EP2388067A1 - Method and device for mixing a liquid by a microfluidic test element, and test element - Google Patents

Abstract

The method involves rotating a test element (2) with the fluid according to a rotational profile. The rotational profile has two cycles. The angular velocity changes within one of the cycles by the rotational profile. The rotation of the test element is accelerated with an acceleration until reaching the angular velocity. Independent claims are also included for the following: (1) a device for mixing a fluid by a microfluidic test element with a substrate and a microfluidic channel structure for receiving the fluid; and (2) a test element with an opening.

Description

The present invention relates to a method for mixing a liquid by means of a microfluidic test element, which has a substrate and a channel structure and rotates at an angular velocity about an axis of rotation. The invention also relates to a device for mixing a liquid and the test element itself.

Microfluidic test elements are used, for example, for analyzing liquid samples and for mixing a liquid, primarily in diagnostic tests (in-vitro diagnostics). In such tests, for example, body fluid samples are examined for an analyte for medical purposes contained therein.

One field of application of microfluidic test elements are so-called microarrays or solid-phase tests based on solid-phase binding reactions. One group of such assays are sandwich assays in which a solid phase-bound first binding partner undergoes a specific binding reaction enters with an analyte in the liquid. The analyte, in turn, can be visualized by "docking" a label molecule. The visualization may be by, for example, luminescence or fluorescence or other forms of labeling, such as by enzymes in enzyme immunoassays.

Since a small concentration of the analyte is often present when small sample volumes are used, so-called mock tests are carried out in which the solid-phase-bound reactant does not cover the entire bottom surface of an incubation chamber but only individual regions or points thereof. The use of spots thus results in a concentration of the analyte and the label at the individual spots in the chamber. In particular, at low concentration, the density of the analyte and the label in the area of the spots is considerably higher than with full-surface application of the solid phase-bound reactant. For many tests, an arrangement of several (three to ten) spots for the same parameter (analyte) has proved suitable. The spots are evaluated individually. The results are averaged. In some tests, another, preferably higher number of spots per parameter is advantageous. For a meaningful mean value evaluation, the analytes and labels must be distributed as evenly as possible over all spots.

As with the microarrays, even in biochemical reaction chambers with surface sensors and a corresponding measuring technique uniform binding of the analytes is necessary, especially if optical evaluation takes place, for example by image recognition. The same applies to fully coated (active) surfaces. These may be, for example, a gel (solid phase reaction partner hydrogel) at the bottom of the chamber or a large pore 3D matrix as an alternative to a planar surface.

In addition, microfluidic test elements are also used in tests in which a reagent in the reagent chamber in liquid form or as a solid is present, which is dried on the test element. Such dry reagents must be dissolved and homogenized prior to analysis. It is necessary in each case a uniform mixing or solving. The term "mixing" in addition to the dissolution of a solid in a liquid (homogenizing with a liquid solvent) also includes the possibility that two liquids are mixed together when the reagent is present for example in liquid form.

An important part of the above-mentioned tests and the analysis of a sample liquid are test carriers on which microfluidic test elements with channel structures for receiving a liquid sample are arranged or integrated. To facilitate the implementation of elaborate, multi-level test routines ("test protocols"), the channel structures often include a plurality of channel sections, chambers, and fluidic valves for sequencing.

Test carriers and microfluidic test elements consist of a carrier material, often a substrate made of plastic material. Suitable materials include COC (cyclo-olefin copolymer) or plastics such as PMMA (polymethyl methacrylate), polycarbonate or polystyrene. The channel structure of the test carrier is enclosed by the substrate and a cover or a cover layer. Such a channel structure is manufactured by a three-dimensional structuring of the plastic parts, for example by injection molding techniques or other methods. It is also possible to introduce the structure by material-removing methods, such as milling or laser ablation.

The control of the liquid transport within the channel structures and the control of the process flow can be done with internal (within the fluidic test element) or with external (outside the fluidic test element) measures. The control can be caused by applying pressure differences or by changing forces, for example by changing the effective direction of gravity.

A targeted control of the liquid flow can be achieved within the channel structure by the rotation of a test element. The generated forces may be made by controlling the change in rotational speed or direction of rotation, or by the distance from the axis of rotation, or by utilizing density differences in the liquid (eg, dissolving a solid). For example, the microfluidic test elements can be arranged in a rotating disk in the form of a compact disc (CD). A comparison of different methods is for example Marc Madou, et al .; Lab on CD; Annual Review of Biomedical Engineering, 2006.8, Page 601 to 628 (online @ http://bioenc.annualreviews.org ) known.

