Microfluidic element for analyzing a liquid sample

A microfluidic element for analyzing a bodily fluid sample for an analyte contained therein is provided, the element having a substrate, a channel structure that is enclosed by the substrate, and a cover layer, and is rotatable around a rotational axis. The channel structure of the microfluidic element includes a feed channel having a feed opening, a ventilation channel having a ventilation opening, and at least two reagent chambers. The reagent chambers are connected to one another via two connection channels in such a manner that a fluid exchange is possible between the reagent chambers, one of the reagent chambers having an inlet opening, which has a fluid connection to the feed channel, so that a liquid sample can flow into the rotational-axis-distal reagent chamber. At least one of the reagent chambers contains a reagent, which reacts with the liquid sample.

TECHNICAL FIELD

The present disclosure relates to diagnostic test devices and, more particularly, to a microfluidic element for analyzing a liquid sample, typically in a bodily fluid sample.

BACKGROUND

Microfluidic elements for analyzing a liquid sample and for blending a liquid with a reagent are used in diagnostic tests (in vitro diagnostics). In these tests, bodily fluid samples are determined for an analyte contained therein for medical purposes. The term blending comprises the possibility that the reagent is provided in liquid form, i.e., that two liquids are mixed with one another. In addition, the term comprises the possibility that the reagent is provided as a solid and is dissolved in a liquid and homogenized. In many applications, the solid dry reagent is introduced in liquid form into the fluidic element and dried in a further step, before the element is used for the analysis.

An important component during the analysis are test carriers, on which microfluidic elements having channel structures for accommodating a liquid sample are provided, to allow the performance of complex and multistep test protocols. A test carrier can comprise one or more fluidic elements.

Test carriers and fluidic elements consist of a carrier material, typically a substrate made of plastic material. Suitable materials are, for example, COC (cyclo-olefin copolymers) or plastics such as PMMA, polycarbonate, or polystyrene. The test carriers have a sample analysis channel, which is enclosed by the substrate and a cover or a cover layer. The sample analysis channel frequently consists of a succession of a plurality of channel sections and interposed chambers, which are expanded in comparison to the channel sections. The structures and dimensions of the sample analysis channel having its chambers and sections are defined by a structuring of plastic parts of the substrate, which is generated by injection-molding technologies or other methods for producing suitable structures, for example. It is also possible to introduce the structure by material-removing methods such as milling.

Fluidic test carriers are used, for example, in immunochemical analyses having a multistep test sequence, in which a separation of bound and free reaction components occurs. A controlled liquid transport is required for this purpose. The control of the process sequence can be performed using internal measures (inside the fluidic element) or using external measures (outside the fluidic element). The control can be based on the application of pressure differences or also the change of forces, for example, resulting from the change of the action direction of gravity. If centrifugal forces occur, which act on a rotating test carrier, a control can be performed by changing the rotational velocity or the rotational direction or through the spacing from the rotational axis.

To perform the analyses, the sample analysis channel of the microfluidic elements contains at least one reagent, which reacts with a liquid introduced into the channel. The liquid and the reagent are mixed with one another in the test carrier so that a reaction of the sample liquid with the reagent results in a change of a measuring variable which is characteristic for the analyte contained in the liquid. The measuring variable is measured on the test carrier itself. Measurement methods which can be optically evaluated and in which a color change or another optically measurable variable is detected, are typical.

For the performance of the analysis, it is decisive that the reagent provided in dried form is dissolved by the sample liquid and is blended therewith. In the prior art, some efforts have been made to improve the blending. For example, in rotating test carriers, which are rotated around a rotational axis in an analysis system, the blending is promoted by rapid changes of the rotational direction. This resulting “shake mode” is described, for example, in a particular embodiment by Markus Grumann, “Readout of Diagnostic Assays on a Centrifugal Microfluidic Platform”, (Dissertation University of Freiburg, 2005, URN (NBN): urn:nbn:de:bsz:25-opus-22723).

Further known methods for improving the blending of sample liquid and reagent comprise the introduction of magnetic particles, which are set into motion by the action of an electromagnet or permanent magnet. The outlay during the production of the test carriers rises through the integration of the particles. In addition, the analysis systems must have further components, namely the magnets, and therefore become expensive.

Other methods include, for example, elements whose capillary channels contain particular flow obstructions. The production of such obstructions, for example, ribs, must be implemented in the microstructure and are therefore expensive and make the production process of the test carrier more difficult. In addition, such structures are not suitable for all mixing processes or for all reagents and sample liquids.

In spite of the manifold efforts to improve mixing procedures in microfluidic elements, in particular the blending of dried solid reagents and sample liquids, there is still a demand for a microfluidic element or test carrier, in which the blending of small amounts of sample liquids in particular is improved. Furthermore, the fluidic element is to be capable of simultaneously dissolving different reagents which are introduced separately and are located at different spatial locations, for example, and to cause the sample liquid to react with different reagents.

SUMMARY

It is against the above background that the embodiments of the present disclosure provide certain unobvious advantages and advancements over the prior art. In particular, the inventors have recognized a need for improvements in microfluidic elements for analyzing liquid samples, typically bodily fluid samples.

Although the embodiments of the present disclosure are not limited to specific advantages or functionality, it is noted that the present disclosure provides a test carrier for analyzing a bodily fluid sample for an analyte contained therein without restriction of the generality of a microfluidic element. In addition to bodily fluids, other sample liquids can also be analyzed.

