce the process time to 45 minutes in the pilot experiment. It is omitted by far the largest part This time on the reaction / incubation between the initially introduced Sample with the detection structure while washing in less than a minute has ended. In general, another, Drastic acceleration of the process time and also a significant Reduction of the chamber volume above the Microarray conceivable.

The usable according to the invention closed reaction chamber allows compared to a non-capped substrate that microarray-based Experiment permanently in a controlled environment, which runs In particular, it can prevent the microarray spots from drying out. In particular, allows the "stirring" in the reaction space by rotating with a time-varying rotary vector one shortened process duration, while the fluidic "siphon structure" a controlled Environment supplies.

preferred embodiments The present invention will be described below with reference to FIG the enclosed drawings closer explained. Show it:

1 and 2 schematically a plan view and a cross-sectional view of an embodiment of a device according to the invention, wherein 2 schematically a cross-sectional view along the line XX of 1 represents.

3 to 5 schematic embodiments of channel structures of embodiments of a device according to the invention;

6a and 6b schematic cross-sectional views for illustrating an embodiment of the method according to the invention;

7 the results of an experiment carried out using the invention;

8th Examples of temporally variable rotation vectors that can be used according to the invention;

9 an exemplary frequency protocol for performing a microarray experiment and the resulting level height in the chamber.

An embodiment of the device according to the invention will now be first on the basis of 1 and 2 explained.

The device comprises a rotor 10 that with a wave 12 connected by a rotary motor 14 is drivable. The rotor 10 can in any way, for example on a step portion of the shaft 12 to be appropriate. The rotary motor 14 is with a controller 16 which is designed to set the rotor in the required manner in rotation.

The rotor has four receiving areas 20 for receiving modules according to the invention containing fluid structures 22 , The modules consist in the embodiment shown of a substrate 24 and a lid 26 wherein fluid structures in the substrate 24 are formed. The fluid structures comprise, as in FIG 1 is shown and later referenced 3 is explained in more detail, a reaction chamber 30 , an outlet channel 32 with a centrifugal siphon structure 32 , a waste reservoir area 34 as well as two inlet channels 36 and 38 , In the embodiment shown, the fluid structures are in the substrate 24 structured. Alternatively, the fluid structures could also be structured in the lid or in the lid and substrate. The combination of substrate and lid creates an arrangement which contains a reaction space and channel systems for the supply and removal of samples or analytes, reagents and / or washing liquids to the reaction chamber.

By the control device 16 becomes the rotary motor 14 controlled such that the rotor, and thus the modules 22 , are subjected to a rotation in order to carry out the method according to the invention.

Exemplary channel structures will now be described with reference to FIGS 3 and 4 described. In preferred embodiments, the fluidic structures or channel structures are thereby formed by using a planar substrate on which the microarray is immobilized, while a lid chip is structured in order to realize the fluidic structures.

At the in 3 In the example shown, the fluidic structures comprise an inlet area 40 for a sample or an analyte and an inlet area 42 for reagents. The inlet area 40 is via an inlet channel 44 with a reaction chamber 46 connected while the inlet area 42 via an inlet channel 48 with the reaction chamber 46 connected is. The inlet channels 44 and 48 are with respect to a centrifugal force field (see arrow F _ν in 3 ) radially inward to the reaction chamber 46 connected. Radially outward is an outlet channel to the reaction chamber 50 connected, in this application example, such a siphon structure 52 which initially comprises a channel section extending radially outward in the direction of the centrifugal force field 52a , Then a against the force field radially inwardly extending channel section 52b and then again from a channel section extending radially outward with the force field 52c is formed. The radial position of the outlet of the channel structure is below the bottom of the reaction chamber 46 , ie Δr <0. A vent 54 is intended for the siphon structure. The vent 54 can optionally be hydrophobized completely or in sections. The outlet channel 50 flows into a waste reservoir 56 , The vent 54 is in the waste reservoir 56 intended. At the end of the exhaust duct 50 Optionally, a hydrophobized section 57 be provided.

The siphon structure 52 is designed to maintain a defined liquid level in the reaction chamber at a given centrifugal force field set at a particular point by the rotational speed, radial position, and density of the fluids used upon initial charging 46 to create.

Preferably, the structure is designed such that the reaction chamber 46 completely filled with liquid at this stage.