1. 1. Peytavi, et al.; Microfluidic Device for Rapid (< 15 Min.) Automated Microarray Hybridization, 2005, Clinical Chemistry; page 1.138 to 1.844 ; (online published at DOI: 10.1373/clin_chem.2005.052845)
2. 2. Guangyao, Jia, et al.; Dynamic Automated DNA Hybridization on a CD (Compact Disc) Fluid Platform; Sensors and Actors B 114 (2006); page 173 to 181; (online at www.sciencedirect.com )
3. 3. Grumann; Readout of Diagnostic Assays on a Centrifugal Microfluidic Platform; Oktober 2005; Dissertation an der Universität Freiburg
4. 4. S. Lutz, et al.; Unidirectional Shake-Mode for Mixing Highly Wetting Fluids on Centrifugal Platforms; 12th International Conference on Miniaturized Systems for Chemistry and Live Science; October 12 to 16, 2008; San Diego, California, USA

Analysis systems with rotating biosensors are also known, for example, from the following publications:

1. 1. Peytavi, et al .; Microfluidic Device for Rapid (<15 min) Automated Microarray Hybridization, 2005, Clinical Chemistry; page 1.138 to 1.844 ; (published online at DOI: 10.1373 / clin_chem.2005.052845)
2. Second Guangyao, Jia, et al .; Dynamic Automated DNA Hybridization on CD (Compact Disc) Fluid Platform; Sensors and Actors B 114 (2006); page 173 to 181; (online at www.sciencedirect.com )
3. Third Grumann; Readout of Diagnostic Assays on a Centrifugal Microfluidic Platform; October 2005; Dissertation at the University of Freiburg
4. 4th S. Lutz, et al .; Unidirectional Shake Mode for Mixing Highly Wetting Fluids on Centrifugal Platforms; 12th International Conference on Miniaturized Systems for Chemistry and Live Science; October 12 to 16, 2008; San Diego, California, United States

In order to improve the mixing of liquids in rotating disks, Grumann (3) proposes two possibilities in his dissertation: In the first concept, paramagnetic polymer beads are introduced into the mixing chamber and periodically deflected by fixed permanent magnets as the disc rotates. The relative movement of the beads with respect to the liquid accelerates the mixing. In the second concept, the disc is not rotated at a constant frequency, but is subjected to a so-called shake mode under periodically changing rotation, so that the mixing is improved and accelerated due to inertial effects of the liquids.

In "shake mode", the rotational frequency is constantly accelerated up to an end angular velocity of the rotating disk and then decelerated to a standstill with a second acceleration (deceleration), which is opposite to the first. Subsequently, the disc is accelerated in the opposite direction until a second end angular velocity is reached. The disc is then braked again and accelerated again in the opposite direction after standstill. The first and the second acceleration and the first and second end angular velocity are equal in magnitude, however, the direction of rotation is opposite. This "shake-mode" is also called "Euler-mixing" in professional circles, because in addition to the centrifugal force and Coriolis force occurring with constantly rotating disks, the force of acceleration also adds an additional force component, the Euler force. The Euler force is proportional to the time change of the angular velocity, while the centrifugal force is proportional to the square of the angular velocity and the Coriolis force is proportional to the angular velocity.

Lutz et al. (4) have found in studies that the Euler mixing can be improved if the acceleration (change of the rotational angular velocity) does not take place around the zero point (frequency = zero or angular velocity = zero) but with an offset. According to Lutz, the rotating disc is also constantly accelerated until a first end angular velocity is reached. The disc is then decelerated until a second final angular velocity is reached, followed by acceleration. The first and second end angular speeds differ in terms of amount. The direction of rotation is not changed. This mixing process, referred to as "unidirectional shake mode," has proven itself at least in systems in which the reaction chamber or mixing chamber is followed by a channel structure having a combination of siphon and liquid valve. The unidirectional shake mode is intended to prevent liquid from being able to pass through the siphon in capillary mode at low rotational speeds or at standstill.

Despite intensive research in various directions and despite the advances made, there continues to be a great need in the art to improve the mixing of liquids and a (homogeneous) distribution of components in a liquid within process chambers of a rotating test element. In this case, the homogeneity within the liquid space should be improved and, if possible, the process duration should be reduced at the same time.

The object of the present invention is thus to propose an improved method and an improved apparatus for mixing liquids.

The present object is achieved by a method having the features of claim 1 and by an apparatus having the features of claim 12 and by a test element having the features of claim 13.

The method according to the invention for mixing a liquid by means of a microfluidic test element comprising a substrate and a microfluidic channel structure with a mixing chamber requires that the microfluidic test element rotate at an angular velocity ω about an axis of rotation which can preferably extend through the test element. For example, the axis of rotation can be a central axis of rotation the center of the test element or by the center of gravity. Preferably, the test element is designed as a test carrier or integrated into a test carrier. The test carrier rotates about an axis of rotation, which preferably extends through the test carrier and is preferably arranged centrally.

