WO2007042208A1

WO2007042208A1 - Device and method for handling a liquid sample using a siphon-structure

Info

Publication number: WO2007042208A1
Application number: PCT/EP2006/009661
Authority: WO
Inventors: Markus Grumann; Jens DUCRÉE; Roland Zengerle; Oliver Gutmann
Original assignee: Albert-Ludwigs-Universität Freiburg
Priority date: 2005-10-07
Filing date: 2006-10-05
Publication date: 2007-04-19
Also published as: DE102005048233A1

Abstract

The invention relates to a device for handling a liquid sample, which comprises a body which comprises a chamber (30) and an outlet channel (32). A detection structure is arranged in the chamber (30) which enables a property of a sample to be detected. The outlet channel (32) is fluidically connected to the chamber (30) and has a siphon-structure, which leads to a defined filling hole of the liquid sample in the chamber (30) in a provided centrifugal force field. Said type of device is particular suitable for carrying out microarray-experiments.

Description

Apparatus and method for handling a liquid sample using a siphon structure

description

The present application relates to a method and apparatus for handling a liquid sample using a centrifugal siphon structure suitable for assisting in the performance of microarray experiments.

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 molecular ^ΛV ), 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 up to several hours and depends on the characteristic times it takes for the complementary molecules to reach the catcher molecules and react there, for example to have hybridized DNA. By means of 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 durations for the reaction, for example the hybridization, can be greatly reduced The molecules with the microarray are further process steps, such as the washing of the substrate and thus the microarray, ie the removal of unbound molecules by exchange of the liquid to be examined by a defined buffer, or supplying reagents, such as fluorescently labeled detection antibodies, 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 waves. 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. One such technique is in MA Bynum et al., "Hybridization Enhancement using Microfluidic Planetary Centrifugal Mixturing ", Analytical Chemistry, Vol. 76, No. 23, 1 December

2004, pages 7,039 - 7,044. According to this technique, samples are applied to seal slides and covered with a microarray to form a chamber. The chamber is then inserted into a planetary centrifuge. The centrifuge rotates the chamber about an axis of rotation external to the chamber at a rotational speed of 1,200 revolutions per minute. In addition, the chamber is still rotated at a rate of 10 revolutions per minute about its own axis.

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

Johan Vanderhoeven et al., "DNA Microarray Enhancement Using a Continuously and Discontinuously Rotating Microcave", Analytical Chemistry, Vol. 77, No. 14, Jul. 15

2005, pp. 4474 to 4480, describe a DNA microarray hybridization in which a set of rotating circular chambers covering the microarray region are placed on the same with a depth of 1.6 to 70 μm. A substrate in which the chambers are formed is rotated while the microarray is held stationary.

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.

The object underlying the present invention is to provide a device and a system for handling a liquid sample, which make it possible to drying out of the detection structure, in particular during the typically on the hourly scale carried out

^"" led reaction is avoided with the sample, even if successively different liquids to one

5 detection structure to be supplied.

The present invention provides an apparatus for handling a liquid sample, having the following features:

0 a body having a chamber and an outlet channel,

wherein in the chamber, a detection structure is arranged, which allows detection of a property of the sample, 5 and

wherein the outlet channel is fluidically connected to the chamber and adjusts the level in the chamber according to the siphon principle. 0

The present invention further provides a method of handling a liquid sample, comprising the steps of:

Introducing the liquid sample into a chamber in which a sensing structure enabling detection of a property of the sample is disposed, wherein an outlet channel having a siphon structure is fluidly connected to the chamber; and

0 Rotate the chamber and the outlet channel to create a centrifugal force field, which results in a defined filling level of the sample in the chamber by a siphon structure.