The inlet areas 40 and 42 have inlet area openings 40a and 42a on. When filling the two inlets, each inactive channel serves as a vent. In 4 An alternative structure is shown where all the fluids pass through a single channel 84 be fed, but this makes the vent difficult. The channel 84 is at its radially inner end with an inlet area 82 connected to an inlet area opening 82a and at its radially outer end with a chamber 80 connected.

In the embodiments of 3 and 4 Corresponds to the coordinate .DELTA.r of the radial height difference relative to the outlet of the chamber in which a detection structure 60 , For example, a microarray is provided. If the initially empty chamber with the detection structure is filled with a volume such that the liquid level in the equilibrium state of the centrifugal field is below the radially innermost position of the siphon channel, this liquid level remains constant at Δr> 0, regardless of the rotational frequency. In this phase, the sample is incubated with the microarray in the shaking mode in this embodiment. On the other hand, if the siphon channel is completely filled with fluid up to its outlet, the fluid from the chamber will continuously flow into the outlet during centrifugation. Such filling can take place for example by the capillary force at low speeds or at rest structure, or by adding a volume of liquid that forces an equilibrium liquid level above the radially innermost point of the Siphonkanals r _max (temporary).

In preferred embodiments of the invention, the channel structures are formed in the cover chip, wherein the cover chip is so merged with a microarray substrate that in the reaction chamber, the microarray 60 is arranged. The walls of the channel structures preferably have hydrophilic properties. The inlet area 40 and the inlet channel 44 serve to sample liquid by centrifugal force on the rotating arrangement of the reaction space 46 supply. The inlet area 42 and the inlet channel 48 serve to reagents by the centrifugal force on the rotating arrangement of the reaction space 46 supply. The outlet channel 50 The purpose of this is to leave the first sample in the chamber at any high speed and during the shaking mode, thus maintaining a constant liquid level in the reaction chamber 46 adjust. Preferably, the reaction chamber 46 always completely filled with liquid in this phase and it is thus reliably prevented drying out of the reaction chamber and thus of the microarray arranged therein. Already in the reaction chamber 46 located liquid is displaced by the subsequently supplied liquid volumes in the waste area. The inlet areas 40 and 42 have the openings 40a and 42a either in the lid chip or in the substrate to allow them to be filled with the respective liquids, either manually or automatically. Furthermore, the optionally hydrophobicized vent comprises 54 an opening in either the lid chip or the substrate to allow venting.

One embodiment a method according to the invention will now be based on performing a microarray experiment described.

First, a sample liquid, ie an analyte, is introduced into the reaction chamber 46 introduced by the inlet area 40 filled with the sample liquid and at the same time or subsequently the channel structure (for example by the rotary motor 14 with the associated control device 16 . 2 ) is subjected to a suitable rotation, by the centrifugal force F _v and optionally also by a capillary force, the liquid through the inlet channel 44 in the reaction chamber 46 contribute. The introduced volume is chosen so that the centrifugal force in the hydrostatic equilibrium initially sets a liquid level above the detection structure, but below r _max , so that the reaction chamber can not yet be centrifugally emptied by the siphon.

After filling the reaction chamber, it is then subjected to a time-variable rotary vector, which has a multiple acceleration and a multiple deceleration, possibly interrupted by resting phases with a constant or vanishing rotational vector. As a result of the rotation of the arrangement with a time-variable rotary vector, hydrodynamic inertial forces generate convection currents of the liquid located there within the reaction chamber. Thus, the length of time for the mixing of complementary molecules in the sample to the capture molecules by a specific "stirring" in the reaction chamber 46 located liquid can be reduced. Due to the convection currents, molecules that can be far away from the capture molecules in the stationary case are also transported past the immobilized and respectively complementary capture molecules. Furthermore, unbound reactants are rapidly removed from the capture molecules.

After a sufficient time for the processing of the microarray time is then over the inlet area 42 a washing liquid in the reaction chamber 46 introduced by centrifugal force through the inlet channel 48 , This will put you in the reaction chamber 46 Sample liquid through the outlet channel 50 displaced and filled the siphon structure centrifugally or by the capillary force at a suitable small centrifugal field. With a completely filled siphon, a continuous evacuation of the reaction chamber takes place in the centrifugal field 46 instead of.