According to one embodiment, a microfluidic element for analyzing a liquid sample is provided comprising a substrate, a channel structure enclosed by the substrate, and a cover layer, wherein the microfluidic element is rotatable around a rotational axis; the channel structure includes a feed channel having a feed opening, a ventilation channel having a ventilation opening, and at least two reagent chambers; the reagent chambers are connected to one another via two connection channels in such a manner that a fluid exchange is possible between the reagent chambers, one of the reagent chambers has an inlet opening, which has a fluid connection to the feed channel, so that a liquid sample can flow into the rotational-axis-distal reagent chamber, which, of the two reagent chambers, is positioned farther away from the rotational axis, and at least one of the reagent chambers contains a reagent, which reacts with the liquid sample.

These and other features and advantages of the embodiments of the present disclosure will be more fully understood from the following detailed description taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by specific discussion of features and advantages set forth in the present description.

Skilled artisans appreciate that elements in the figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions of some of the elements in the figures may be exagerated relative to other elements to help improve understanding of the embodiment(s) of the present disclosure.

DETAILED DESCRIPTION

In the context of the present disclosure, a microfluidic element is understood as an element having a channel structure, in which the smallest dimension of the channel structure is at least 1 μm and its largest dimension (for example, length of the channel) is at most 10 cm. Because of the small dimensions and the capillary channel structures, laminar flow conditions predominantly prevail in the channels or channel sections. The poor conditions resulting therefrom for blending of liquid and solid in such capillary channels are significantly improved by the microfluidic element according to the embodiments of the instant disclosure.

The microfluidic element rotates around a rotational axis. The rotational axis typically extends through the microfluidic element. It extends through a predetermined position, e.g., typically through the center of gravity or the center point of the element. In a typical embodiment, the rotational axis extends perpendicularly to the surface of the fluidic element, which typically has a flat, disc-like form and can be a round disc, for example. For this purpose, the microfluidic element is held in a holder of an analysis device, for example, the rotational axis being formed by a rotating shaft of the device.

Through corresponding structuring of a substrate of the element, a channel structure is formed, which comprises a feed channel having a feed opening and a ventilation channel having a ventilation opening as well as at least two reagent chambers. A reagent is contained in at least one of the reagent chambers, which is typically provided in solid form as a dry reagent and which reacts with the liquid sample, which is introduced into the channel structure. Each two adjacent reagent chambers are connected to one another via at least two connection channels in such a manner that a fluid exchange is made possible between the two reagent chambers. One of the reagent chambers has an inlet opening, which has a fluid connection to the feed channel so that a liquid sample can flow from the feed channel into the reagent chambers. According to one embodiment, the liquid sample flows out of the feed channel into the reagent chamber which, of the (two) reagent chambers, is farther away from the rotational axis. The liquid thus flows into the rotational-axis-distal reagent chamber.

The expressions used in the meaning of the disclosure, “rotational-axis-distal” and “rotational axis-proximal”, do not represent absolute area specifications, of where a structure is located, but rather specify how far away a structure is from the rotational axis. The rotational axis is understood as the zero point of a distance scale, which extends radially outward from the rotational axis. A rotational-axis-distal (rotational-axis-remote) structure is farther away from the rotational axis in this meaning than a rotational-axis-proximal structure. A rotational-axis-distal reagent chamber (reagent chamber which is distal to the rotational axis) is thus the reagent chamber which is farther away from the rotational axis in relation to another reagent chamber. In the case of two reagent chambers, the rotational-axis-distal reagent chamber is the chamber which is farthest away from the rotational axis in comparison to other chambers, i.e., the most distal of the reagent chambers. The term “rotational-axis-proximal” is to be understood in a similar manner. In this meaning, a rotational-axis-proximal reagent chamber is to be understood as the reagent chamber which—in comparison to the other reagent chambers—is located closest to the rotational axis.

In the context of the present disclosure, it has been recognized that—in contrast to the microfluidic elements known in the prior art—multistep reaction protocols are possible using the reagent chambers, which are connected via at least two connection channels, and the arrangement offers manifold control capabilities. In particular, the arrangement allows different reagents which are introduced separately from one another, without mixing during drying, to be dissolved in a single processing step, the dissolving not being fluidically obstructed.

The at least two connection channels between the two reagent chambers allow an unobstructed and rapid fluid exchange. In the context of the disclosure, it has been recognized that more than two connection channels are typical. Three connection channels are particularly typically used, which may be positioned essentially parallel to one another, for example. The reagent chambers are fluidically connected one behind another by the two connection channels in such a manner that a fluid series circuit results. The reagent chambers are geometrically independent component structures and have a separate receptacle volume. However, they are fluidically jointly a single fluid chamber. The positive properties of individual reagent chambers are therefore combined with the properties of a single fluid chamber. The solid dry reagents are introduced in liquid form into the chambers and then dried. This drying is performed either by heating or freezing, which typically occurs at temperatures of less than about −60° C., particularly typically at approximately −70° C. The test carrier is typically pre-cooled, in order to improve the drying of the liquid reagent. In particular, in the case of “surfactant-containing” reagents, “cold drying” by freezing is typical.