A siphon structure generally refers to a structure exposed to the gravitational field, in the case of the present invention the centrifugal field, comprising a reservoir and a connecting channel, one of which End is connected to the reservoir and the other end 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 outlet channel is designed to be completely filled with liquid according to the principle of communicating tubes, if the position of the outlet is below the liquid level in hydrostatic equilibrium, liquid flows out of the outlet until the liquid level in the reservoir is at the level of the liquid Outlet is located. 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 should generally control the level in a reservoir through the course and the Position of the connecting channel can be understood on the principle of communicating tubes.

By providing the outlet channel with a centrifugal siphon structure, it is possible according to the invention to ensure that the detection structure does not dry out, i. is always covered with liquid, even if successively different liquids are introduced into the fluid chamber. For example, when microarray experiments are carried out, it is necessary to successively pass different liquids past the microarray, which is advantageously possible by the inventive approach, without running the risk of the microarray drying out.

In embodiments of the present invention, the method may further include a step of rotating the chamber with a time varying rotary vector having multiple accelerations and multiple decelerations to produce convective currents of the sample in the chamber by hydrodynamic inertial effects around the sample passing the collection structure. Optionally, phases with a temporally constant or vanishing rotation vector can also be inserted.

Accordingly, the apparatus of the present invention may include drive means configured to subject the chamber to rotation and control means configured to control the drive means to rotate the chamber with a time-varying rotational vector which causes multiple accelerations and having multiple decelerations to generate, by hydrodynamic inertial forces, convection currents of a sample in the chamber to pass the sample past the detection structure. Such embodiments are based on the recognition that convection currents generated by rotation with a temporally variable rotation vector can be advantageously exploited to a large proportion of a sample arranged in a sample chamber to lead past a detection structure, or to change the parts of a sample which is exposed to the detection structure in a simple and fast manner.

In preferred embodiments of the invention, the detection structure is defined by a microarray, i. H. a parallel arrangement of a plurality of different, but defined capture structures or capture molecules, which are immobilized on a carrier formed.

In this regard, the present invention is particularly useful for processing microarrays, wherein drying out of the microarray can be prevented, and in preferred embodiments, a quasi-instantaneous active delivery of sample and reagents to the reactants in the microarray points by rotating the chamber with one time-varying rotary vector, which may be referred to as a shake mode, takes place. Further, driven by the centrifugal field and possibly capillary forces, 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 closed reaction chamber. By alternating rotational movements of the reaction chamber convection in the chamber can be brought about, which accelerates the coupling (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 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 used in 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 in the cover chip a channel structure having at least the reaction chamber and the outlet channel is structured. 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 may be a planar 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.

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 interaction of centrifugal force and capillary force with synchronized sample addition and reagent addition makes it possible, for example, to fully process a microarray experiment. In the centrifugal force field, the siphon structure generates a course of the filling level defined by the time curve of the rotational frequency and the liquid volumes added at specific times, which allows precise control of the reaction conditions in the reaction chamber. In particular, when carrying out a microarray experiment, the siphon structure can cause the reaction space to always be filled with liquid at least so far that the microarray is covered by the latter when the sample, a washing liquid and reagents are fed successively into the reaction space. 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 siphon principle also shows that the newly introduced volume in an already completely filled siphon channel corresponds to the displaced volume. In order to ensure a complete exchange of liquid in the chamber, the newly introduced volume must therefore at least correspond to the chamber volume. Under laminar conditions, which are typical in the very shallow chambers (at typical aspect ratios well below 1:10) and cause straight mixing problems, the volume displacement can be hydrodynamically designed so that the newly introduced is not flushed out with. Ideally, a complete volume exchange takes place in the chamber 46. In embodiments of the invention, the inlet is in the siphon structure of the outlet channel in a radially outer region of the chamber, so that the supplied fluids initially remain in the chamber and displaces only the volume that has already been in the chamber, out of the chamber becomes. The method may include sequential introduction of different liquids into the chamber in embodiments of the invention. According to the invention, one or more liquids can be supplied to the chamber in different ways, eg centrifugally or by pipetting or pumping systems. Furthermore, according to the invention, instead of a centrifugal pre-filling of the siphon structure, a purely capillary pre-filling of the siphon structure can also take place.

