WO2007042219A1 - Method and device for handling a liquid sample using a rotation having a temporally modifiable rotational vector - Google Patents

Abstract

The invention relates to a method for handling a liquid sample wherein the sample is inserted into a chamber (46), wherein a detection structure (60) is arranged enabling a property of the sample to be detected, and the chamber which has a temporally modifiable rotational vector can be rotated, and which comprises multiple acceleration and multiple braking, in order to produce sample convection flows in the chamber by means of hydrodynamic inertial effects in order to bypass the sample on the detection structure (60). Said method is particularly suitable for carrying out microarray experiments.

Description

 Method and device for handling a liquid

Sample using a rotation with a time-varying rotation vector

description

The present application relates to a method and apparatus for handling a liquid sample using rotation with a time-varying rotary vector suitable for passing the sample past a detection structure, such as a microarray.

A microarray is a parallel arrangement of a plurality of different but defined "capture structures", which may also be referred to as reaction sites, in a miniaturized grid (for example 500 μm) on a mostly flat substrate molecules or "capture probes") such as DNA, cDNA, proteins, antibodies, biological cells or the like A "microarray-based test" allows the detection of the molecules complementary to the capture molecules. Microarray-based assays are of great commercial interest, for example in gene expression analysis, single nucleotide polymorphism (SNP) analysis or personalized medicine.

A "microarray-based test" consists of a multistep procedure, in which a defined microarray, ie the substrate with the immobilized capture molecules, is brought into contact with the liquid to be examined, so that the complementary molecules can react with the capture molecules Reaction can take many hours and depends on the characteristic times it takes to complete the complementary ones Molecules have arrived at the capture molecules and have reacted there, for example, have hybridized a DNA.

By continuous "stirring" or suitable fluid guidance, the complementary molecules can be brought very close to the capture molecules very quickly, so that the diffusion lengths can be greatly shortened and thus the time periods for the reaction, for example the hybridization can be greatly reduced Cells with the microarray perform further process steps, such as the washing of the substrate and thus the microarray, ie the removal of unbound or non-specifically bound molecules by exchange of the liquid to be examined by a defined buffer, or the supply of reagents, such as fluorescently labeled detection antibody to the microarray.

For example, in the prior art, microarrays are processed by manually pipetting the sample and the relevant reagents. Due to the planar surface of the substrate, typically in the form of a microscope slide, the consumption of sample and reagents is high. In addition, the overflow of the microarray reaction points with the sample to be examined is not controlled. Due to the non-continuous closed substrate it can very quickly lead to Eindrocknungseffekten at individual reaction points, which adversely affect the outcome of the experiment.

From the prior art, it is also known to process microarrays in a closed fluidic structure. Here, there are technical solutions, for example, by a targeted mixing of the sample in reaction chambers using surface acoustic wave (SAW = SAW = surface acoustic wave). Such techniques have been disclosed, for example, by Advalytix AG (www.advalytix.de). An alternative technique was to perform a targeted mixing of the sample in reaction chambers by a coupled rotational movement about two axes. Such a technique is described in MA Bynum et al., "Hybridization Enhancement using Microfluidic Planetary Centrifugal Milling", Analytical Chemistry, Vol. 76, No. 23, December 1, 2004, pages 7,039-7,044 The chamber is then placed in a planetary centrifuge and the centrifuge rotates the chamber about an axis of rotation external to the chamber at a rotational speed of 1,200 revolutions per minute still rotated around its own axis at a rate of 10 revolutions per minute.

Systems utilizing pressure-driven or electrically-driven convection are described in Rinie van Beuningen et al., "Fast and Specific Hybridization Using Flow Through Microarrays on Porous Metal Oxides", Clinical Chemistry, 47 (10), pp. 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), p.2412-2420, 2001.

Chaotic advection is described in Mark K. McQuain et al., "Chaotic mixer improved microarray hybridization," Analytical Biochemistry, 325 (2), pp. 215-226, 2004.

