WO2025162946A1

WO2025162946A1 - In vitro diagnostic test system, ivd test apparatus and a method of performing a multiplexed diagnostic assay at an improved degree of efficiency and eco-friendliness

Info

Publication number: WO2025162946A1
Application number: PCT/EP2025/052151
Authority: WO
Inventors: Oscar GUTIÈRREZ-SANZ; Eloisa Lopez-Calle; Christopher Probst
Original assignee: F Hoffmann La Roche AG; Roche Diagnostics GmbH; Roche Diagnostics Operations Inc
Current assignee: F Hoffmann La Roche AG; Roche Diagnostics GmbH; Roche Diagnostics Operations Inc
Priority date: 2024-02-01
Filing date: 2025-01-29
Publication date: 2025-08-07
Anticipated expiration: 2026-08-01

Abstract

The invention allows an increased throughput of diagnostic assays and doubles, triples or even further increases the number of assays per cartridge and may therefore be considered as very environmental- and eco-friendly. At the same time, multiple potentially life-saving test results may be provided for one or more patients. The invention relates to an In Vitro diagnostic (IVD) test system (1a, 1b) for performing a multiplexed diagnostic assay, wherein the IVD test system (1a, 1b) comprises: a test carrier (2) comprising a sample application port (3) configured to receive a sample fluid (30); a test zone (4) comprising a shared recessed assay membrane area (4a) and a sample release port (4b) for releasing at least one portion (30, 30a) of the sample fluid (30) to the shared recessed assay membrane area (4a); and a microfluid sample channel system (5) configured to guide the at least one portion (30, 30a) of the sample fluid (30) from the sample application port (3) to the sample release port (4b); the IVD test system (1a, 1b) further comprising: a first assay membrane (6) positioned in the shared recessed assay membrane area (4a) and configured to receive a first part of the at least one portion (30, 30a) of the sample fluid (30) from the sample release port (4b) and to indicate at least one first analyte (31a) in the first part of the at least one portion (30, 30a) of the sample fluid (30); and a second assay membrane (7) positioned in the shared recessed assay membrane area (4a) next to the first assay membrane (6) and configured to receive a second part of the at least one portion (30, 30a) of the sample fluid (30) from the sample release port (4b) and to indicate at least one second analyte (31b) in the second part of the at least one portion (30, 30a) of the sample fluid (30) and/or to indicate the at least one first analyte (31a) in the second part of the at least one portion (30, 30a) of the sample fluid (30) in a different sensitivity range as the first assay membrane (6).

Description

In Vitro diagnostic test system, IVD test apparatus and a method of performing a multiplexed diagnostic assay at an improved degree of efficiency and eco-friendliness

Field of the Invention

The invention relates to an In Vitro diagnostic (IVD) test system, an IVD test apparatus and a method of performing a multiplexed diagnostic assay to detect several parameters or at least one parameter in different measurement range, i.e. sensitivity ranges, specifically comprising cardiac parameters, without limitation. The invention allows an increased throughput of diagnostic assays and may double, triple or even further increase the number of assays per cartridge and may therefore be considered as very eco-friendly, as waste material can be reduced tremendously. At the same time, multiple potentially life-saving test results may be provided in a short time for one or more patients.

Background of the Invention

In medical diagnostics, it is often crucial to provide test results instantaneously or in a very short term, specifically if a patient is in a life-threatening health situation. To identify the acute health condition and/or the severity of the situation, it may often be required to quickly and reliably determine several parameters and/or properties of a sample of the patient at the same time, such that a decision to apply appropriate and efficient therapeutic measures can be met. In addition, it may be required to quickly provide test results for several patients who urgently require medical help at the same time. Therefore, medical point-of-care (POC) tests, which are used in emergency rooms, medical practices or the like, need to efficiently provide reliable test results for multiple parameters, such as the qualitative or quantitative determination of analytes in sample fluids/liquids of one or multiple patients.