Thus, in accordance with preferred embodiments, the present invention is an apparatus and method for processing microarrays on a planar substrate, which invention enables the integration of the typical microarray-based experiment typical steps described below. These steps include adding the sample to a reaction chamber and blocking it, optionally washing the reaction chamber several times, and thus the microarray, to detect, i. H. the dyeing, the microarray and a following, again optionally multiple washing of the microarray. 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 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 chamber had a volume of about 50 μL, the total volume consisting from sample, reagents and buffers performed in the

Experiment at only 500 μL. This generally represents a considerable reduction in the consumption of the Tei-1 very expensive fluids. Due to the optimized hydrodynamic conditions, the by the application of the

Rotation can be achieved with a time-varying rotary vector and can be achieved alternatively or supportive by a meandering reaction chamber, the process time could be reduced to 45 minutes in the pilot experiment. 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 a considerable reduction of the liquid volumes are 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 prevents the microarray spots from drying out. In particular, the optional "stirring" in the reaction space by rotating with a temporally variable rotation vector allows a shorter 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:

Fig. 1 and Fig. 2 schematically shows a plan view and a

Cross-sectional view of an embodiment of a device according to the invention, wherein FIG. 2 schematically shows a cross-sectional view along the

Line XX of Fig. 1 represents. 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. 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 exemplary 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 will be explained in more detail below with reference to FIG. 3, a reaction chamber 30, an outlet channel 32 with a centrifugal siphon structure 32, a waste reservoir area 34 and two inlet channels 36 and 38. In the embodiment shown, the fluid structures in the substrate 24 are patterned. Alternatively, the fluid structures could also be patterned in the lid or lid and substrate. The combination of substrate and lid produces 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 embodiments, the fluidic structures or channel structures are formed using a planar substrate on which the microarray is immobilized while a lid chip is patterned 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 connected radially inwardly to the reaction chambers 46 with respect to a centrifugal force system (see arrow F _v in FIG. 3). Radially outward, an outlet channel 50 is connected to the reaction chambers, which has such a siphon structure 52, which initially consists of a radially outwardly extending channel section 52a, then a radially inwardly extending channel section 52b and then again extending radially outward Channel section 52c is formed. The radial position of the outlet of the channel structure lies below the bottom of the reaction chambers 46, that is to say Δr <0. A vent 54 is provided for the siphon structure. If desired, the vent 54 can be rendered completely hydrophobic or partially hydrophobic. 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 define at a given centrifugal force field defined by the rotational velocity which results in the centrifugal force F _v , the dimensions of the fluidic structures, as well as the liquids used, upon initial filling with the sample Liquid level in the reaction chamber 46 to produce. Preferably, the structure is designed such that the reaction chamber 46 is always 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, then so this liquid level remains constant at Δr> 0, regardless of the rotation frequency. In this phase, in this embodiment, the sample is incubated with the microa-rray in the Schütel mode. By contrast, if the siphon channel is completely filled with liquid up to its outlet, the liquid from the chamber runs continuously into the outlet during centrifugation. Such filling can be done, for example, by the capillary force at low speeds or at rest, or by adding a volume of fluid which (temporarily) forces an equilibrium fluid level above the radially innermost point of the siphon channel r 1.

In preferred embodiments of the invention, the channel structures are formed in the cover chip, wherein the cover chip is brought together with a microarray substrate such that the microarray 60 is arranged in the reaction chamber. The walls of the channel structures preferably have hydrophilic properties. The inlet portion 40 and the inlet channel 44 serve to supply sample liquid by centrifugal force on the rotating assembly to the reaction space 46. 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 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 applied by the centrifugal force F _{v 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 the reaction chamber has been filled, it is then subjected to a time-variable rotary vector which has a multiple acceleration and a multiple deceleration. As a result of the rotation of the arrangement with a temporally variable rotary vector, hydrodynamic inertial forces within the reaction chambers produce convection currents of the liquid located there. Thus, the time for the mixing of complementary molecules in the sample to the capture molecules can be reduced by a specific "stirring" of the liquid present in the reaction chamber 46. Due to the convection currents, molecules are also removed which are far removed from the capture molecules in the stationary case may be near the immobilized capture molecules unbound reactants are rapidly removed from the capture molecules.