Sample oscillation using microfluidic hybridization and a meandering array channel is described in 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 is described in K. Pappaert et al., "Enhancement of DNA micro-array analysis using a shear-driven micro-channel flow system", Journal of Chromatography A, 1014 (1-2), p. 1-9, 2003; Johan Vanderhoeven et al., "Exploiting the benefits of miniaturization for the enhancement of DNA microarrays," 25 (21-22), pp. 367-3686, 2004, and Johan Vanderhoeven et al., "DNA microarray enhancement using a continuously and discontinuously rotating microcage ", Analytical Chemistry, 77 (14), pp. 4474-4480, 2005.

Cavitation-induced microflows are described in Robin Hui Liu et al., "Hybridization enhancement using cavitation micro-streaming", Analytical Chemistry, 75 (8), pp. 1911-1917, 2003.

Finally, acoustically induced convection is described by Andreas Toegl et al., "Enhancing results of microarray hybridizations through microagitation", Journal of Bio- molecular Techniques, 14 (3), p.197-204, 2003, Achim Wixforth, "Fiat fluidics: acoustically driven planar microfluidic devices for biological and chemical applications ", in Proceedings of the ^{th th} International Conference on Solid-State Sensors, Actuators & Microsystems (Transducers' 05), June 5-9, Seoul, Korea, S. 143-146. IEEE, 2005.

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

Disadvantages of the known state of the art for processing microarrays are the necessarily active and complex systems for excitation and control in order to achieve targeted mixing of the sample into reaction chambers. Also, some of the alternatives require Systems, such as pumped systems, inlets and outlets, whose additional dead volumes have a negative effect on the sensitivity of the arrays.

The minimum lateral dimensions of the sensing structure are dictated by the lateral dimensions of the 2-dimensional microarray (typically in the range of one square centimeter) through the manufacturing process. At the same time, the dead volume of the chamber should be minimized. Technically speaking, during the litigation, it is mainly a question of passing a liquid sample past a detection structure in a wide and as flat as possible chamber (in the case of the present invention, aspect ratios far below 1:10) with volumes in the microliter range.

The object underlying the present invention is to provide an apparatus and a method for handling a liquid sample which, in a simple structure, make it possible to guide an increased proportion of a sample arranged in a sample chamber past a detection structure.

This object is achieved by a method according to claim 1 and an apparatus 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 convective currents of a sample within the chamber by hydrodynamic inertial forces; to pass the sample past the detection structure.

The present invention is based on the recognition that convection currents generated by rotation with a time-varying rotary vector can be advantageously utilized to pass a large portion of a sample arranged in a sample chamber past a detection structure or around the parts of a sample who is exposed to the collection structure to change in a simple and fast way.

In embodiments of the invention, the acceleration phases may be interrupted by several intervals with a stationary or constantly rotating chamber, which may be advantageous for reactions to be effected.

In preferred embodiments of the invention, the sensing structure is a microarray, ie, a parallel array of a plurality of different but defined capture structures or capture molecules, which are immobilized on a support formed.

In this regard, the present invention is particularly useful for processing microarrays, where quasi-instantaneous active delivery of sample and reagents to the reactants in the microarray points may be referred to as rotating the chamber with a time-varying rotational vector, which may be referred to as a shake mode. takes place. Further, this results in the rapid removal of unbound reactants from the microarray points, i. the individual catcher structures or catcher molecules.

In preferred embodiments of the present invention, a microarray body and a lid together form a reaction chamber whereby convective rotation in the reaction chamber causes convection in the chamber which accelerates mixing (between sample and microarray) in a microarray experiment. Thus, due to forced convection of a sample placed in a reaction chamber, for example, the time for DNA hybridization can be reduced.

In preferred embodiments, the reaction chamber may be completely filled with liquid.

In embodiments of the present invention, the chamber may be formed in a body, the body having a substrate and a lid chip. One or more such bodies can be inserted into a rotor which can be set in rotation by means of a rotary motor. The detection structure, for example the microarray, can be immobilized on the substrate, while a channel structure with inlet region, reaction cavity and outlet region is structured in the cover chip. Alternatively, the sensing structure may be immobilized on the part of the body, such as the lid, in which the channel structures are formed while the lid can be a plainer member. The channel structure may include one or more inlet regions, one or more sample chambers, such as reaction chambers, and one or more outlet regions.