The microfluidic test element rotates according to a rotation profile that comprises at least two cycles and in which the angular velocity changes within one cycle. The rotation profile is to be understood as a time sequence of several Winkelendgeschwindigkeiten, which are divided into cycles. One cycle comprises accelerating the rotation of the test element with at least a first acceleration a1 until reaching a first end angular velocity ω1 and, after reaching the first end angular velocity ω1, accelerating the rotation of the test element with at least one second acceleration a2 until reaching a second one End angular velocity ω2. The two accelerations a1 and a2 are opposite. One cycle is defined to define the timing of angular velocity, for example, where the first end angular velocity is contained twice in a cycle. If the change in the angular velocity takes place around the zero point (standstill; f = 0), then a cycle can be defined by the zero crossings. One cycle then connects three zero crossings, with two consecutive cycles Z ₁ , Z ₂ having a common zero crossing. The cycle thus corresponds to a period, ie the smallest temporal interval, after a process repeats itself. The end angular velocities and / or the accelerations need not be the same or constant.

The rotation profile has a plurality of cycles, wherein the rotation profile can also be repeated periodically, so that the sequence of the cycles is repeated after a predetermined number of cycles (greater than 2). However, this is not mandatory.

According to the invention, at least one of the accelerations a1, a2 and / or at least one of the end angular velocities ω1, ω2 are changed from one cycle to the next in order to produce a (preferably homogeneously) mixed liquid. It was recognized that the homogeneity of the mixed liquid can be significantly improved by the juxtaposition of different cycles. Also, the use of different cycles has positive effects on the mixing time, which is reduced.

In addition to the objects set out above, the method according to the invention also solves the problem that the analyte becomes "depleted" in the analyte if the analyte in the liquid sets, for example, on the solid phase of the microarray and therefore the portion of the liquid near to the solid phase has few or fewer analyte molecules , By means of the method according to the invention, it is ensured that (continuously) analyte is replenished from the liquid phase regions of the chamber which are remote from the phase to the depleted liquid regions. Thus, therefore, the mass transport within the liquid is optimized by the method as well as the mixing.

It has been found that in the case of rotation profiles, which have the same accelerations and the same end angular velocities in all cycles, repetitive patterns form in the channel structure, in particular in a liquid chamber or mixing chamber. These recurrent flow patterns are repeated with each cycle (oscillation) and are characteristic of the selected acceleration and end frequency of the shake mode and the end angular velocity, respectively. Particularly in a periodic shake mode with constant acceleration and constant end frequency with change of direction these patterns are very pronounced.

In biochemical assays with a plurality of detection spots (array spots), the constant shaking causes the flow pattern on the array surface to "burn in". In this case, the array spots (Detection locations) are not introduced at any location in the chamber, but would have to be selected depending on the flow pattern and thus depending on the rotation profile. The coupling of an equal amount of analyte in the liquid at different locations on the chamber surface is thus different efficient. To eliminate systemic variations, multiple array spots are evaluated by averaging the actual actual spot values to increase biosensor precision in array format. However, the formation of flow patterns distorts the measurement result, so that this method has significant disadvantages here.

Based on the realization that in microfluidic conditions, in contrast to macrofluidics, there are always laminar flow conditions, it was recognized that partial filling of the chamber has a negative influence on the flow conditions of a (round) chamber and thus also has a negative influence on the mixing ratios. With the small dimensions of the mixing chambers and channel structures of the microfluidic test element and the typical achievable flow rates of several mm / sec can be assumed that laminar conditions. Thus, it has been recognized within the scope of the invention that the best mixing effects are achieved with a round chamber shape in which the walls of the chamber are also substantially smooth. Stirring elements or similar elements projecting into the room do not have a measurable or negative effect. As part of the experiments, it was further recognized that the round chamber is preferably circular and not elliptical. Since the shape of a flat disk is selected for the rotating test elements and an optical evaluation is carried out over the surface of the disk, it was recognized that a chamber in the form of a cylindrical disk is optimal. Particularly preferably, this has a circular floor plan. A ratio of chamber diameter to chamber height of 1 to 1 has been found to be ideal. This "aspect ratio" should therefore preferably be close to 1 and, taking into account the typical systemic boundary conditions, be at most 4. The quotient from surface A to volume V should be based on the investigations at constant volume have a value between 1 and 3.5. In this case, the liquid chamber is to be arranged such that the rotation axis (preferably extending through the test element) around which the test element rotates does not extend through the liquid chamber. Rather, the liquid chamber is preferably spaced from the axis of rotation.

In a preferred embodiment of the method according to the invention, the amounts of the accelerations a1 and a2 are different in one cycle. The formation of constant flow patterns in the liquid chamber is prevented within a cycle.

Preferably, at least one of the accelerations a1, a2 is variable during a cycle. The one or both accelerations a1, a2 thus change within the cycle. In the context of the invention it has been recognized that alternatively at least one of the accelerations a1, a2 can be constant during one cycle, preferably both accelerations. The change in the angular velocity within the cycle thus changes continuously (constant). Since a cycle is relatively short in relation to the rotation profile and thus to the entire mixing time, no stationary, constant flow patterns are formed within the period (cycle duration). However, it has been found that with a constant acceleration within one cycle, the mixing results are of (nearly) the same quality as with changing accelerations during the cycle. However, the control of the rotation with a constant acceleration is much easier to realize.