In the field of In Vitro diagnostics, medical POC tests for the qualitative and/or quantitative determination of analytes in a liquid sample are often based on immunoassays that may use lateral flow assay strips. In most cases, lateral flow assay (LFA)-strips which comprise porous immunoassay membranes (e.g. nitrocellulose membrane) or paper sheets (e.g. cellulose) with the capability to passively transport fluids by means of capillary forces are employed for such applications, such as assay membranes and/or functional pads for example. Typically, such an LFA-strip is coated with a capture reagent for capturing and detecting the analyte of interest. This capture and detection zone is also known as the test line or test zone. To provide test results for multiple parameters at the same time, multiplexing POC tests may be used. Such multiplexing POC tests allow simultaneous determination of more than one analyte from the same sample in a single measurement run using a single test carrier. This type of test is often called a “panel test”. A POC platform should offer the possibility for single-testing and multiplex-testing on the same system in order to allow the healthcare professionals to perform the most possible convenient and fast measurements. From the manufacturer's point of view, this platform flexibility for single- and panel-testing may be ideally implemented without requiring major changes in design of the test carrier and instrument as well as the possibility of using the same sample volume.

As an example, Fig. la and Fig. lb illustrate the concept of a known POC test using an immunoassay membrane 6 (also denoted “assay membrane”). A sample fluid 30, such as a whole blood sample in the present case, is received from the patient. As shown in Fig. la, the sample fluid 30 is indicated to contain an analyte 31 of interest, for which the sample fluid 30 should be tested. Plasma 30a is extracted from the whole blood sample 30, for example by centrifugation. The plasma 30a may be considered a component of the whole blood sample 30 and after separation, i.e. extraction from the whole blood sample 30, it contains the analyte 31. The plasma 30a is afterwards mixed and incubated with dried reagents 32, 33 that are designed to specifically couple to the binding sites of the analyte 31 and therefore label the analyte 30a. The dried reagents 32, 33 comprise a detection reagent 32 and a capture reagent 33. The detection reagent 32 comprises a detection antibody for coupling to a binding site of the analyte 31. The detection reagent 32 comprises and/or is coupled to a detection element for providing a detection signal, which may for example correspond to a fluorescent label and/or a JG9-Latex bead. The capture reagent 33 comprises a capture antibody 33 for coupling to another binding site of the analyte 31. The capture reagent 33 comprises and/or is coupled to a capture element for realizing/performing a capture process on the assay membrane 6, which may for example be biotinylated.

Fig. lb illustrates the capture process of the labeled/label-sandwiched analyte 31c and the detection principle of an LFA-strip 6, which corresponds to and/or comprises an assay membrane 6 that is indicated in Fig. lb. A test line 37 of the assay membrane 6 is coated with analyte-specific antibodies for specifically capturing the analyte 31 of interest. The assay membrane 6 has a general flow axis 6a, a flow start side 6b and a flow end side 6c.- The flow start side 6b and the flow end side 6c may be defined by the functions of the elements on the assay membrane 6. By allowing a fluid to enter an assay membrane 6 from the flow start side 6b, the correct function of the assay membrane 6 may be ensured. In other words, the (sample) liquid should be provided from the flow start side 6b to the assay membrane 6 such that the assay can be properly used. The assay membrane 6 may in some cases be contacted with a waste pad (which may be a waste fleece) at the flow end side 6c (not shown in this figure). The upper most scheme of Fig. lb shows the assay membrane 6 before the serum 30a is applied and the central scheme of Fig. lb shows the situation after the serum 30a is applied to the assay membrane 6 and after the labeled analyte 31c is bound on the assay membrane 6. The lower most scheme shows a CCD image of a real assay membrane 6 as indicated in the central scheme.

The plasma 30a with the labeled analyte 31 is applied to the assay membrane 6 at the flow start side 6b. At least a portion, specifically a part and/or a component of the plasma 30a is passively transported by capillary forces substantially along the general flow axis 6a from the flow start side 6b to the flow end side 6c. The assay membrane 6 comprises three lines 35, 36, 37, a control line 35, a calibration line 36 and a capture and test result line 37 (previously denoted “test line”). The control line

35 (assay control) is pre-coated with analyte-specific antibodies to the analyte 31 of interest. The calibration line 36 (instrument calibration) is pre-coated with the fluorescent label 32’ comprised in the detection reagent with the detection antibody 32. The capture and test result line 37 is coated with an agent/ capture mechanism 34 that captures/binds the capture reagent 32 and therefore is the line, where the capture process is performed. The capture mechanism 34 may for example correspond to and/or comprise streptavidin.

The serum 30a is applied to the assay membrane 6 on the flow start side 6b and therefore firstly passes the capture and test result line 37, where the analyte 31c that is sandwiched between the two reagents 31, 32 is specifically bound/ captured via the capture antibody that binds to the capture mechanism 34 of the capture and test result line 37, namely streptavidin in the present case, if the labelled analyte 31 is present in the serum 30a. The residual portion of the serum 30a passes the calibration line

36 where no further binding process takes place and then it passes the control line 35, where residual unbound detection reagent 32 comprising the detection antibody may at least partially specifically bound.