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

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 and the outlet channel 50 constitute the flow-determining channels, ie. H. have a cross section through which the hydrodynamic flow resistances are defined.

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 extending portion 70b that terminates in a waste reservoir 76 provided with a vent 76a. This structure cut off at the highest point of the siphon r _max is based in the T / EP2006 / 00966!

fugal field directly on the principle of communicating tubes. In hydrostatic equilibrium, a liquid level is .l _Δr> .JD. above _r _max the complete filling as well as at the same time also the determination of the equilibrium liquid level on r _max safe. The end of the siphon channel at r _max is preferably above the detection structure and thus ensures a complete filling thereof, as indicated again by the 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 chambers 92 are patterned and to 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 lid, wherein, according to FIG. 6 a, an unstructured lid 98 is used, while according to FIG. 6 b a reaction chamber 100 is structured in the lid 102. In this case, the lid 98 is a PDMS lid while the lid 102 is a COC lid. Hereinafter, the sample having a known target concentration.ti.an. _cB.SÄ__ of mouse AntirBSA antibodies, __in_ die_

Reaction chambers introduced, as can be seen under point 2 of Fig. 6a and 6b. Hereafter, sheep anti-mouse

Cy3 antibodies supplied, see point 3 in Fig. 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 acting on the channel structures with a temporally variable rotation vector, which has multiple acceleration and multiple deceleration and possibly also phases with a constant rotation vector, 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. Fig. 8b shows switching between a maximum rotational frequency f _max and a rotational 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 rotational frequency a short rotation at this maximum rotational frequency takes place.

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. 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 Δti and At 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 emptied during rotation, as can be seen from section 120a of the curve 120.

Notwithstanding the illustrated preferred embodiments, numerous variations of the present invention are possible. Thus, instead of the described disc-shaped rotor 10, a four-armed rotor could be used. Again alternatively, the body in which the fluoride structures are formed could be formed as a body of revolution, 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 preferred embodiments of the invention, the channel structures comprise one or more inlet channels, to supply liquid by centrifugal force to the reaction chamber. Alternatively, however, the liquid could also be on. Any other species can reach the Reaktip_ns_karηme_r_, for example via a feed channel while exerting a pressure or by manual filling.

For example, the rotary drive may be designed to perform a frequency controlled PC-controlled protocol to generate a time-dependent centrifugal pressure to precisely control flow rates and timing for each step to be performed.

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. 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 performed with reduced time, a fast and homogeneous reaction, an automated washing process, a reduced consumption of sample and reagents, and an integrated waste handling. Furthermore, by eliminating uncontrolled drying steps through the siphon structure, the sensitivity of the microarray experiment can be increased. In this way, the use of separate devices such as slide cleaners and the like becomes obsolete and handling steps are automated. After all the symmetry of the rotor or the rotary body potentially allows that several be microarray experiments paral- _"lel _ d_ur_chge_führt.

Claims

claims

] Device for "._ Handhabe_n. eiηe_r ___ flüs_s_i_gen _J robe, with the following features:

a body having a chamber (30; 46; 80; 92; 100) and an outlet channel (32; 50; 70),

wherein in the chamber a detection structure (60; 94) is arranged, which allows detection of a property of the sample, and

wherein the outlet channel (32; 50; 70) is fluidically connected to the chamber and adjusts the level in the chamber according to the siphon principle.

2. Apparatus according to claim 1, further comprising drive means (14) for rotating the body to produce the given centrifugal force field.