It has also been shown that the mixing efficiency with higher amplitudes (larger end angular velocities ω1, ω2) is more effective than the mixing efficiency with low amplitudes (ω1, ω2). Consequently, with the same acceleration, the mixing efficiency with a longer period duration and fewer cycles (assay interval) is more effective than with a consequently short period duration and many cycles per examination, ie with the same total mixing time. According to the method according to the invention, when the first end angular velocity ω1 is reached, the direction of rotation of the test element is equal to or opposite to the direction of rotation of the test element when the second end angular velocity ω2 is reached.

In a preferred embodiment, the direction of rotation is opposite. Consequently, a reversal of the direction of rotation takes place while an acceleration is exerted on the rotating test element between the first and second end angular speeds. The "vibration" thus takes place around the zero point of the frequency. After reaching the first end angular velocity ω1, the rotating test element is consequently decelerated by reaching a second acceleration a2 until it comes to a standstill and then accelerated further with the same second acceleration a2 until the second final angular velocity ω2 is reached. In the context of the invention, it has been recognized that a better mixing occurs when the direction of rotation of the test element changes. At the time of zero crossing (frequency f = 0, rotational angular velocity ω = 0) no centrifugal force and no Coriolis force act on the liquid in the test element. The capillary force as a predominant component, which is given by the arrangement and geometry of the capillary structures, is added to the continuing euler force. Due to the short-term (dominant) action of the capillary force, the mixing result is improved overall. It is important, however, that the geometry, in particular a siphon adjoining the mixing chamber, is designed accordingly, so that a break-through of the siphon is prevented.

In the context of the invention, the term "mixing" means not only the dissolution of a solid in a liquid and the mixing of several liquids, but also the uniform transport of dissolved in the liquid components, for example, a depletion of the solution with reagent or analyte to avoid a binding phase (solid phase). After the reaction of individual analyte molecules with capture molecules of the binding phase, the analyte in the liquid is depleted in the Area of the binding phase. By mixing, the transport of analyte molecules within the liquid is ensured such that a continuous subsequent delivery of the analyte molecules from remote areas to the liquid zones near the binding phase takes place in order as possible all possible in the context of the relevant equilibrium reaction analyte molecules to the binding phase (or their catcher molecules), which increases the sensitivity of the detection method. The goal thus achieved is enrichment of all possible analyte molecules from the liquid phase at the binding phase. The term "mixing" thus also includes this balancing of the analyte molecules within the liquid.

Fig. 1

eine Vorrichtung zum homogenen Durchmischen einer Flüssigkeit;

Fig. 2

einen Testträger mit einem erfindungsgemäßen Testelement;

Fig. 3

ein Diagramm mit der zeitlichen Abfolge der Winkelgeschwindigkeit im "Standard-Shake-Mode";

Fig. 4

ein Diagramm mit der zeitlichen Abfolge der Winkelgeschwindigkeit bei geänderten End-Winkelgeschwindigkeiten;

Fig. 5a-c

je ein Diagramm mit der zeitlichen Abfolge der Winkelgeschwindigkeit bei unterschiedlichen Beschleunigungen;

Fig. 6

ein weiteres Diagramm der zeitlichen Abfolge der Winkelgeschwindigkeit im "Zufalls-Shake-Mode";

Fig. 7

eine Tabelle zur Gegenüberstellung verschiedener Shake-Modi;

Fig. 8

ein Diagramm zum Vergleich zweier Shake-Modi;

Fig. 9

ein Diagramm mit dem zeitlichen Verlauf zweier "Zufalls-Shake-Modes".

The invention will be explained in more detail with reference to specific embodiments shown in the figures. The peculiarities illustrated therein may be used alone or in combination to provide preferred embodiments of the invention. The described embodiments do not limit the invention defined by the claims in their generality. It shows:

Fig. 1

a device for homogeneously mixing a liquid;

Fig. 2

a test carrier with a test element according to the invention;

Fig. 3

a diagram with the time sequence of the angular velocity in the "standard shake mode";

Fig. 4

a diagram with the time sequence of the angular velocity with changed end angular velocities;

Fig. 5a-c

one diagram each with the temporal sequence of the angular velocity at different accelerations;

Fig. 6

another diagram of the time sequence of the angular velocity in the "random shake mode";

Fig. 7

a table to compare different shake modes;

Fig. 8

a diagram comparing two shake modes;

Fig. 9

a diagram with the time course of two "random shake modes".