In embodiments of the present invention, the body itself may be formed as a body of revolution, for example a disk, which is rotatable about an axis of rotation. Such a rotary body can likewise be formed from substrate and cover chip and have a channel structure required for implementing the present invention or for parallelization a plurality of corresponding channel structures which are arranged in a star shape on the pane.

According to embodiments of the present invention, the channel structure may include an outlet channel fluidly connected to the chamber and having a siphon structure which in a given centrifugal force field results in a defined fill level of the liquid sample in the chamber. This makes it possible, especially in processes in which successively different liquids are to be supplied to a detection structure, to reliably avoid drying out of the detection structure.

A siphon structure is generally understood to mean a structure subjected to the gravitational field, in the case of the present invention the centrifugal field, comprising a reservoir and a connecting channel, one end of which is connected to the reservoir and the other end of which has an outlet. The system is at least partially filled with a liquid.

According to the principle of communicating tubes, the system now aims for a hydrostatic equilibrium state, in which all menisci take the same position in the direction of the force field. If the exhaust duct is so forms that it completely filled with liquid according to the principle of communicating tubes, flows when the position of the outlet is below the liquid level in the hydrostatic equilibrium, as long as liquid from the outlet until the liquid level in the reservoir is at the level of the outlet. In sanitary engineering, the continuous liquid column of the connecting channel acts as an odor stop, which remains odor-proof even after rinsing.

Once the connection channel is completely filled with liquid, the liquid level can also be adjusted independently of the course of this connection channel only by the height of its outlet. By lowering the outlet below the reservoir bottom, the reservoir is emptied as long as the continuous column of liquid is not interrupted. For example, if the connection of the connection channel is at the bottom of the reservoir, the reservoir is completely emptied.

The complete filling of the siphon can be done either by the principle of communicating tubes by pouring into the reservoir until the common liquid level is in the hydrostatic equilibrium state above the highest point of the connecting channel, and / or by the capillary driven filling of the channel.

As a siphon effect, the regulation of the filling level in a reservoir by the course and the position of the connecting channel according to the principle of the communicating tubes should be understood here in general.

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, C0 ₂ sensors or biosensors that use biomolecules to detect properties of a sample.

The channel structures are preferably designed such that the interaction of centrifugal force and capillary force with synchronized sample addition and reagent addition, for example, allows complete processing of a microarray experiment. In this case, a siphon structure in the centrifugal force field can generate a course of the filling level defined by the time curve of the rotational frequency and the liquid volumes added at specific times, whereby the reaction conditions in the reaction chamber can be precisely controlled. In particular, when carrying out a microarray experiment, the siphon structure can cause the reaction space to always be filled with liquid at least to such an extent that the microarray is covered by the latter when successively introducing the sample, a washing liquid and reagents into the reactor. be supplied onsraum. Thus, the microarray can be processed under continuously controllable fluidic conditions, since in the centrifugal force field of the siphon channel determines the filling level in the reaction chamber.

The present invention thus consists, in accordance with preferred exemplary embodiments, of an apparatus and a method for processing microarrays on a preferably planar substrate, the invention making it possible to integrate the typical steps of a microarray-based experiment described below by way of example. These steps include adding the sample to a reaction chamber and blocking it, optionally washing the reaction chamber multiple times, and thus the microarray, detecting, ie, staining, the microarray and subsequently washing the microarray again, optionally several times. Also reading the microarray, which can be done in any conventional manner, could be integrated. Thus, the present invention allows for nearly complete integration of all necessary steps to process a microarray-based experiment, with the reduction in manual operations also helping to increase the reproducibility of microarray-based experiments. The body in which the fluidic structures are formed, which preferably consists of a substrate and a lid, can be produced simply and inexpensively and can therefore be used as a disposable item.

In a pilot experiment, the reaction chambers had a volume of about 50 μL, the total volume consisting of sample, reagents and buffers in the experiment carried out was only 500 μL. This generally represents a considerable reduction in the consumption of the sometimes very expensive liquids. These volumes, in contrast to experiments that are carried out on a free, so not limited by a chamber microarray surface, very low and could be through a targeted optimization of the chamber shape to reduce considerably.