Since the reagent chambers are geometrically separated from one another, different reagents may be introduced into each of the reagent chambers, without mixing of the reagents occurring before or during the drying. This is supported by a corresponding geometric design of the reagent chambers. For example, the chambers can be separated by sharp delimitations such as webs or edges, in order to prevent blending (“crosstalk”) by creeping effects. The sharp-edged delimitations do also form a barrier for the transport of the fluid out of a reagent chamber. However, this can be easily overcome by the occurring external forces (centrifugal force, hydrostatic force). Multiple (different) reagents can be dissolved and homogenized in only one processing step through the possibility of filling each chamber with a different reagent.

The arrangement of the reagent chambers of the channel structure is implemented in such a manner that one of the chambers is positioned farther away from the rotational axis than the other chamber, i.e., the distance of the rotational-axis-distal reagent chamber from the rotational axis is greater than the distance of the other chamber. Through the rotation of the microfluidic element, the liquid introduced into the channel structure is first conducted into the rotational-axis-distal chamber, so that this chamber is filled first and the reagent accumulated in the chamber is dissolved. The liquid reacts with the reagent. The liquid quantity and the volume of the first reagent chamber are adapted to one another.

The second (and possibly further more rotational-axis-proximal) reagent chamber is only filled when a larger liquid quantity is introduced into the channel structure or flows out of the feed channel into the reagent chambers. In this manner, reagents can also be dissolved very well using small sample quantities. The reagent chamber which is farthest away from the rotational axis is therefore filled first. The chambers positioned closer to the rotational axis (in relation to the most distal (remote) reagent chamber) are only filled in one or more further steps, the sequence of the filling being dependent on the distance to the rotational axis. The reagent chamber having the smallest distance to the rotational axis is filled last. In the context of the disclosure, it was recognized that dissolving of the reagents occurs more reliably, completely, and rapidly in completely filled reagent chambers than in only partially filled chambers. Through the arrangement of a plurality of reagent chambers having relatively small partial volumes, e.g., 2, 3, 4, 5, 6, 8, 10, 12, 15, etc. chambers, good blending can be achieved for a large number of different volumes. For example, three or five of the 12 reagent chambers, for example, can be filled with the volume to be assayed, all (e.g., three or five) chambers being completely filled. If all reagent chambers are filled with the same reagent or the same composition of reagents, very good blending with the reagents can be achieved in this manner for different volumes of sample liquid.

In a typical embodiment, the respective two (or more) connection channels between two adjacent reagent chambers are positioned parallel. The spaced-apart (separate) connection channels are typically formed by linear channel sections. The length of at least one of the connection channels is typically smaller than the smallest dimension of the reagent chambers in the test carrier plane. The test carrier plane is the plane which extends perpendicularly to the surface normals of the test carrier, for example, perpendicularly to the rotational axis.

One of the at least two connection channels is advantageously positioned centrally between adjacent reagent chambers. It is aligned with the centers of the two reagent chambers which it connects. The (other) connection channel is typically connected laterally to the reagent chambers in such a manner that it extends outside the central axis connecting the centers. It is particularly typically positioned tangentially on the reagent chambers, so that its outer side (outer wall) aligns with outer walls of the reagent chambers. The central connection channel is typically wider (it has a greater cross section at equal channel height) than the laterally positioned channel.

The connection channels between two adjacent reagent chambers are implemented so that upon filling of the reagent chamber arrangement, the liquid can flow through the connection channels from one chamber into the second. The liquid typically flows through one of the connection channels. The air contained in the not yet filled chamber can simultaneously escape through the other of the two channels, i.e., the channel which is not wetted by the liquid, typically through the central connection channel.

An embodiment having three connection channels between two adjacent reagent chambers is particularly typical. One connection channel extends along the central axis, which connects the centers of two adjacent reagent chambers. The two other connection channels are typically positioned tangentially on the reagent chambers. Upon filling of the reagent chambers, the rotational-axis-distal reagent chamber is filled first. For this purpose, liquid is conducted through the (tangential) connection channel, which is adjacent to the inlet opening, into the rotational-axis-distal chamber. Upon filling of the rotational-axis-distal reagent chamber, air escapes through the two other connection channels (the middle channel and the second tangential channel) until the reagent chamber is filled. Upon further filling, the air in the two other connection channels is displaced by liquid, so that further filling of the rotational-axis-proximal reagent chamber first occurs through the two other connection channels and finally also via the first connection channel, which is adjacent to the inlet opening.

In an arrangement of a fluidic element having three or more reagent chambers, the at least two connection channels (typically three connection channels) are each positioned between two adjacent reagent chambers. Two reagent chambers are adjacent if no further reagent chamber is positioned between them and a fluid exchange occurs between them directly via the at least two connection channels, without further fluidic structures being connected between them.