The control line 35 provides a control of whether or not the serum 31 was mixed with the reagents 32, 33 by capturing the unbound detection reagent 32. The control line 35 is specifically significant if the test result is negative as in that case, the result might be negative due to the absence of the analyte 31, but it may also be negative (specifically false-negative) due to the absence of the detection reagent 32. The calibration line 36 serves for calibration purposes and provides a pre-coated area with the fluorescent label 32’ that is identical to the fluorescent label 32’ used in combination with the detection antibody 32 to label the analyte 31 for detection.

Finally, the assay membrane 6 is washed with a buffer solution to remove unbound analytes 31, reagents 32, 33 and/or other molecules from the assay membrane 6 and a CCD image is recorded to read out the test result. The lower most scheme of Fig. lb shows such a CCD image of a real assay membrane 6, as indicated in the central scheme. The three lines 35, 36, 37 provide a visible and/or detectable signal due to the bound fluorescent molecules/ fluorescent labels 32’ either being present on the label-sandwiched analyte 31c on the capture and test result line 37 or being already present in the pre-coated calibration line 36 or being bound to the pre-coated control line 35. The lower most scheme of Fig. lb therefore reveals from the visible signal that the analyte 31 is present in the serum 30a and was apparently properly mixed with the reagents 32, 33.

As can be seen in Fig. lb, the assay membrane 6 does not provide plenty of space for a multiplexed assay, which would require at least one or even three more lines for the detection of one additional analyte. Further, unspecific binding events and/or crosstalk between the fluorescent labels captured on closely neighbored lines may falsify the test result, which is problematic for a patient that suffers a life-threatening condition and correct test results are required to choose an adequate treatment. Therefore, reliable, sensitive and/or effective multiplexed assays are required.

Further, US 2021/154662 Al describes an apparatus for directing a liquid through a porous medium includes a fluidic module rotatable about a center of rotation and including a fluid chamber and an inflow structure. A porous medium is disposed in the fluid chamber to allow centrifugal force-effected flow of the liquid impinging on a radially inner portion of the porous medium, to a radially outer portion of the porous medium. The porous medium is laterally at least partially spaced apart from chamber walls of the fluid chamber with respect to the flow, so that a fluid connection exists between the radially inner portion of the porous medium and the radially outer portion of the porous medium outside the porous medium. The inflow structure is configured to limit a centrifugal force-effected inflow of the liquid to the radially inner portion of the porous medium to a first flow rate, wherein a ratio of the first flow rate to a maximum possible flow rate through the porous medium is not greater than two. US 2011/293489 Al describes systems and methods including a microfluidic chip having a plurality of microfeatures interconnected to provide a configurable fluid transport system for processing at least one reagent. Inserts are provided to removably interfit into one or more of the microfeatures of the chip, wherein the inserts include sites for interactions with the reagent.

US 2013/184188 Al describes devices and methods for performing optical and electrochemical assays and, more particularly, to testing devices having an optically readable microspot array and/or an electrochemical detector and to methods of performing microspot arrays and electrochemical assays using such devices. This is used for performing immunoassays and/or electrochemical assays at the point-of- care.

US 2010/120173 Al describes a strip-assembled immunochromatographic disc, containing: a base, a lid engaged with the base and a draining piece disposed between the strips on the base and the lid, wherein a sampling opening is disposed on the lid directly facing to the draining piece, and the said sampling opening intercommunicates to a draining groove provided on the inner side of the lid which is formed by a plurality of draining channels; several strip stages are provided on the base with their location and number corresponding to those of the draining channels provided on the lid, and the edge of the draining piece laps to the sample pads of the strips carried on the stage adjacent to one end of the sampling opening.

Lutz, Sascha et al., “A fully integrated microfluidic platform for highly sensitive analysis of immunochemical parameters”, ANALYST, 22 September 2017, pages 4206-4214, XP093171974, UK ISSN: 0003-2654, DOI: 10.1039/C7AN00547D describes a microfluidic platform for highly sensitive analysis of immunochemical parameters.