The siphon principle also shows that the newly introduced volume corresponds to the displaced volume. To complete a fluid exchange in the reaction chamber 46 To ensure this, the newly introduced volume must therefore at least correspond to the chamber volume. Under laminar conditions, which are typical in the very shallow chambers and just cause the mixing problems dealt with in this document, the volume displacement can be hydrodynamically designed so that the newly introduced is not flushed out with it. Ideally, takes place in the chamber 46 So a complete volume exchange instead.

After feeding the washing liquid can over the inlet area 42 and the inlet channel 48 taking advantage of the centrifugal force further reagents, such as fluorescently labeled detection antibodies in the reaction chamber 46 be introduced. This is followed by feeding a washing liquid and its centrifugally driven outflow through the siphon. Subsequently, an evaluation of the microarray can take place in any suitable manner.

With regard to the channel structures, it should be noted that in 3 the inlet channels 44 and 48 as well as the outlet channel 50 represent the essential part of the hydrodynamic flow resistance of the entire structure.

In 5 is schematically shown a channel structure whose outlet channel 70 an alternative siphon structure 72 having. The siphon structure includes a radially outwardly extending portion in the flow direction 70a and a radially inwardly extending portion 70b in a waste reservoir 76 that with a vent 76a is provided, flows. This structure, cut off at the highest point of the siphon r _max, is based directly on the principle of communicating tubes in the centrifugal field. In hydrostatic equilibrium provides a liquid level .DELTA.R> 0 above r _max complete filling and thus also the determination of the equilibrium liquid level to r _max safe. The end of the siphon channel at r _max is preferably above the detection structure and thus ensures a complete filling of the same, as again by the level 58 is displayed.

With the described apparatus and the described method, an experiment was carried out, the course of which in the 6a and 6b is shown schematically and its results in 7 are shown. More specifically, two experiments were carried out with the in 6a experiment shown a microarray substrate 90 was used in which the reaction chamber 92 is structured and on which the microarray 94 is immobilized. The substrate used was a polymer substrate, and in particular a substrate made of COC (cyclic olefin copolymer).

According to the experiment 6b became a non-structured substrate and in particular a PDMS substrate 96 (Polydimethylsiloxane substrate) used without channel structures.

at In both experiments, BSA (bovine serum albumin) was used the well-documented EDC-NHS affinity ligand coupling to the respective substrate immobilized to the surface density increase the immobilized capture molecules.

The corresponding microarray substrates are below point 1 in the 6a and 6b shown. The substrates were then provided with a respective lid, according to 6a an unstructured lid 98 is used while according to 6b a reaction chamber 100 in the lid 102 is structured. In doing so, the lid provides 98 a PDMS lid while the lid 102 represents a COC lid. Subsequently, the sample having a known target concentration c (BSA) of mouse anti-BSA antibodies was introduced into the reaction chamber as in point 2 of the 6a and 6b you can see. Subsequently, sheep anti-mouse Cy3 antibodies were added, see item 3 in 6a and 6b , The specific antigen-antibody complexes are formed. This is followed by a washing step, followed by fluorescent detection antibodies 104 be supplied with which the said complexes are marked.

The results obtained are in 7 shown, with the curve 110 the COC microarray substrate with channel structure affects while the curve 112 relates to the PDMS microarray substrate without channel structure. 7 shows that a clear and reproducible correlation for varying target concentrations cBSA and the resulting fluorescence signal I over a working range of at least 3 decades is obtained. The entire microarray experiment was completed in 45 minutes, with all steps fully automated.

Possible rotation protocols for applying the channel structures with a temporally variable rotation vector, which has a multiple acceleration and multiple deceleration are in the 8a . 8b and 8c shown. 8a shows a switching between maximum rotational frequencies f _max with different directions of rotation. The maximum rotational frequency may be 8 Hz, for example, while the maximum rotational acceleration may be, for example, ± 32 Hz / s. 8b shows a switching between a maximum rotational frequency f _max and a rotational frequency of 0, so that the rotation protocol as shown in FIG 8th can be performed using centrifuges, which are operable in one direction only, can be performed. Finally shows 8c a rotation protocol in which after accelerating to the respective maximum rotational frequency, a short rotation takes place at this maximum rotational frequency. Not shown in the 8a to 8c is an occasional suspension of the acceleration phases with a constant or vanishing rotational vector.