The channel structure according to an embodiment of the disclosure having at least two reagent chambers, which are directly connected to one another by at least two connection channels, offers high flexibility, a space-saving and compact arrangement, and an array of functional advantages:1. A two-step reaction protocol is possible using two reagent chambers connected to one another. In a first step, a liquid quantity which corresponds to the volume of the first reagent chamber is conducted into the first rotational-axis-distal reagent chamber. The dry reagent contained therein is dissolved, so that the first reaction can occur. In a further step, a second liquid quantity is filled into the arrangement of the reagent chambers, the second partial quantity corresponding to the volume of the second reagent chamber. This second partial quantity of the liquid can be a buffer medium, for example. The filling procedure occurs in that the additional second partial quantity is first pressed into the first chamber by the centrifugal force and mixes with the fluid present therein and only then flows into the second reagent chamber. Through a corresponding control of the rotational velocity and rotational direction, a mixing procedure begins, in which the reagent in the second reagent chamber is dissolved and a second reaction with the second reagent occurs. Since both reagent chambers are completely filled during the dissolving in each case, good homogenization and blending in the different phases is achieved in each of the two chambers.2. The reagent chamber arrangement offers the advantage that optimized dissolving of a dry reagent in the rotational-axis-distal first chamber occurs in that this chamber has the entire filling volume flow through it multiple times, two times if two reagent chambers are provided. A flow through the first reagent chamber first occurs upon the filling of the chamber. The second flow through occurs upon emptying of the structure. In this manner, particularly good dissolving of the dry reagent is achieved. This has the further advantage that the agglomerates resulting during drying of reagents, which are pressed radially outward into the first chamber by the centrifugal force, are also “flushed out” with the fluid from the radially inner chamber during the subsequent emptying. Losses on the inner surface of the first reagent chamber are prevented.3. Dilution series may be implemented in a simple manner using the arrangement according to another embodiment of the disclosure. Since the arrangement of the reagent chambers allows a very compact channel structure, a plurality of channel structures may be implemented on one test carrier. To perform a dilution series, only the respective rotational-axis-distal first reagent chamber is equipped with reagents in the channel structures positioned in parallel. To perform a dilution series, the parallel structures are filled with different volumes, so that different dilutions can be generated in only one processing step for a defined reagent quantity. The advantage of such a sequential microreactor cascade using a so-called pearl necklace structure (series circuit of multiple chambers) is that the complete reaction can be performed using variable volumes, without having to perform changes in the geometry of the channel structure. The smallest volume of the sample liquid is as large as the volume of the first reagent chamber. The volumes to be assayed are typically a multiple of the typically equal volumes of the individual reagent chambers.4. A further advantage of the reagent chamber structure is that the individual reagent chambers can be adapted to the partial volumes to be assayed. In the context of the disclosure, during experiments on the dissolving and mixing behavior, it has been recognized that the mixing procedures run optimally with completely filled chambers. For example, if only a partial quantity of the fluid is available in a first filling step, for example, a dilution buffer, which is only filled up later with a sample liquid, in a “single chamber system”, the homogenization with the first partial quantity would only run very poorly, since air inclusions would be formed. In a reagent chamber arrangement having multiple reagent chambers, the chambers are each designed for the partial volume of the liquid to be assayed and thus allow optimum dissolving and mixing, since the individual reagent chambers are completely filled by the liquid partial volumes. Foaming of the solution is also prevented.

To further improve the mixing procedures in the reagent chamber arrangement, in a typical embodiment, the channel structure comprises a mixing chamber, in which the reagent chambers and the connection channels between the reagent chambers are integrated. In this manner, the properties of the individual reagent chambers are combined still better with the properties of a fluidic individual chamber. The reagent chambers are typically positioned in the mixing chamber in the radial direction in series in such a manner that the series of the chambers encloses an angle of at most 80° to the radial direction, particularly typically at most 60°. The radial direction is to be understood as a straight line which extends outward from the rotational axis of the microfluidic element or the test carrier. Therefore, the reagent chambers do not have to be oriented directly radially outward, but rather can enclose an angle to the radial direction which is different from 90°.

In a further typical embodiment, the reagent chambers are implemented so that filling with a liquid and dissolving of a solid dry reagent contained in the reagent chamber occur without the liquid flowing into the adjacent reagent chamber. As long as the liquid quantity does not exceed the volume of the reagent chamber, the liquid remains in the reagent chamber into which it flows. During the first filling, this is always the rotational-axis-distal reagent chamber. It typically has an inlet opening, which has a fluid connection to the feed channel in such a manner that a liquid sample can flow into the rotational-axis-distal reagent chamber.

The reagent chambers typically have a round design. Their footprint is implemented as circular. The base of the individual chambers is rounded so that the base merges continuously into the chamber walls, i.e., without an edge. The reagent chambers are typically implemented in the form of a hemisphere or a hemispherical segment. A web which separates the two chambers is implemented between two adjacent chambers. An edge is provided at the upper edge of the chamber, so that a capillary stop is formed, which prevents an exit of liquid from one of the reagent chambers. This web-like barrier is designated in technical circles as a plate edge. Of course, the edge in the transition does not have to be sharp-edged. It can also have a small radius. However, the radius is to be selected as sufficiently small that the barrier function is maintained.

The reagent chambers, which are each connected to one another by at least two connection channels, are typically integrated in a mixing chamber. The mixing chamber consists of the reagent chambers, the connection channels, a feed opening, through which the liquid can enter the mixing chamber from a feed channel, and a ventilation opening, which is positioned at the end of a ventilation channel, which has an air exchange connection with the mixing chamber. In addition, the mixing chamber can also comprise a transport channel, which is led laterally along the reagent chambers.

Reagent chambers having a rounded base or a rounded depression are also suitable as a structure, independently of the use in rotating test carriers and centrifugal devices, for introducing two or more reagents individually into the structure and only mixing them jointly at a later point in time upon dissolving with a liquid. This is true in particular for reagents which react to one another, but may only be mixed with one another at an analysis point in time (for example, upon dissolving with plasma), but not beforehand. They are only to dissolve jointly in the analysis. The statements made in the description of the figures herein with respect to rotating test carriers can therefore also be transferred to nonrotating test carriers, in which the reagent chambers have a rounded base and typically have a hemispherical design.