FIG. 1 shows a device 1 for homogeneously mixing a liquid with a test element 2, which is held in the device 1. The device 1 comprises a holder 3 for receiving the test element 2, which is rotatable about a rotation axis 4. The two test elements 2 are integrated in a test carrier 16. The rotatable holder 3 with its shaft 3 a is moved by a drive 5 in such a way that the holder 3 together with the held test carrier 16 rotates about the rotation axis 4 at an adjustable variable angular velocity. The drive 5 is controlled by a control unit 6, wherein a movement sequence can be defined, which is preferably stored as a rotation profile in the control unit 6. For example, the rotation profile may consist of a plurality of control commands which set the rotation speed (angular velocity), the acceleration, the hold time during which the end angular velocity is kept constant, and the rotation direction. The rotation profile can either be stored in a memory of the device 1 or adjusted by manual adjustment of the above parameters on the device or generated from the parameters.

In the embodiment shown, the device 1 comprises an optical measuring and evaluation unit 7 with an optical sensor 8. A liquid recorded in the test element 2 can be analyzed and measured. In this case, the known in the prior art investigation can be applied.

The microfluidic test element 2 comprises a microfluidic channel structure 10, which has a channel section 11 which extends from an opening 12 to a microfluidic fluid chamber 13. The liquid chamber 13 is fluidically connected via a siphon channel 14 with a collection chamber 15, which is also referred to as waste chamber. The test elements 2 are embedded in the test carrier 16, which is formed as a round disk (disc) and through which the axis of rotation 4 extends. The channel structure 10 is enclosed by a substrate and a cover layer, not shown, which covers the test carrier 16 from above.

The holder 3 in the device 1 is designed as a shaft 3 a, which runs concentrically to the axis of rotation 4. Of course, it is also possible to provide other holders 3, for example, a retaining disk, a rotor or a clamping device with outer brackets, in which the test element is clamped. In addition to the central shaft 3a, it is possible to rotate the test carrier with one or more test elements about an eccentric (eccentric) axis of rotation. The axis of rotation may extend, for example, through the center of gravity of the test carrier 16, in order to take into account spatial structures in the test carrier 16 or the test elements 2 and to avoid an imbalance during rotation. The rotation axis does not necessarily have to be aligned vertically. It can also run obliquely in space at a solid angle θ ≠ 0 with respect to the vertical.

The test carrier 16 in FIG. 2 for homogeneous mixing of a liquid comprises a test element 2 with a channel structure 10. The channel structure 10 has two openings 12a, 12b, to which two adjacent channel sections 11a, 11b extend up to two intermediate chambers 17a, 17b. For example, two liquids can be supplied simultaneously. The intermediate chambers 17a, 17b are in fluid communication via a further channel 18a or 18b, each with an opening 19a, 19b. Through the openings 19a, b, the liquid receiving channel section can be reliably vented in multiple use. An adjoining the intermediate chambers 17a, 17b Channel section 20a, 20b leads to a fluidic valve 21, through which liquids from the intermediate chambers 17a, 17b can be directed into the liquid chamber 13. The fluidic valve 21 has an air outlet 22 for venting, which provides an air channel 23 for the venting of the fluidic valve 21 and connected thereto fluidic regions (eg, chamber 13).

As soon as liquid has passed from the intermediate chambers 17a, 17b into the liquid chamber 13, thorough mixing can take place. The liquid chamber 13 is adjoined by a siphon channel 14, which connects the liquid chamber 13 to a collecting chamber 15.

If two different liquids stored separately in the intermediate chambers 17a, 17b and placed in the liquid chamber 13, there is a mixing of the two liquids due to diffusion instead. However, this process takes a long time, often several hours. To speed this up, a standard shake mode (standard Euler mixing) is used in the prior art in which the rotational speed of the test carrier 16 or the test element 2 is changed, FIG. 3 , The change in frequency describes a "zero-crossing" oscillation (f = 0) between a first end frequency of f1 = +40 Hz and a second end frequency f2 = -40 Hz. The applied accelerations a1, a2 are equal in magnitude. The period is thus constant. The cycle duration (period duration) is understood to mean the time between the first reaching of the first end frequency f1 and the next reaching of the first end frequency f1.

In the context of the invention, it was recognized with this "standard Euler mixing" that the shaping of the liquid chamber 13 has an influence on the mixing speed. Thus, using the example of a liquid chamber 13 with a constant volume of 10 microliters (10 μl), which is arranged in the disk-shaped test carrier 16 with a height of 2.7 to 3 mm, it was recognized that a round liquid chamber 13 is advantageous. Preferably, the liquid chamber 13 is smaller than a round cylindrical disk with a height the height of the test carrier 16th Particularly preferred is a circular floor plan of the cylinder disc. The quotient of the diameter of the circular cylindrical disk and the height of the cylinder should be as close as possible to one. At a quotient of 1.25, a chamber diameter r1 of 2.5 mm and a height h1 of 2 mm, homogeneous mixing of two liquids containing plasma is achieved after just 5 seconds, while with a diameter r2 of 4 mm and a height h2 of 0.8 mm a homogeneous mixing takes place only after 10 seconds. It was recognized that the ratio of surface A to volume V is crucial. Preferably, the quotient is close to 1. The first chamber has a quotient Q ₁ = A ₁ / V ₁ = 2.6 while the second chamber has a quotient Q ₂ = A ₂ / V ₂ = 3.5. The chamber with the smaller quotient achieves homogenous mixing faster.