9 shows by way of example the respective frequency protocols ν (t) and level heights in the reaction chamber Δr as a function of the time t for the siphons in the manner of 3 and 5 shown. Regarding the level heights the curve refers 120 to the siphon structure after 3 while the bend 122 to the siphon structure after 5 refers. The time scale is not linearly scaled here, for example the sample incubation time in the shake mode is typically at least a factor of ten longer than the subsequent wash-through phase of the wash buffer. In the resting phases Δt ₁ and Δt ₂ liquid volumes are added.

Like the curve 120 in 9 it can be seen, the microarray structure is after 3 completely covered during shaking mode. Upon the addition of a subsequent volume and the associated complete filling of the siphon, the chamber is continuously deflated during rotation, such as the section 120a the curve 120 can be seen.

Notwithstanding the illustrated preferred embodiments, numerous variations of the present invention are possible. So could instead of the described disc-shaped rotor 10 a likewise in 1 indicated four-armed rotor can be used. Again alternatively, the body in which the fluid structures are formed could be formed as a rotational body, for example as a disc, which is rotatable about an axis of rotation thereof. In this body, a plurality of channel structures could be arranged radially star-shaped, comparable to the arrangement of the modules 22 in the rotor 10 ,

at alternative embodiments of the present invention beyond the siphon structure of the outlet channel beyond the outlet channel with a hydrophobic barrier or a high flow resistance be provided, which is a dependent of a rotational frequency liquid switch represent.

at preferred embodiments In accordance with the invention, the channel structures comprise one or more inlet channels for liquid by centrifugal force into the reaction chamber. alternative could but the liquid get into the reaction chamber in any other ways, for example about a feed channel under exercise of a Pressure or by manual filling, for example with a pipetting system. Also the outlet channel or the siphon structure of the same is not a mandatory feature of Invention, especially if not several liquids in succession be guided through the reaction chamber but using a sensor structure only the properties of a liquid should be recorded. The liquids could also through a lid opening or removing the lid in the meantime by suction or rinsing.

moreover could the mixing process still by the in the above-mentioned publication M. Grumann u. a., "Batch-Mode Mixing on Centrifugal Microfluidic Platforms ", Lab Chip, 2005, 5, pages 560-565, by the addition of (para) magnetic beads in combination with Permanent magnets, which are arranged fixedly along the chamber orbit are, support. Especially in shake mode could also Beads whose density differs from the corresponding liquid density, the Accelerate mixing process. By centrifugation or a appropriately positioned magnets, the beads were finally out again be removed from the detection window of the microarrays.

Of the Rotary drive, for example, can be designed to be PC-controlled to carry out a frequency protocol, around a time-dependent To generate centrifugal field in order for the individual to be carried out Steps to precisely control the flow rates and timing. Control software may also include, for example, one or more Dispensers control which at rest or even with appropriate Synchronization with rotating substrate the reagents on the inlets give up. For example, by a suction device could be also the discharge of liquids automate.

The present invention thus provides apparatus and methods that are particularly suitable for performing microarray experiments in an increasingly automated manner. in particular In particular, the present invention provides a means to efficiently deliver sample liquid and the molecules contained therein to capture molecules of the microarray and to prevent the microarray from drying out throughout the experiment. As a result, reliable, reproducible results can be obtained with only a very small dead volume and high sensitivity. In particular, the speed and sensitivity of the processing can be improved by a chamber with very low volume and also very low dead volumes in the feeders. The present invention is also particularly suitable for use in so-called "lab-on-a-disk" systems, which provide a flexible platform for fully integrated and fast processing of experiments.

The present invention enables the implementation of microarray experiments with a reduced amount of time, one fast and homogeneous reaction, an automated washing process, a reduced consumption of sample and reagents, as well as a integrated waste handling. By the elimination of uncontrolled drying steps The sensitivity of the microarray experiment can be further improved by the siphon structure elevated become. In this way, the use of separate devices, like slide-washing devices and the like become obsolete and handling steps are automated. Finally, the Symmetry of the rotor or the body of revolution potentially that multiple microarray experiments carried out in parallel become.

According to the invention Chamber preferably designed to be completely, i. without gas inclusion, filled with the sample to become. For this purpose, the chamber can be completely closed or with the exception of inlet channel and outlet channel completely closed be. The present invention makes it possible even with completely filled chamber and very small sample volumes efficiently pass the Sample on a detection structure.