Hemispherical reagent chambers, which are typically combined in a mixing chamber, also have a large advantage during the introduction and during the drying of reagents. The reagents are introduced in liquid form into the reagent chambers and dried therein. The surface tension acts during the drying procedure, so that the dosed liquid reagent wets the surroundings of the application point and is slowly distributed. If it hits edges or similar points which have a higher capillarity, it dries in concentrated form thereon. Such concentration is prevented by the rounded base. Since only one reagent is applied per reagent chamber, flowing together and mixing is also prevented. This is assisted by the sharp-edged upper boundaries of the chambers. The reagent chambers having rounded base also prove to be particularly advantageous during the dissolving of the reagents.

FIG. 1shows a microfluidic element1having three identically constructed channel structures2, which extend essentially radially outward. The smallest dimension of the channel structure2is typically at least 0.1 mm, particularly typically at least 0.2 mm in size. The microfluidic element1is a test carrier3, which is implemented as a rounded disc and through which a rotational axis4extends centrally, around which the disc-shaped test carrier3rotates. The channel structure2is enclosed by a substrate5and a cover layer (not shown), which covers the test carrier3on top.

The microfluidic element1is suitable for use in an analysis device or a similar device, which has a holder, in order to accommodate the microfluidic element and cause it to rotate. The device is typically implemented so that the microfluidic element is rotated around a rotating shaft of the device, the axis of the rotating shaft aligning with the rotational axis4of the microfluidic element1. The rotating shaft of the device can extend through a hole4aof the test carrier3for this purpose. The rotational axis4typically extends through the center point or the center of gravity of the element1.

The channel structure2of the microfluidic element1includes a feed channel6, which comprises a U-shaped channel section7and a linear channel section8. A feed opening9is provided at each of the ends of the two U-legs of the U-shaped channel section7, through which a liquid sample, typically a bodily fluid such as blood, for example, can be introduced into the feed channel6. For example, a sample liquid can be dosed by an operator manually (using a pipette) into a feed opening9. Alternatively, the feed channel can also be equipped with a liquid by means of a dosing station of an analysis device. During the dosing of a liquid into the feed channel6, the liquid is introduced through one of the two feed openings9, while the air contained in the channel can escape through the second feed opening.

Furthermore, the channel structure2comprises a ventilation channel10having a ventilation opening11as well as two reagent chambers13, which are connected to one another via three connection channels14so that a fluid exchange occurs between the two reagent chambers13. The channel structure2is implemented in a typical embodiment according toFIG. 1as an analysis function channel15, which comprises a measuring chamber16, a measuring channel17between the measuring chamber16and the reagent chambers13, and a waste chamber18, which is connected via a disposal channel19to the measuring chamber16. The measuring chamber16is ventilated via a separate ventilation channel. The waste chamber18, which is implemented as a collection basin20, has a ventilation channel21having an outlet valve at the end, through which air can escape from the channel structure2.

In a typical embodiment, as shown inFIG. 1, for example, the channel structure2comprises a mixing chamber22, in which the two reagent chambers13and the three connection channels14are integrated. The mixing chamber22has an inlet opening23, which has a fluid connection to the feed channel6, so that a liquid sample can flow into the rotational-axis-distal reagent chamber13a. The rotational-axis-distal reagent chamber13ahas a greater distance to the rotational axis4than the other reagent chamber13b. The rotational-axis-proximal reagent chamber13b(closer to the rotational axis4than the reagent chamber13a) is in fluid contact via an air outlet33with the ventilation channel10, so that air can escape from the reagent chamber arrangement and the mixing chamber22.

If a liquid is introduced into the U-shaped channel section7and the test carrier3is then rotated around a rotational axis4, the centrifugal force presses the liquid through the linear channel section8of the feed channel6until the liquid reaches the mixing chamber22through the inlet opening23. The liquid is then collected in the rotational-axis-distal reagent chamber13auntil it is filled. A dry reagent which is dried in the reagent chamber13ais dissolved. If further liquid is introduced into the mixing chamber22, the liquid flows through the three connection channels14into the more rotational-axis-proximal reagent chamber13b, the connection channel14located farthest radially outside first being filled with liquid. The air contained in the mixing chamber22escapes outward through an air outlet33in the ventilation channel10.

Optimum dissolving of the reagents can occur in the reagent chambers13through suitable control of the rotational velocity, the rotational direction, and the acceleration, which is supported by the rounded reagent chambers13.

FIG. 2ashows a section along line IIA fromFIG. 1through the two reagent chambers13a,13b. The reagent chambers13a,13bare typically implemented as hemispherical, the open opening surface of the hemispheres24being terminated by the cover layer. The reagent chambers13are rounded on their base so that no sharp edges occur. The rounded chamber base thus ensures uniform distribution of the reagent and also uniform dissolving and uniform flow velocity. The transitions to the connection channels are typically not rounded, but rather sharp-edged, i.e., a sharp edge25is implemented at the upper boundary of the hemispheres24, the edge25typically enclosing an angle of 90°. A type of geometrical valve results in this manner, which forms an overflow protection, since the edge represents a physical barrier for the transport of the liquid.