Summary of the Invention

It is therefore desirable to provide an IVD test system, test apparatus and method to perform reliable, sensitive and/or effective multiplexed diagnostic assays. It is further desirable to provide an IVD test system that is based on and/or compatible with a platform, which allows for single analyte testing and multiplexed testing using some or even almost all or all of the same components of the IVD test system. In other words, an IVD test system is required that can be used for single analyte testing and multiplexed testing or that can be easily adapted from single analyte testing to multiplexed testing is required. Further, it is desirable to provide an IVD test system to perform multiplexed diagnostic assays that may be produced in a similar or the same manner as an IVD test system to perform single analyte testing. Moreover, it is desirable to provide an IVD test system, test apparatus and method to achieve an increased throughput of diagnostic assays and/or a more quantitative diagnostic assay. Further, it is desirable to provide a versatile IVD test system, test apparatus and method to perform multiple diagnostic assays. It is also desired to provide a compact IVD test system that is compatible with an IVD test method that is based on a rotational operation.

At least one of the described problems is respectively addressed by the IVD test system, the IVD test apparatus comprising the same and the method of performing a multiplexed diagnostic assay.

According to a first aspect of the invention an IVD test system is provided for performing a multiplexed diagnostic assay, wherein the IVD test system comprises: a test carrier comprising: a sample application port configured to receive a sample fluid; a test zone comprising an shared and/or common recessed assay membrane area (specifically one single shared assay membrane area and/or which may at least partially correspond to a flat plane that can receive the assay membranes and which may be confined at least partially by recess walls and being recessed with respect to an outer and/or upper surface of the test carrier and/or a cover plate of the test carrier) and a sample release port (specifically one single sample release port) for releasing at least one portion of the sample fluid to the shared recessed assay membrane area; and a microfluid sample channel system configured to guide the at least one portion of the sample fluid from the sample application port to the sample release port, wherein the IVD test system further comprises: a first assay membrane positioned in the shared recessed assay membrane area and configured to receive a first part of the at least one portion of the sample fluid from the sample release port and to indicate at least one first analyte in the sample fluid, specifically in the first part of the at least one portion of the sample fluid; and a second assay membrane positioned in the shared recessed assay membrane area next to the first assay membrane and configured to receive a second part of the at least one portion of the sample fluid from the sample release port and to indicate at least one second analyte in the sample fluid, specifically in the second part of the at least one portion of the sample fluid and/or to indicate the at least one first analyte in the sample fluid, specifically in the second part of the at least one portion of the sample fluid in a different sensitivity range and/or measurement range as the first assay membrane. The sensitivity range may in some cases comprise or be considered a measurement range. The invention allows an increased throughput of diagnostic assays and doubles, triples or even further increases the number of assays per cartridge and may therefore be considered as very eco-friendly (may be understood as a green invention). At the same time, multiple potentially life-saving test results may be provided for one or more patients. Further, the invention allows decreasing the cost pressure for clients to run (multiple) assays and/or many assays in a short time.

A very compact IVD test system is provided for multiplexing tests, wherein, in the production of different IVD test systems for single testing and different multiplexing techniques, the number of individual/customized elements can be reduced. This makes it very efficient to produce and use the IVD test system.

The IVD test system may be considered a cartridge, e.g. a centrifugal cartridge that can be used with an IVD test apparatus, e.g. an IVD test apparatus, which comprises a centrifuge. The IVD test system allows performing reliable, sensitive and/or effective multiplexed diagnostic assays. The IVD test system may be based on and/or compatible with a platform, which allows for single analyte testing and multiplexed testing using some of the same components, specifically using the same test carrier format for single analyte testing and multiplexed testing. Further, the IVD test system for multiplexed testing may be produced in a similar or the same manner as an IVD test system to perform single analyte testing. Moreover, the IVD test system allows achieving an increased throughput of diagnostic assays and/or a more quantitative diagnostic assay. The IVD test system is versatile in performing multiple diagnostic assays.

Specifically, the IVD test system may allow performing a multiplexed diagnostic assay, in which at least one analyte can be detected at two different sensitivity ranges and/or measurement ranges to better quantify the amount of analyte in the sample or determine at least a range. A first measurement range and/or a second measurement range may lie between approximately 0,5 and 15mmol/L, specifically 2,0 and 7,3mmol/L for Potassium, without limitation, and between approximately 0,05 and 50, specifically 0,2 and 20 and more specifically between approximately 0,3 and 10 mg/dL for Creatinine, without limitation. Alternatively or in addition, the IVD test system may allow performing a multiplexed diagnostic assay, in which at least one first analyte may be reliably detected and at least one second analyte may be reliably detected given that the according analyte is present in the original sample fluid. The multiplexed diagnostic assay, i.e. the multiple assays are respectively performed on two assay membranes and are therefore spatially separated from each other.