Due to the optimized hydrodynamic conditions, which can be achieved by applying the rotation with a time-varying rotary vector and can be achieved alternatively or supportive by a meandering reaction chamber, in the pilot experiment, the process time could be reduced to 45 minutes. By far the largest part of this time is spent on the reaction / incubation between the initially introduced sample and the detection structure, while a washing step is completed in less than a minute. In general, a further drastic acceleration of the process time and also a considerable reduction of the chamber volume over the microarray is conceivable. The closed reaction chamber useful in the present invention allows the microarray-based experiment to run permanently in a controlled environment compared to a non-capped substrate, which in particular can prevent the microarray spots from drying out. In particular, the "agitation" in the reaction space by rotating with a time-varying rotational vector allows a shortened process time, while the fluidic "siphon" structure provides a controlled environment.

Preferred embodiments of the present invention will be explained in more detail below with reference to the accompanying drawings. Show it:

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

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

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

Fig. 7 shows the results of an experiment conducted using the invention;

8 shows examples of temporally variable rotation vectors that can be used according to the invention;

9 shows an exemplary frequency protocol for carrying out a microarray experiment and the resulting fill level in the chamber. An embodiment of the device according to the invention will now be explained first with reference to FIGS. 1 and 2.

The device comprises a rotor 10 which is connected to a shaft 12 which is drivable by a rotary motor 14. The rotor 10 may be mounted in any manner, for example on a step portion of the shaft 12. Of the

Rotary motor 14 is provided with a controller 16 which is designed to set the rotor in the required manner in rotation.

The rotor comprises four receiving regions 20 for accommodating modules 22 containing fluid structures according to the invention. In the embodiment shown, the modules consist of a substrate 24 and a cover 26, wherein fluid structures are formed in the substrate 24. The fluid structures comprise, as shown in FIG. 1 and later explained in more detail with reference to FIG. 3, a reaction chamber 30, an outlet channel 32 with a centrifuge siphon structure 32, a waste reservoir region 34 and two inlet channels 36 and 38. In the embodiment shown, the fluid structures in the substrate 24 are 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, the rotary motor 14 is 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. In preferred exemplary embodiments, the fluidic structures or channel structures are In this case, a planar substrate is used, on which the microarray is immobilized, while a lid chip is structured in order to realize the fluidic structures.

In the example shown in FIG. 3, the fluidic structures comprise an inlet region 40 for a sample or an analyte and an inlet region 42 for reagents. The inlet region 40 is connected to a reaction chamber 46 via an inlet channel 44, while the inlet region 42 is connected to the reaction chamber 46 via an inlet channel 48. The inlet channels 44 and 48 are radially inwardly connected to the reaction chamber 46 with respect to a centrifugal force field (see arrow F _v in FIG. 3). Radially outward, an outlet channel 50 is connected to the reaction chamber, which in this application example has such a siphon structure 52, which initially consists of a radially outwardly extending channel section 52a in the direction of the centrifugal force field, then a channel section 52b running radially inwardly against the force field, and then in turn, is formed from a channel section 52c extending radially outward with the force field. 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 provided for the siphon structure. The vent 54 may optionally be completely or partially hydrophobized. The outlet channel 50 opens into a waste reservoir 56. The vent 54 is provided in the waste reservoir 56. Optionally, a hydrophobized section 57 may be provided at the end of the outlet channel 50.

The siphon structure 52 is designed to generate a defined liquid level in the reaction chamber 46 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. Preferably, the structure is designed such that the reaction chamber 46 is completely filled with liquid in this phase.

The inlet regions 40 and 42 have inlet area openings 40a and 42a. When filling the two inlets, the respective inactive channel serves as a vent. In Fig. 4, an alternative structure is shown, in which all liquids are supplied through a single channel 84, but this complicates the venting. The channel 84 is connected at its radially inner end to an inlet region 82 which has an inlet region opening 82a and to a chamber 80 at its radially outer end.

In the exemplary embodiments of FIGS. 3 and 4, the coordinate Δr corresponds to 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, independent 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 be, for example, by the capillary force at low speeds or at rest structure take place, or by adding a volume of liquid that forces (temporary) an equilibrium liquid level above the radially innermost point of the Siphonkanals r _max.