In order to place the reagents in the chamber, the reagents provided in liquid form are introduced into the open test carrier3without cover layer, for example, by pipetting. The sharp edges are then used as a delimitation, which prevents creeping of the liquid reagents during the drying. The structure therefore becomes independent with respect to interfering effects during the automatic processing upon the drying. An overflow protection26adjoins the reagent chambers13at the upper boundary, which prevents reagents from being able to exit from the mixing chamber22. The surface enlargement by the overflow protection26can additionally lengthen the mixing time during the mixing or dissolving of the dry reagents.

FIG. 2bshows the section through the channel structure2fromFIG. 2a, but with dry reagents35and cover layer34shown. The reagent chambers13and the mixing chamber22are implemented here so that the depth t of the overflow protection26is approximately one-third of the depth T of the mixing channel22. The depth t of the overflow protection26is approximately 400 μm. Two-thirds of the depth T of the mixing channel22is formed by the reagent chambers13. The dried reagent35covers the base and the inner surfaces of the hemispheres24, the fill level h of the dry reagent35on the base corresponding to approximately half of the height H of the hemisphere24. At the boundaries, the reagent35flows further upward during the drying; however, it is prevented by the physical barrier and the edge25from creeping further over the web27formed between the two chambers13a,13b. The web27typically extends between two adjacent reagent chambers13in the direction toward the cover layer34and thus separates the two reagent chambers13a,13bof the mixing chamber22.

FIG. 2cshows a three-dimensional view in the area of line11cfromFIG. 1through the connection channels14of the channel structure2. The feed channel6has a return barrier28, which is implemented as a microfluidic valve29. The depth of the feed channel6from the surface30of the microfluidic element1is in the same order of magnitude as the depth of the connection channels14. However, it is significantly greater than the depth of the rotational-axis-distal reagent chamber13a. The depth of the feed channel6is thus also approximately 400 μm. A liquid which flows due to rotational force from the feed channel6into the overflow protection26of the mixing chamber22flows over the edge25into the hemispherical reagent chamber13a. Through rotation of the test carrier3, the inflowing liquid is moved into the reagent chamber13aand thus dissolves the dry reagent (not shown here) contained therein.

Upon the inflow of further liquid, it is also conducted through the connection channels14a,14b, and14cinto the further reagent chambers13(not shown). In the context of the disclosure, it has been established that the outgoing transitions from the reagent chamber13, which is implemented as a hemisphere24, into the capillary connection channels14a,14b,14ctypically cannot be smaller than 0.4×0.4 mm in cross section (or its diameter cannot be smaller than 0.4 mm) and can only gradually taper later. In connection channels14having a smaller cross section, the applied capillary force is so great that overflow (“crosstalk”) occurs, in particular of the liquid reagents before the drying.

The channel structure2having reagent chambers13which are rounded on the base may also be used in nonrotating test carriers. A liquid driven by an (external) force first flows in the case of a nonrotating microfluidic element1into the first reagent chamber13a, fills it completely, and dissolves the reagent contained therein. Not only uniform distribution of the reagent is ensured by the rounded base of the chamber. The dissolving of the reagent also occurs in an optimized manner. Only the inflow of further (force-driven) liquid may overcome the edge25, so that it can flow through the connection channels14into the adjacent reagent chamber. The reagent contained therein is therefore only dissolved in a second step.

FIG. 3shows an example of a further embodiment of a test carrier3, having five identical channel structures2. The feed channel6also has a U-shaped channel section7and a linear channel section8. The mixing chamber22also has, on its rotational-axis-proximal end, a ventilation channel10having a ventilation opening11. The channel structure2is also implemented as an analysis function channel15and comprises a measuring chamber16in this arrangement.

FIG. 4shows a detail view of the mixing chamber22fromFIG. 3having the three reagent chambers13a, b, cconnected in series and two connection channels14in each case, namely a central connection channel14aand a lateral (rotational-axis-proximal) connection channel14bin each case. The mixing chamber22typically has a rotational-axis-proximal inlet opening23, through which liquid enters the mixing chamber22from the feed channel6. A capillary transport channel31is typically positioned on the rotational-axis-distal long boundary36of the mixing chamber22. The transport channel31extends laterally and radially outside on the reagent chambers13positioned in series. Its depth (considered from the surface30of the test carrier3) is, at approximately 150 to 200 μm, less than the depth of the connection channels. The entering liquid is conducted through the transport channel31into the reagent chamber13a.

The ventilation channel10is wider than the feed channel8and wider than the connection channels14between the reagent chambers13. In this manner, a smaller capillary force is generated by the ventilation channel10, so that no liquid penetrates into the ventilation channel10. In addition, the ventilation channel10is always positioned rotational-axis-proximal, so that the liquid cannot reach the ventilation channel10from the reagent chambers13during the rotation. During the filling of the reagent chamber13a, the air contained therein already escapes through the connection channels14aand14binto the closest reagent chamber13c. As soon as the reagent chamber13ais completely filled, liquid flows through the two connection channels14aand14binto the reagent chamber13c. The filling of the second reagent chamber13cthus also initially occurs at least partially through the connection channels14a,14band through the transport channel31.

The air contained in the second reagent chamber13cescapes through the connection capillaries14aand14b, which form the connection to the rotational-axis-proximal reagent chamber13b. It is ensured in this manner that no air is enclosed in the reagent chambers13a,13b, and13c. The air escapes from the reagent chamber13bvia the ventilation channel10. Typical filling of the reagent chambers13from radially outside to radially inside is made possible in this manner.

The arrangement according to the embodiments of the disclosure already allows mixing of the liquids upon dissolving of the reagents, in particular upon dissolving of the reagents in the second and further reagent chambers13. The degree of dissolving is therefore particularly high and effective.