By using a rotation profile according to the invention with different cycles, the mixing efficiency can be further increased. It has thus been recognized that, according to the invention, at least one of the accelerations a1, a2 and / or at least one of the end angular velocities ω1, ω2 has to be changed from one cycle Z ₁ to the next cycle Z ₂ . The incubation time with mixing over the solid phase of the microarray can thus be reduced to a few minutes, while the quality of the subsequent delivery of analytes to the detection surface and the homogeneity of the setting on the solid phase are markedly improved.

FIG. 4 shows a rotation profile in which only the end angular velocities ω1, ω2 have been changed from cycle to cycle. The accelerations a1, a2 are the same in all cycles. In this preferred embodiment, the first and second accelerations a1, a2 are equal in magnitude.

The FIGS. 5a to c each show a section of a rotation profile for the angular velocity. For all profiles, both the accelerations a1, a2 and the final angular velocities ω1, ω2 change in the respective cycles. In each case, two cycles Z ₁ , Z _{2 are shown} .

In FIG. 5a the first acceleration a11 in the first cycle Z _{1 is} different from the first acceleration a12 in the second cycle Z ₂ . At the same time, the first end angular velocity ω11 of the first cycle is different from the first end angular velocity ω12 of the second cycle. The same applies to the second acceleration a21, a22 and the second end angular velocity ω21 and ω22. Within one cycle, the respective acceleration a1, a2 is constant and does not change.

In a preferred embodiment, at least one of the accelerations a1, a2 is variable during a cycle Z. For example, the respective acceleration a1, a2 consist of two partial accelerations a1 _a , a1 _b and a2 _a , a2 _b . For example, the respective accelerations a _a , a _{b may} each be constant. Preferably, they are different from each other. However, the accelerations a _a , a _b can also be variable, so that in the diagram no straight line, but a curve would be shown.

Preferably, a cycle consists of a first partial cycle Z _T1 until reaching the first end angular velocity ω1 and from an adjoining second partial cycle Z _T2 until reaching the second final angular velocity ω2. The rotation of the test element takes place in at least one of the two partial cycles Z _T1 , Z _T2 with at least two accelerations a _a , a _b .

FIG. 5b It can be seen that the first cycle Z ₁ consists of the two partial cycles Z _T11 and Z _T21 . In the first sub-cycle Z _T11 , the rotation of the test element takes place first with the acceleration a11 _a and then with a second acceleration a11 _b , which in this embodiment is different from the first partial acceleration a11 _a . The second subcycle Z _{T21 of} the first cycle also has two accelerations a21 _a and a21 _b .

In a preferred embodiment, at least one of the two partial accelerations a _a , a _b in at least one of the partial cycles Z _T1 , Z _{T2 is} equal to zero. For the given period T _P (plateau time, hold time) during which the Acceleration a _a , a _{b is} equal to zero, therefore, no change in the angular velocity takes place, so that for the time period T _P, the test element rotates at a constant speed. However, at least one of the partial accelerations a _a , a _{b is} not equal to zero. Such a rotation profile has proven to be advantageous, in particular in biochemical tests and tests in immunology, when setting takes place during the phases with a constant rotation (acceleration equal to zero).

Of course, the acceleration in a subcycle a1, a2 also consist of more than two partial accelerations a _a , a _b , a _c ... exist. It is conceivable that even before reaching the final angular velocity, the rotation of the test element with a constant angular velocity (not equal to the final angular velocity), ie with an acceleration equal to zero, and then again the rotation is accelerated until the final angular velocity is reached ,

Preferably, at least one of the partial accelerations when reaching one of the end angular velocities is equal to zero. The test element is then constantly rotated at the final angular velocity of the cycle for a predetermined period of time T _P , ie not accelerated. FIG. 5c shows such a rotation profile in which the accelerations a11 _b and a21 _b in the first cycle are equal to zero. The rotation of the test element at a constant speed takes place here when the final angular velocities ω11, ω21 are reached in the first cycle Z ₁ . In the context of the invention, it has been recognized that the rotation for a given time interval T _P at the final angular velocity has positive effects on the binding of the molecules on the active surface. The durations T _P1 and T _P2 within one cycle may be the same or different from each other. They can vary from cycle to cycle or repeat themselves.