The test carrier may be comprised of one or more pieces. If the test carrier comprises several pieces, for example, one piece may comprise the microfluid sample channel system as recesses, specifically recessed trace in the one piece. At least one other piece may correspond to a cover plate, specifically one that is configured to close the microfluid sample channel system at least partially. One of these pieces or another piece may comprise at least a portion of the test zone. The test carrier may specifically comprise a test carrier core piece being sandwiched between at least two cover plates to cover and/or seal the test carrier and/or the components being housed by the test carrier such as for example dried reagents. One or both of the cover plates may correspond to a film and/or sheet. At least one of the film(s) and/or sheet(s) may be flexible. At least one of the film(s) and/or sheet(s) may provide stability to the test carrier. At least one of the film(s) and/or sheet(s) may provide information regarding the type of test, the type of cartridge, the brand etc. Instead of being sandwiched, the test carrier core piece may be covered only on one side by a cover plate or by several plate-like elements for covering the test carrier core piece. The microfluid sample channel may correspond to a recessed trace in a center and/or core piece and/or one or more cover plates. Alternatively, the microfluid sample channel may correspond to a channel, channel system, tunnel and/or pipe inside a block of one material piece - in other words, the microfluid sample channel may be surrounded and/or enclosed mostly by one element, which may be a single-piece element and a cover plate may not be required in that case. The test carrier and/or the test carrier core piece may comprise one single piece and/or may be produced in a method of additive manufacturing. Alternatively, the test carrier and/or the test carrier core piece may be produced by injection molding. The test carrier and/or the test carrier core piece may comprise two or more pieces, which are assembled with each other, e.g. two or three or even more plates. The test carrier may for example comprise three pieces, which are assembled with each other, specifically three plates, in which a center and/or core piece is sandwiched between two cover plates. The test carrier may comprise more than three pieces, which are assembled with each other. The test carrier may be at least partially transparent to let pass visible light and/or the test carrier may be at least partially transparent for light that can be detected by a detector. One or all of the pieces of the test carrier may be at least partially transparent. This allows tracking a sample fluid that flows inside and/or along the microfluid sample channel system.

The test carrier and/or the test carrier core piece and/or any component of the test carrier may comprise, without limitation, at least one of the following materials: polystyrene (PS), polycarbonate (PC), polypropylene (PP) polyethylene (PE), etc. One or more cover plates may, without limitation, comprise at least one of the following materials: polystyrene (PS), polycarbonate (PC), polypropylene (PP) polyethylene (PE), etc. At least one of the described plates may correspond to and/or comprise a foil. A sealing element may be used to seal the microfluid sample channel system. Alternatively, a sealing element may not be required to seal the microfluid sample channel system.

The sample application port, which is configured to receive a sample fluid, may correspond to an opening and/or access to the microfluid sample channel system such that a fluid sample, which is applied to the sample application port, can enter the microfluid sample channel system. The sample application port may for example correspond to a tapered opening that is configured to collect and guide the sample fluid towards the microfluid sample channel system. The sample application port may specifically correspond to a recess and/or a hole, specifically a tapered hole and/or cut-out in an element of the test carrier, for example a center piece of the test carrier and/or a cover plate (first or second cover plate) to allow access to the microfluid sample channel system, specifically a microfluid sample channel system of a test carrier core piece positioned underneath the sample application port. Besides the sample application port and the sample release port, the microfluid sample channel system may be provided with further openings and/or holes to allow a fluid, a liquid and/or a gas to escape the microfluid sample channel system.