In preferred embodiments of the invention, the channel structures are formed in the lid chip, wherein the Lid 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 portion 40 and the inlet channel 44 serve to supply sample liquid to the reaction space 46 by centrifugal force on the rotating assembly. The inlet region 42 and the inlet channel 48 serve to supply reagents to the reaction space 46 by the centrifugal force on the rotating assembly. The outlet channel 50 serves to leave the first supplied sample at arbitrarily high speeds and during the shaking mode in the chamber and so to set a constant liquid level in the reaction chamber 46. Preferably, the reaction chamber 46 is always completely filled with liquid in this phase and thus drying out of the reaction chamber and thus of the microarray arranged therein is reliably prevented. Already in the reaction chamber 46 located liquid is displaced by the subsequently supplied liquid volumes in the waste area. The inlet regions 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. Further, the optionally hydrophobized vent 54 includes an opening in either the lid chip or the substrate to allow venting.

An embodiment of a method according to the invention will now be described by performing a microarray experiment.

In this case, first of all a sample liquid, ie an analyte, is introduced into the reaction chambers 46 by filling the inlet region 40 with the sample liquid and simultaneously or subsequently with the channel structure (for example by the rotary motor 14 with the associated control device 16, FIG. 2) suitable rotation is acted upon by the centrifugal force F _x , and optionally also by a capillary force to introduce the liquid through the inlet channel 44 in the reaction chamber 46. 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 time for mixing complementary molecules in the sample to the capture molecules can be reduced by deliberately "agitating" the liquid in the reaction chamber 46. Due to the convection currents, molecules that can be far away from the capture molecules in the steady state also become Close to the immobilized and complementary catcher molecules, non-bound reactants are rapidly removed from the capture molecules.

After sufficient time for the processing of the microarray, a washing liquid is then introduced into the reaction chambers 46 via the inlet region 42, and centrifugally through the inlet channel 48. The sample liquid in the reaction chamber 46 is thereby displaced via the outlet channel 50 and the siphon structure is centrifugally displaced or filled by the capillary force in a suitably small centrifugal field. With complete filled siphon takes place in the centrifugal field, a continuous emptying of the reaction chamber 46 instead.

The siphon principle also shows that the newly introduced volume corresponds to the displaced volume. In order to ensure a complete exchange of liquid in the reaction chamber 46, the newly introduced volume must thus 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, a complete volume exchange takes place in the chamber 46.

After the washing liquid has been supplied, further reagents, for example fluorescently labeled detection antibodies, can be introduced into the reaction chamber 46 via the inlet region 42 and the inlet channel 48 by utilizing the centrifugal force. 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 FIG. 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 overall structure.

FIG. 5 schematically shows a channel structure whose outlet channel 70 has an alternative siphon structure 72. The siphon structure includes a radially outwardly extending portion 70a and a radially inward portion 70b that terminates in a waste reservoir 76 provided with a vent 76a. These at the highest point of the siphon -T _max truncated structure is based in the centrifugal field directly on the principle of communicating tubes. 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 complete filling thereof, as indicated again by the fill level 58.

With the described apparatus and the described method an experiment was carried out, the sequence of which is shown schematically in FIGS. 6a and 6b and whose results are shown in FIG. More specifically, two experiments were performed, and in the experiment shown in Figure 6a, a microarray substrate 90 was used in which the reaction chamber 92 is patterned 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).

In the experiment of FIG. 6b, a non-structured substrate, and in particular a PDMS substrate 96 (polydimethylsiloxane substrate) without channel structures was used.

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

The corresponding microarray substrates are shown at point 1 in Figs. 6a and 6b. The substrates were then provided with a respective cover, wherein, according to FIG. 6a, an unstructured cover 98 is used, while according to FIG. 6b a reaction chamber 100 is structured in the cover 102. is ruled. In this case, the lid 98 is a PDMS lid, while the lid 102 is 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 seen at point 2 of Figs. 6a and 6b. Subsequently, sheep anti-mouse Cy3 antibodies were supplied, see item 3 in Figs. 6a and 6b. The specific antigen-antibody complexes are formed. This is followed by a washing step, whereupon fluorescent detection antibodies 104 are added, with which the said complexes are labeled.