In a preferred embodiment of the method according to the invention, the accelerations of the rotation profile are coded with a random number; they are formed from the random number. In addition or optionally, the end angular velocities ω1, ω2 are preferably formed from a random number. For example, for each applicable acceleration and end angular velocity a random number is used which is within the systemic limits of the device. In the examples shown, the amount of the final angular velocity is limited to 100 Hz due to the system, so that the final angular velocities determined by a random number can likewise not be greater than 100 Hz in terms of magnitude. At the same time, in the test element 2 used here, the magnitude of the final angular velocity must be greater than or equal to 20 Hz as a result of the system, since otherwise the siphon channel 14 adjoining the liquid chamber 13 breaks through and liquid escapes.

The random number is preferably a "true random number". For example, the circle number π or the Euler number e = 2.718281828459 ..... can be used. These numbers, known as true random numbers, are particularly suitable for a "chaotic eulogy". The random number is used as a matrix for choosing the "random" process parameters. For example, values of the accelerations and / or the end angular velocities may be formed from two consecutive digits of the random number. Alternatively, any number may be used that is constant with a factor, e.g. B. 10, is multiplied.

Using the example of the number π = 3.141592653589793 ... it follows that the test element 2 in the first cycle Z _{1 is} accelerated by a first end angular velocity ω1 = 31 Hz (initial velocity) with a second acceleration a2 = -41 Hz / sec until reaching a second end angular velocity ω2 = -59 Hz. The test element 2 is thus first braked and then accelerated with a changed direction of rotation. Then it is accelerated with the first acceleration a1 = 26 Hz / sec. This process also includes braking, a (short) standstill with reversal of the direction of rotation and an acceleration. The first acceleration a1 is opposite to the second acceleration a2 and consequently has a positive sign. The test element 2 is accelerated until reaching the first end angular velocity ω1 = 53 Hz with the first acceleration a1. This first end angular velocity ω1 already belongs to the second cycle Z _{2 of} the rotation profile.

For the second cycle Z _{2 of} the rotation profile, the following digits of the number π are selected so that the second acceleration of the second cycle is a2 = -58 Hz / sec. All subsequent accelerations and final angular velocities of the individual cycles are selected accordingly.

This rotational profile of a "π-shake mode" (π-Euler mixing) is in FIG. 6 shown. In the rotation profile, both the end angular velocities ω1, ω2 and the accelerations a1, a2 will be changed from cycle to cycle. The amount of the maximum occurring end angular velocity ω1, ω2 in this application example is 100 Hz due to the system. It can be clearly seen that the cycles have different cycle times, which follows from the different accelerations or end angular velocities.

Evidence of improved mixing of an analyte in a liquid by using a chaotic Euler rotation profile in which both the accelerations and the final angular velocities are varied from one cycle to the next is shown in the table in FIG Fig. 7 shown. For this purpose, three spots of a solid phase-bound reaction partner were placed in a liquid chamber 13. An analyte (in this case, a protein) contained in a liquid was detected by an optical evaluation with an exposure time of 10 seconds. Shown is the mean value MW of the measured signals via different test elements. The signal is the mean value from the spot integrals (= spot signals). The higher the mean and the smaller the coefficient of variation, the more advantageous the type of mixing for the microarrays.

In FIG. 7 the results of two measurements are shown. In both the first (1st) and second (2nd) measurements, a standard shake (standard Euler mix) with constant accelerations and constant end angular velocities over all cycles is compared to one "Chaotic Eulemic" in which the accelerations a1, a2 are constant and the final angular velocities ω1, ω2 vary within the incubation period and time intervals of the constant rotation alternate with active mixed phases during the incubation. In the first measurement, about 5% more signals (counts) are detected in chaotic Eulemian, while the coefficient of variation VK has fallen from 27% to 21%. Also in the second measurement with a changed protein density in the liquid results in an increase of the mean value of the detected signals (counts). In this case, the increase is 11%. At the same time, the coefficient of variation VK improves from 26% to 17%. From both values it can be seen that a significantly improved homogeneity was achieved.

In FIG. 8 the standard Euler mixing (curve A) is compared with the chaotic Euler mixing (curve B) on the basis of the number π (π-Euler mix). To demonstrate the homogeneity, a BI-DIG model system was used in which the bottom of the liquid chamber 13 is coated with a TRSA-BI streptavidin. On the floor, individual spots are placed with a BI-RPLA-DIG (BI-bovine plasma albumin DIG) with the bottom surface blocked (coated) around the spots with biotin. An anti-DIG latex is detected as a model analyte in a sample fluid. In FIG. 8 in each case the median of the measured, signaling fluorescence signals (FS) (counts) over the individual (numbered) spots (S) of the chamber 13 is shown. The comparison of the two curves shows that in the chaotic Euler mix significantly lower outliers of the individual spots can be detected than with standard mixing. Also in this case, the variation of the individual spots is smaller. Thus, there is a better distribution of the analyte contained in the liquid, which is an indicator of a better mixing of the liquid.