The test zone comprises one single shared recessed assay membrane area corresponding to a flat plane (with no substantially no walls, bumps, and/or irregularities inside the recessed assay membrane area) confined at least partially by recess walls (recess edges), which is the area, in which the one assay membrane and/or the several assay membranes may be positioned. In other words, the shared recessed assay membrane area is a single area defined by the recess edges and being shared by several assay membranes at once, such that the sample fluid that reaches the sample release port and that is released to the shared recessed assay membrane area can be received by the multiple assay membranes substantially at the same time. Again in other words, the shared recessed assay membrane area does not comprise any walls except for the recess edges which would separate the assay membranes from each other. The recessed area may be considered a plane that is defined by the recess edges forming the walls of the shared recessed assay membrane area and that is lower than the top surface of the cartridge from which a portion of material is left out and/or cut out to establish the recess. The shared recessed assay membrane area has a depth with respect to the top surface of the cartridge that is reflected by the depth of the recess edges and that is described further below in this description. The fact that the shared recessed assay membrane area can be used by one single assay membrane or shared by two, three, four or more assay membranes has the advantage, that no further recessed assay membrane areas need to be added when providing more than one assay membrane. In other words, the same cartridge that is used for one single test (providing only one assay membrane) can also be used for several tests (providing more than one assay membrane). Further, only one single sample release port to the shared recessed assay membrane area is required and may therefore provided. Several assay membranes may be received by the shared recessed assay membrane area such that two neighboring assay membranes have a distance d to each other between approx. 0 and 5 mm, specifically between approx. 0, 1 and 2mm and more specifically between approx. 0,3 and 1mm. In other words, two neighboring assay membranes may either be in physical contact along the length edges or they are very close to each other - they are not apart from each other and/or in different chambers and they are not being spatially separated. A chamber may be considered a volume being enclosed by walls by around 60-100%, specifically by around 70-98% and more specifically by around 80-97%. Specifically, there may be no wall between two assay membranes in an embodiment. The test zone may comprise the waste pad area, which is the area, in which one waste pad and/or several waste pads may be positioned. The test zone comprises a sample release port for releasing at least one portion of the sample fluid to the shared recessed assay membrane area. The sample release port may be an opening to fluidically connect the microfluid sample channel system with the test zone on the opposite side of the sample application port in terms of the flow direction. In other words, a fluid that passes the microfluid sample channel system is guided towards the test zone via the sample release port to control the position where the sample fluid is released in the test zone. The test zone may provide a reservoir volume that is large compared to the microfluid sample channel system.

The test zone may corresponds to a zone that is recessed substantially in the shape of one assay membrane and/or several assay membranes oriented next to each other, i.e. side by side. In all cases, the test zone may be configured to receive one assay membrane and/or several assay membranes. In other words, the test zone may provide a space to position one assay membrane and/or several assay membranes therein. The test zone may match at least in one dimension the size of the assay membrane or the assay membranes such that the assay membrane(s) do(es) not shift and the position with respect to the test carrier is exactly defined. The test zone may be optically accessible, such that a detector can detect test results provided on the assay membrane(s) and/or a viewer can read the result. The test zone may be at least partially open, i.e. not covered by a cover plate or any other element of the test carrier. Alternatively, the test zone may be covered by an at least partially transparent portion of a cover plate and/or another element of the test carrier. The recess may define the test zone having the shared recessed assay membrane area and potentially a waste pad area. The shared recessed assay membrane area may be recessed substantially in the shape of one or more assay membranes arranged side-by-side and/or defining a position, in which the according assay membrane(s) can be positioned. The shared recessed assay membrane area may comprise one or more barrier(s) (wall(s)) each between two or more assay membranes such that liquid flow between the barriers may be stopped and/or the assay membranes are restricted in their movement such that a predefined fixed position can be attributed to the assay membranes. A recess, which may define and/or correspond to the test zone may have a length between approximately 0,5mm and 300mm, specifically 1mm and 100mm. The length of the recess may be measured along the general flow direction of the assay membrane, when being positioned in/on the recess and the recess may substantially correspond to the added/combined lengths of the waste pad and the assay membrane or may correspond substantially to a length that is slightly smaller than the combined lengths of the waste pad and the assay membrane when they are supposed to overlap or may correspond substantially to a length that is slightly larger than the combined lengths of the waste pad and the assay membrane when they do not overlap. The recess, which may define and/or correspond to the test zone may have a width between about 0,05mm and 50mm, specifically between about 1mm and 20mm. The recess, which may define and/or correspond to the test zone may have a depth between about 0,05mm and 20mm, specifically between about 1mm and 15mm and may or may not vary in different areas of the test zone. If varying the depth, a flow direction may be predefined from a less deep position to a deeper position for example.

Two or more assay membranes may be positioned next to each other, i.e. side by side in the shared recessed assay membrane area. This may refer to a parallel orientation of their length axes and/or general flow directions. Alternatively, it may refer to an assembly of the assay membranes having an angle between their length axes and/or general flow directions. The shape of the shared recessed assay membrane area and/or the position of the barrier(s) may define such an orientation of the assay membranes, which is either parallel or non-parallel, i.e. having an angle between at least two assay membranes.