The apparatus of claim 1 or 2, further comprising control means (16) configured to control the drive means (14) to rotate the body with a time-varying rotational vector including multiple acceleration and multiple deceleration has the same or different directions of rotation.

Apparatus according to any of claims 1 to 3, wherein the sensing structure (60; 94) comprises a microarray of

Carrier structures for trapping molecules from the sample.

5. Device according to one of claims 1 to 3, wherein the detection structure comprises a sensor structure.

6. Apparatus according to claim 5, wherein the sensor structure is a biosensor, an oxygen sensor or a

having.

The apparatus of any of claims 1 to 6, wherein the body further includes an inlet channel (36; 44; 84) for

Introducing the sample into the chamber (30; 46; 80; 92, 100) by centrifugal force.

The apparatus of claim 7, wherein the body has a separate inlet channel (38; 48) for introducing reagents into the chamber (30; 46; 80; 92; 100).

The apparatus of any one of claims 1 to 8, wherein the body further comprises a waste reservoir (34; 56; 76) into which the outlet channel (32; 50; 70) opens.

The apparatus of any one of claims 1 to 9, wherein the body comprises a substrate (24; 90; 96) and a lid (26; 98; 102), the chamber, the outlet channel and, if present, the inlet channels and the waste reservoir is structured in the lid, the substrate, or both.

The device of claim 10, wherein the sensing structure (60; 94) is immobilized on the substrate.

12. A method for handling a liquid sample, comprising the following steps:

Introducing the liquid sample into a chamber (30; 46;

80; 92; 100), in which a detection structure (60; 94) is arranged, which allows detection of a property of the sample, wherein an outlet channel (32; 50; 70) having a siphon structure (52; 72) with the chamber is fluidly connected; and Rotating the chamber and the outlet channel to produce a centrifugal force field that the filling height _in. of the chamber according to the j3iphpn principle, so that the detection structure is covered by the sample.

13. The method according to claim 12, wherein the chamber and the outlet channel are rotated with a time-varying rotary vector having a multiple acceleration and a multiple deceleration in the same or different directions of rotation.

14. The method of claim 12 or 13, wherein the sensing structure (60; 94) comprises a microarray of capture structures for trapping molecules from the sample.

15. The method of claim 12 or 13, wherein the detection structure comprises a sensor structure.

16. The method of claim 15, wherein the sensor structure comprises a biosensor, an oxygen sensor or a CO ₂ sensor.

The method of any one of claims 12 to 16, wherein the step of introducing the sample comprises introducing the sample through an inlet channel (36; 44; 84) by centrifugal force.

A method according to any one of claims 12 to 17, comprising the steps of sequentially introducing different liquids into the chamber (30; 46; 80; 92; 100), the siphon structure causing the sensing structure to not act during said steps dry out.

19. A method according to any one of claims 12 to 18, further comprising a step of introducing a further liquid into the chamber (30; 46; 80; 92; 100). has thereby the sample through the outlet channel (32; 70) to be emptied, while the chamber and the outlet channel are rotated wp_bei _.. JPress siphon "structure

(72) causes the sensing structure (60; 94) to be covered by the sample or other liquid during that time.

The method of any one of claims 12 to 18, further comprising a step of introducing another liquid into the chamber (30; 46; 80; 92; 100) to thereby empty the sample through the outlet channel (32; 50) while the chamber and the outlet channel are rotated, the siphon structure (52) causing the introduction of the further liquid to empty the sample and subsequently the further liquid from the chamber.

21. The method of claim 19 or 20, wherein the further liquid is a washing liquid.

22. The method of claim 21, further comprising a step of introducing reagents into the chamber (30; 46; 80; 92; 100) after the step of introducing the wash liquid, whereby the wash liquid is passed through the outlet channel (32; ) while the chamber and the outlet channel are rotating, the siphon structure (72) causing the sensing structure to be covered by the wash liquid or reagents during that time.