FIG. 9 shows the comparison of two measurements in a "chaotic Euler-mixing". Here, too, the median of the measured, signaling fluorescence signals (FS) (counts) are shown above the individual (numbered) spots (S) of a chamber. Except for one outlier each to one Spot shows the measurement result a low variance across all spots. There is thus a very good uniform distribution, since the coefficient of variation is low. The two outliers were expected in this attempt because they are systemic. The fact that both outliers were detected at different spots is due to the fact that no ideal matrices (coated liquid chambers) were available. However, there is a clear trend that by changing the accelerations and the end-to-cycle angular velocities within a rotation profile, the mixing of a liquid can be significantly improved.

Claims (15)

Method for mixing a liquid by means of a microfluidic test element (2) with a substrate and a microfluidic channel structure (10) for receiving the liquid, wherein the test element (2) rotates about an axis of rotation (4) at an angular velocity ω,
comprising the following steps: Rotating the test element (2) with the liquid according to a rotation profile comprising at least two cycles and in which the angular velocity changes within one cycle, whereby the following steps are carried out in one cycle: Accelerating the rotation of the test element (2) with at least one acceleration a1 until reaching a first end angular velocity ω1, after reaching the first end angular velocity ω1, accelerating the rotation of the test element (2) with at least one acceleration a2 until reaching a second final angular velocity ω2, wherein the acceleration a1 and the acceleration a2 are opposite,
characterized in that
at least one of the accelerations a1, a2 and / or at least one of the final angular velocities ω1, ω2 changes from one cycle to the next cycle. A method according to claim 1, characterized in that the amounts of the accelerations a1, a2 are different in one cycle. Method according to claim 1 or 2, characterized in that at least one of the accelerations a1, a2 is constant during one cycle. Method according to claim 1 or 2, characterized in that at least one of the accelerations a1, a2 changes during one cycle. Method according to one of the preceding claims, characterized in that a cycle consists of a first subcycle until reaching the first end angular velocity ω1 and from an adjoining second subcycle until reaching the second final angular velocity ω2 and the rotation of the test element in at least one of the two partial cycles with at least two accelerations a _a , a _b takes place, wherein the accelerations a _a , a _b are different from each other. A method according to claim 5, characterized in that at least one of the accelerations a _a , a _{b is} equal to zero in at least one of the subcycles. Method according to Claim 6, characterized in that at least one of the accelerations a _a , a _b is zero when one of the final angular velocities ω 1, ω 2 is reached, so that the test element remains constant for a predetermined time period T _p at the final angular velocity ω 1, ω2 rotates. Method according to one of the preceding claims, characterized in that the direction of rotation of the test element (2) on reaching the first end angular velocity ω1 of the direction of rotation when reaching the second end angular velocity ω2 is opposite. Method according to one of the preceding claims, characterized in that the rotation profile comprises a plurality of cycles and the acceleration a1, a2 and / or end angular velocity ω1, ω2 of the rotation profile are coded with a random number such that at least one of the accelerations a1, a2 and / or end angular velocities ω1, ω2 are formed from the random number. A method according to claim 9, characterized in that the random number is a true random number and the values of the accelerations a1, a2 are formed from successive digits of the random number. A method according to claim 9 or 10, characterized in that the random number is a true random number and the final angular velocities ω1, ω2 are formed of successive digits of the random number. Device for mixing a liquid with a microfluidic test element (2), which has a microfluidic channel structure (10) with a liquid chamber (13), comprising an analyzer with a holder (3) for receiving and rotating the test element (2), - With a drive (5) for rotating the holder (3) about an axis of rotation (4) with an angular velocity ω - With a control unit (6) for controlling the drive (5) such that the test element (2) according to a multi-cycle rotation profile is rotated, wherein - the angular velocity changes within one cycle, within one cycle the rotation is accelerated with at least one first acceleration a1 up to a first final angular speed ω1 and then accelerated with at least one oppositely directed second acceleration a2 up to a second final angular speed ω2, and at least one of the accelerations a1, a2 and / or at least one of the final angular velocities ω1, ω2 within the rotation profile is changed from one cycle to the next cycle. A test element for mixing a liquid comprising an opening (12) for receiving liquid and a fluid chamber (13) in fluid communication with the opening (12), in particular suitable for carrying out a method according to one of claims 1 to 11 or especially suitable for use in a device according to claim 12,
characterized in that the liquid chamber (13) has the shape of a round cylindrical disk and the test element (2) rotates about an axis of rotation (4) which is spaced from the liquid chamber (13). Test element according to claim 13, characterized in that the liquid chamber (13) has a circular floor plan. Test element according to claim 13 or 14, characterized in that the liquid chamber (13) is formed such that the quotient of the surface of the liquid chamber (13) to the volume of the liquid chamber (13) is between 1 and 3.5, preferably close to 1 ,

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