Claims (25)

Method for handling a liquid sample, comprising the following steps: introducing the sample into a chamber ( 30 ; 46 ; 80 ; 92 ; 100 ), in which a collection structure ( 60 ; 94 ), which enables detection of a property of the sample, is arranged; Rotating the chamber with a time-varying rotary vector having multiple accelerations and multiple decelerations to generate convection currents of the sample in the chamber by hydrodynamic inertial effects to cause the sample to be exposed to the sensing structure (FIG. 60 ; 94 ) pass by. The method of claim 1, wherein the chamber is completely with filled the sample becomes. Method according to Claim 1 or 2, in which the detection structure ( 60 ; 94 ) is a microarray of capture structures for trapping molecules from the sample. Method according to claim 1 or 2, wherein the detection structure has a sensor structure. The method of claim 4, wherein the sensor structure comprises a biosensor, an oxygen sensor or a CO ₂ sensor. Method according to one of claims 1 to 5, wherein the rotation vector a multiple acceleration and deceleration in one direction having. Method according to one of claims 1 to 5, wherein the rotation vector a multiple acceleration and deceleration in different Has directions of rotation. Method according to one of claims 1 to 5, wherein the step of introducing the introduction of the sample through an inlet channel ( 36 ; 44 ; 84 ) by centrifugal force. The method of any one of claims 1 to 8, further comprising a step of emptying the sample from the chamber (10). 30 ; 46 ; 80 ; 92 ; 100 ) by centrifugal force through an outlet channel ( 32 ; 50 ; 70 ) having. Method according to one of claims 1 to 9, further comprising a step of introducing a washing liquid into the chamber ( 30 ; 46 ; 80 ; 92 ; 100 ), thereby separating the sample from the chamber by a siphon structure ( 52 ; 72 ), which generates a defined fill level in the chamber in a given centrifugal force field, to empty from the chamber. Method according to one of claims 1 to 10, further comprising a Step of introducing reagents into the chamber. The method of claim 11, wherein the sample and reagents are separated via separate inlet channels ( 36 . 38 ; 44 . 48 ) are introduced into the chamber. Device for handling a liquid sample, comprising: a chamber ( 30 ; 46 ; 80 ; 92 ; 100 ), in which a collection structure ( 60 ; 94 ), which enables detection of a property of the sample, is arranged; a drive device ( 14 ) configured to subject the chamber to rotation; and a control device ( 16 ) configured to control the drive means to rotate the chamber with a time-varying rotational vector having multiple accelerations and multiple decelerations to generate convection currents of a sample within the chamber by hydrodynamic inertial forces around the sample at the collection structure ( 60 ; 94 ) pass by. Apparatus according to claim 13, wherein the chamber is designed to be complete filled with the sample to become. Device according to Claim 13 or 14, in which the detection structure ( 60 ; 94 ) is a microarray of capture structures for trapping molecules from the sample. Apparatus according to claim 13 or 14, wherein the Detection structure has a sensor structure. Apparatus according to claim 16, wherein the sensor structure comprises a biosensor, an oxygen sensor or a CO ₂ sensor. Device according to one of claims 13 to 17, wherein the Rotation vector a multiple acceleration and deceleration in one direction of rotation having. Device according to one of claims 13 to 17, wherein the Drehvektor a multiple acceleration and deceleration in different Has directions of rotation. Device according to one of claims 13 to 19, in which the chamber ( 30 ; 46 ; 80 ; 92 ; 100 ) is formed in a body. Apparatus according to claim 20, wherein the body further comprises an inlet channel (16). 36 ; 44 ; 84 ) for centrifugally feeding a sample into the chamber ( 30 ; 46 ; 80 ; 92 ; 100 ) and / or an outlet channel ( 32 ; 50 ; 70 ) for centrifugally emptying a sample from the chamber. Device according to Claim 21, in which the outlet channel has a siphon structure ( 52 ; 72 ), which adjusts the level in the chamber according to the siphon principle. Device according to one of claims 20 to 22, in which the body is a substrate ( 24 ; 90 ; 96 ) and a lid ( 26 ; 98 ; 102 ), wherein the chamber and, if present, the inlet channel and the outlet channel in the substrate, the lid or both are structured. Device according to Claim 23, in which the detection structure ( 60 ; 94 ) is immobilized on the substrate. Device according to one of Claims 20 to 24, in which the substrate ( 24 ; 90 ; 96 ) a separate inlet channel ( 38 ; 48 ) for introducing reagents into the chamber.