The filling of the reagent chambers13a, b, cof the mixing chamber22will be explained in greater detail on the basis ofFIGS. 5ato5c. Liquid entering the mixing chamber22is conducted via the capillary-active transport channel31, which is adjacent to the inlet opening23, past the two rotational-axis-proximal reagent chambers13b,13cand flows into the rotational-axis-distal reagent chamber13a(arrow direction F). The inflowing liquid is held by capillary action in the transport channel31. During the rotation of the test carrier3(in the arrow direction R, clockwise here), the liquid is then pressed at the rotational-axis-distal end of the mixing chamber22into the reagent chamber13aand dissolves the dry reagent contained therein. Upon filling, air escapes from the reagent chamber13avia the connection channels14a,14band the chambers13c,13band the ventilation channel10. As soon as further liquid flows after, it is conducted through the transport channel31into the reagent chamber13aand conducted therefrom at least partially through the central connection channel14aand the tangential connection channel14binto the middle reagent chamber13c. The further filling is performed directly via the transport channel31until the reagent chamber13cis filled. Upon further inflow of liquid from the feed channel6, the rotational-axis-proximal reagent chamber13bis finally also filled, in that the liquid first flows through the central and tangential connection channels14a, band also through the transport channel31and later directly into the chamber13b. The air contained in the reagent chambers13finally escapes through the air outlet33and the ventilation channel10.

In the example according toFIGS. 4 and 5, the reagent chambers13have an individual volume of 3 μL, so that the three reagent chambers jointly have a volume of approximately 9 μL. The volumes of the individual reagent chambers13are typically between 3 μL and 10 μL. Reagent chambers having a volume of 2 μL or only 1 μL are also conceivable, as are reagent chambers13having a volume of 20 μL, 50 μL, 100 μL, or 500 μL.

FIG. 6shows a further typical embodiment having a mixing chamber22, in which two reagent chambers13a,13bare integrated. A capillary transport channel31is also provided here, through which liquid entering the mixing chamber22is guided to the rotational-axis-distal reagent chamber13a, which is the reagent chamber farthest away from the rotational axis of the two reagent chambers13a,13b. The rounded reagent chambers13, having the rounded base, which are typically implemented as hemispheres24, do not only ensure a homogeneous reagent application of the still liquid reagent. They have also been shown to be extremely suitable if the test carrier3is operated in a shake mode, in which the rotational velocity and rotational direction are changed according to a typically serrated activation curve. In this method, which is known as “Euler mixing”, and which guarantees good homogenization and dissolving of the dry reagents, the mixing can be increased further by the rounded geometry. It has been recognized in the context of the disclosure that the most effective exchange and the most effective blending occurs at the boundaries and walls of the reagent chambers13. Therefore, the connection channels14are positioned on the boundary, e.g., tangentially to the reagent chambers13, in at least one of the two adjacent reagent chambers13. It has proven to be advantageous to form the connection channels14without edge transitions on the reagent chambers13.

In addition, it has been recognized in the context of the disclosure that a plurality of reagent chambers having connection channels transport the fluid through the connection channels14from chamber13to chamber13during the “Euler mixing” and diffuse exchange and good mixing efficiency can be provided in combination with the rounded surfaces.

Since the reagent chambers13are typically positioned adjacent in such a manner that their spacing is smaller than the smallest dimension of the reagent chambers13in the test carrier plane, a rapid fluid transport from one chamber13into the other is also possible. The smallest spacing is defined in the context of the disclosure as the smallest distance between the reagent chambers13or between the reagent chamber outer walls, respectively. At least the centrally located connection channel14abetween two reagent chambers13is therefore shorter than the smallest dimension of the reagent chambers13. In the example shown inFIG. 6, the central connection channel14ais approximately 0.2 mm long. Its width and depth are each 0.4 mm. The reagent chambers13have a height of 1.4 mm. The diameter of the reagent chambers is 1.95 mm. Through this geometrical arrangement, an unobstructed fluid transport between two adjacent reagent chambers13is possible. The fluid can be transported rapidly from one chamber to the other through the short connection channels14. The transport occurs directly without interposed valve structures, lever arrangements, or siphon-like channel structures, whose length is a multiple of the reagent chambers. In this manner, the processing sequence using the reagent chambers according to the disclosure is very rapid and saves time. In addition, controlled and defined dissolving of different dry reagents which are contained in individual reagent chambers13may be performed.

Through the modular construction having small reagent chambers13, it is possible to provide test carriers3, which may be expanded arbitrarily based on this principle. Therefore, not only two or three, but rather also a plurality of chambers may be connected in series.

In addition to the round hemispherical reagent chambers, other forms of the reagent chambers are also possible, for example, droplet-shaped reagent chambers or, if two reagent chambers are used, which are integrated in a mixing chamber22, e.g., so-called “Yin Yang embodiments”. These reagent chambers are typically also rounded on the base. Oval and round chamber forms prove to be advantageous above all.

FIG. 7shows a star-shaped arrangement of three reagent chambers13in a mixing chamber22. The rotational-axis-distal mixing chamber13ais also filled first via the transport channel31in this arrangement. As further liquid flows in, the two rotational-axis-proximal reagent chambers13b,13care then filled jointly. Only one central connection channel14ais provided between the reagent chambers13aand13b, since the capillary transport channel31is used as the second connection channel14b.