The results obtained are shown in Figure 7, where curve 110 relates to the COC microarray substrate with channel structure, while curve 112 relates to the PDMS microarray substrate without channel structure. Fig. 7 shows that a clear and reproducible correlation is obtained for varying target concentrations C _BSA and the resulting fluorescence signal I over a working range of at least 3 decades. 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 multiple acceleration and multiple deceleration, are shown in FIGS. 8a, 8b and 8c. Fig. 8a shows a switching between maximum rotational frequencies f _max with different rotational direction. For example, the maximum rotational frequency may be 8 Hz, while the maximum rotational acceleration may be ± 32 Hz / s, for example. Fig. 8b shows a switching between a maximum rotation frequency f _max and a rotation frequency of 0, so that the rotation protocol as shown in Fig. 8 can be performed by using centrifuges operable in only one direction of rotation. Finally, Fig. 8c shows a rotation protocol in which after accelerating to the respective maximum Rotation frequency short-term rotation takes place at this maximum rotation frequency. Not shown in FIGS. 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 v (t) and filling levels in the reaction chamber Δr as a function of the time t for the siphons in the manner of FIGS. 3 and 5. With regard to the level heights, the curve 120 refers to the siphon structure of FIG. 3, while the curve 122 relates to the siphon structure of FIG. 5. The time scale is not linearly scaled here, for example the sample incubation time in the shake mode is typically at least ten times longer than the subsequent phase of the wash buffer washout. In the resting phases .DELTA.ti and .DELTA.t ₂ liquid volumes are added.

As can be seen from the curve 120 in FIG. 9, the microarray structure according to FIG. 3 is completely covered during the shaking mode. Upon the addition of a subsequent volume and the associated complete filling of the siphon, the chamber is continuously lent during rotation, as shown in section 120a of the curve 120.

Notwithstanding the illustrated preferred embodiments, numerous variations of the present invention are düng possible. Thus, instead of the described disk-shaped rotor 10, a four-armed rotor, also indicated in FIG. 1, could 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. In alternative embodiments of the present invention, instead of the siphon structure of the outlet channel, moreover, the outlet channel could be provided with a hydrophobic barrier or a high flow resistance, representing a rotational frequency dependent liquid switch.

In preferred embodiments of the invention, the channel structures comprise one or more inlet channels to supply liquid by centrifugal force into the reaction chamber. Alternatively, however, the liquid could also enter the reaction chamber in any other ways, for example via a feed channel while exerting a pressure or by manual filling, for example with a pipetting system. The outlet channel or the siphon structure of the same does not constitute a mandatory feature of the invention, in particular if several liquids are not to be successively passed through the reaction chamber, but only the properties of a liquid are to be detected using a sensor structure. The liquids could also be sucked or flushed out through a lid opening or intermediate removal of the lid.

In addition, the mixing process could be further improved by the (para) magnetic in the above-mentioned publication M. Grumann et al., "Batch Mode Mixing on Centrifugal Microfluidic Platforms", Lab Chip, 2005, 5, pages 560-565 Beads in combination with permanent magnets, which are fixedly arranged along the chamber orbit, assist In particular in the shaking mode, beads whose density differs from the corresponding liquid density could also accelerate the mixing process by means of a centrifugation or a correspondingly positioned magnet finally be removed from the detection window of the microarrays. For example, the rotary actuator may be designed to perform a frequency-controlled PC-controlled protocol to generate a time-dependent centrifugal field to precisely control flow rates and timing for the individual steps to be performed. A control software can also control, for example, one or more dispensers, which give the reagents to the inlets at rest or even with appropriate synchronization with a rotating substrate. For example, by a suction device could also automate the removal of fluids.

The present invention thus provides apparatuses and methods that are particularly suitable for performing microarray experiments in an increasingly automated manner. In particular, the present invention provides a way to efficiently deliver sample liquid and the molecules contained therein capture molecules of the microarray and to prevent during the entire experiment that the microarray dries out. 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 microarray experiments to be carried out with reduced time, a fast and homogeneous reaction, an automated washing process, a reduced consumption of sample and reagents, and an integrated waste handling. The elimination of uncontrolled drying steps by the siphon structure also allows the sensitivity of the microarray experiment. In this way, the use of separate devices such as slide cleaners and the like becomes obsolete and handling steps are automated. Finally, the symmetry of the rotor or body of revolution potentially allows multiple microarray experiments to be performed in parallel.

According to the invention, the chamber is preferably designed to be completely, i. without gas inclusion, to be filled with the sample. For this purpose, the chamber may be completely closed or with the exception of inlet channel and

Drain channel be completely closed. The present

Invention allows an efficient even with completely filled chamber and very small sample volumes

Passing the sample past a detection structure.

Claims

claims 1. A method for handling a liquid sample, comprising the following steps: Placing the sample in a chamber (30; 46; 80; 92; 100) in which a sensing structure (60; 94) enabling detection of a property of the sample is disposed; Rotating the chamber with a time-varying rotary vector having multiple accelerations and multiple decelerations to generate convective currents of the sample in the chamber by hydrodynamic inertial effects to bypass the sample at the sensing structure (60; 94). 2. The method of claim 1, wherein the chamber is completely filled with the sample. The method of claim 1 or 2, wherein the sensing structure (60; 94) is a microarray of capture structures for trapping molecules from the sample. 4. The method of claim 1 or 2, wherein the detection structure comprises a sensor structure. 5. The method of claim 4, wherein the sensor structure comprises a biosensor, an oxygen sensor or a C0 ₂ sensor. 6. The method according to any one of claims 1 to 5, wherein the rotation vector has a multiple acceleration and deceleration in one direction of rotation. 7. The method according to any one of claims 1 to 5, wherein the rotation vector has a multiple acceleration and deceleration in different directions of rotation. 8. The method of any of claims 1 to 5, wherein the introducing step comprises introducing the sample through an inlet channel (36; 44; 84) by centrifugal force. 9. The method of any one of claims 1 to 8, further comprising a step of draining the sample from the chamber (30; 46; 80; 92; 100) by centrifugal force through an outlet channel (32; 50; 70). 10. A method according to any one of claims 1 to 9, further comprising a step of introducing a washing liquid into the chamber (30; 46; 80; 92; 100) to thereby separate the sample from the chamber by a siphon structure (30; 52, 72) which, in a given centrifugal force field, produces a defined fill level in the chamber to empty from the chamber. The method of any of claims 1 to 10, further comprising a step of introducing reagents into the chamber. 12. The method of claim 11, wherein the sample and the reagents via separate inlet channels (36, 38; 48) are introduced into the chamber. 13. Device for handling a liquid sample, having the following features: a chamber (30; 46; 80; 92; 100) in which a sensing structure (60; 94) enabling detection of a property of the sample is disposed; drive means (14) configured to subject the chamber to rotation; and control means (16) 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 to pass the sample past the detection structure (60; 94). 14. The apparatus of claim 13, wherein the chamber is adapted to be completely filled with the sample. The device of claim 13 or 14, wherein the sensing structure (60; 94) is a microarray of capture structures for capturing molecules from the sample. 16. The apparatus of claim 13 or 14, wherein the detection structure comprises a sensor structure. 17. The apparatus of claim 16, wherein the sensor structure comprises a biosensor, an oxygen sensor or a C0 ₂ sensor. 18. Device according to one of claims 13 to 17, wherein the rotary vector has a multiple acceleration and deceleration in one direction of rotation. 19. Device according to one of claims 13 to 17, wherein the rotational vector has a multiple acceleration and deceleration in different directions of rotation. The apparatus of any one of claims 13 to 19, wherein the chamber (30; 46; 80; 92; 100) is formed in a body. The apparatus of claim 20, wherein the body further comprises an inlet channel (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. 22. The apparatus of claim 21, wherein the outlet channel has a siphon structure (52; 72) which adjusts the level in the chamber according to the siphon principle. The apparatus of any one of claims 20 to 22, wherein the body comprises a substrate (24; 90; 96) and a lid (26; 98; 102), the chamber and, if present, the inlet channel and the outlet channel in the substrate, the lid or both are structured. 24. The device of claim 23, wherein the detection structure (60; 94) is immobilized on the substrate. The apparatus of any of claims 20 to 24, wherein the substrate (24; 90; 96) has a separate inlet channel (38; 48) for introducing reagents into the chamber.