Three-dimensional views of such a star-shaped reagent chamber arrangement are shown inFIGS. 8aand8b. The rounded connection channels14between the reagent chambers13and the rounded hemispherical reagent chambers13themselves are clearly recognizable. It can be recognized in this embodiment that the transport channel31also functions fluidically as a connection channel14.

FIG. 9shows that a star-shaped or circular arrangement of reagent chambers13can also be expanded. Thus, as shown here, six reagent chambers13can be fluidically interconnected, the principle being maintained that the reagent chamber13amost distal to the rotational axis (rotational-axis-remotest reagent chamber) is filled first. Filling of the further chambers then begins from the rotational-axis-distal chamber13a, which is located farthest away from the rotational axis4.

During the rotation of the test carrier, the fluid is moved through all reagent chambers, in the star-shaped arrangement precisely as in the serial arrangement. Very efficient dissolving and mixing as well as targeted control of the liquid quantities may be achieved in this manner. The very compact and small arrangement obtained in this case has the advantage that a plurality of cascaded channel structures2may be positioned on one test carrier3.

The drying process of two reagents in a microfluidic element1at different points in time will be explained on the basis ofFIGS. 10ato10c, a view from below and also a section being shown in each figure.

The drying of the initially liquid reagents will be explained based on two reagent chambers13, which are separated from one another and have a fluid connection to one another via connection channels14. The two reagent chambers13a,13bare integrated in a mixing chamber22. A web27is positioned between the two reagent chambers13a,13b, so that the two chambers13are spatially spaced apart from one another. The connection channels14are introduced into the web27. The embodiment shown here has three connection channels14a,14b,14c, the connection channel14abeing a central channel and the two further connection channels14band14ceach being positioned laterally.

FIG. 10ashows that a liquid reagent is introduced into the hemispherical reagent chambers13a,13b. One reagent chamber13is used per reagent, which is also referred to as a “pearl” because of its shape. Therefore, a “pearl necklace structure” is provided overall in the mixing chamber22. The reagent is applied in the middle of the reagent chamber13a,13bin each case. During the following drying procedure, the reagent wets the surroundings of the dosing point and forms a uniform film. Since the reagent chambers are free of edges or corners, in which the reagent could concentrate, very uniform distribution occurs. If the liquid reagent reaches the connection channels14, it enters therein. However, it is decelerated by the flow resistance of the connection channels14and does not flow up to the transition into the adjacent reagent chamber13. If the liquid reagent reaches the upper boundary of the reagent chamber13, which forms the termination to the surface of the microfluidic element1, the reagent stops at the edge and does not flow further. The cross-sectional elevation performed therefore has a capillary stop effect.

The connection channels14typically have a cross section such that the liquid is decelerated in the connection channels14and is not transported into the adjacent reagent chamber13because of capillary forces. On the one hand, the cross section must therefore be sufficiently large that the occurring capillary forces are sufficiently small so that the connection channels are not completely filled with the reagent and the reagents do not mix in the connection channels. On the other hand, the cross section of the connection channels must be sufficiently small that the flow resistance is sufficient to decelerate inflowing reagent in the connection channels14.

The suitable selection of the cross section of the connection channels14does not only influence the drying process if solely capillary forces are active. The cross sections also influence the mixing efficiency and the exchange of liquids between two reagent chambers13. In order that sufficiently high flow velocities are achieved, which allow a fluid exchange between the chambers13, the cross section of the connection channels is at least 0.1 mm2, typically 0.4×0.4 mm2in size. Cross sections of less than 0.05 mm2have been shown to be unsuitable.

The reagent chambers13which are hemispherical or rounded on the base show that drying of the reagents without problems is possible upon filling with a liquid reagent using a volume of at most 70% of the chamber volume. Mixing of two reagents in two adjacent chambers13is reliably prevented. The volume of the liquid reagent to be applied is typically less than 60% of the chamber volume, particularly typically less than 55%.

FIG. 10cshows the two reagent chambers13after the liquid reagent has spread out. The connection channels14are each only wetted with liquid at their beginning. The largest part of the respective connection channels14is free of liquid, so that mixing of the two reagents is reliably prevented.

It has been shown in the context of the disclosure that the reagent chambers13having a rounded base, in particular if they are typically integrated into a mixing chamber22, are not only particularly suitable for the drying of two different reagents, but rather such reagent chambers13may be used in non-rotating microfluidic elements1. The force required for controlling the liquids and dissolving the reagents is generated by an external force. Alternatively to the centrifugal force or rotational force, pressure forces may be generated, which are induced by an external pump, for example. This force may also be based on a hydrostatic pressure. The statements made for rotating test carriers in the context of this disclosure therefore also apply for non-rotating microfluidic elements. The features described on the basis ofFIGS. 2 to 9may also be used accordingly in non-rotating arrangements and channel structures.

It is noted that terms like “preferably”, “commonly”, and “typically” are not utilized herein to limit the scope of the claimed subject matter or to imply that certain features are critical, essential, or even important to the structure or function of the embodiments disclosed herein. Rather, these terms are merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment of the present disclosure.

It will be apparent to those skilled in the art that various modifications and variations can be made to the embodiments described herein without departing from the spirit and scope of the claimed subject matter. Thus it is intended that the specification cover the modifications and variations of the various embodiments described herein provided such modifications and variations come within the scope of the appended claims and their equivalents.