The microfluid sample channel system is configured to guide the at least one portion of the sample fluid from the sample application port to the sample release port. The sample fluid may be transported passively through at least a portion of the microfluid sample channel system driven by capillary forces, Van-der-Waals forces and/or adhesive forces. Alternatively or in addition, the sample fluid may be transported passively through at least a portion of the microfluid sample channel system driven by external forces, specifically by rotation of the cartridge/test carrier generating centripetal and centrifugal forces.

At least one portion of the sample fluid may refer to at least one component of the sample fluid and/or at least one part of the sample fluid. For example, if whole blood corresponds to the sample fluid, the serum may be considered a component of the whole blood. At the same time, the serum is a part of the whole blood. If the whole blood is aliquoted, i.e. partitioned, inside the microfluid sample channel system, the aliquoted volume that is further transported may be considered a part of the whole blood. The term at least one portion may therefore be interpreted in the sense of “a part” and/or “a component”.

The microfluid sample channel system may transport pL-volumes of a sample fluid, for example capillary blood samples. Capillary blood samples are simple to be collected from a finder or a foot, which is specifically convenient when taking blood from newborns or patients who generally do not appreciate the usual procedure of taking higher amounts of blood. Further, taking a capillary blood sample from a patient can be performed quickly compared to the usual procedure of taking higher amounts of blood.

The microfluid sample channel system may be integrated in the test carrier by the recessed trace or by a tunnel-like system in the plastic core of the test carrier (test carrier core piece). The microfluid sample channel system may for example be formed by two elements, such as two plates wherein one or both plates comprise the recessed trace and respectively form an upper wall and a lower wall of the microfluid sample channel system. Therefore, the two elements may at least partially enclose the microfluid sample channel system. The microfluid sample channel system may be sealed by a sealing element that is sandwiched between the two plates. If the microfluid sample channel system is integrated in the test carrier by a tunnel-like and/or tube-like system in the plastic core of the test carrier (test carrier core piece), it may be substantially enclosed by only one piece and therefore, no sealing element may be required. In other words, the test carrier core piece may have a microfluid sample channel system integrated and in this case, it may be possible that the test carrier core piece represents and/or corresponds to the entire test carrier and does not require any cover plate to (en)close the microfluid sample channel system.

The tunnel- and/or tube-like system may comprise portions with a round and/or oval cross-section. Alternatively, the cross-section may have different shapes at least in a section and/or portion thereof, such as a polygonal shape. The microfluid sample channel system may constitute tunnel-like portions and/or functional elements, which differ in geometric shape from a typical tunnel. The microfluid sample channel system may integrate and/or may be connected to other elements, which are not considered microfluid channels and/or part of the microfluid sample channel system, such as tubes, needles, pads, capillaries and/or elements in the test carrier, which are either not microfluidic or specific elements of the microfluid sample channel system. Further, some elements, which are integrated with the microfluid sample channel system, may be considered as part of the microfluid sample channel system but differ in shape from a channel, a tunnel and/or a tube. Such elements may be or are already fluidically connected with other elements of the microfluid sample channel system, specifically with the sample application port and the sample release port. Further, such elements may have specific functions, which exceed the function of merely guiding and/or passively transporting (e.g. by capillary forces) a fluid, as described further below.

A first assay membrane and/or a second assay membrane may be configured, specifically appropriately sized, to be positioned in the shared recessed assay membrane area and/or may be already positioned and/or fixed in the shared recessed assay membrane area. The first assay membrane is configured to receive a first part of the at least one portion of the sample fluid, i.e. a sub-part of a component and/or a part of the sample fluid, such as for example a portion of the serum extracted from a whole blood sample that is released to the test zone/area via the sample release port. The first assay membrane is configured to indicate at least one first analyte in the sample fluid, i.e. in the first part of the at least one portion of the sample fluid.

The second assay membrane may be configured, specifically appropriately sized, to be positioned in the shared recessed assay membrane area and/or may be already positioned in the shared recessed assay membrane area next to the first assay membrane. The second assay membrane may be positioned in parallel to the first assay membrane. Alternatively, the second assay membrane may be positioned having a tilted orientation with respect to the first assay membrane. A flow start side of the first assay membrane and a flow start side of the second assay membrane may be positioned close to the sample release port, such that the sample that is released via the sample release port firstly arrives at the flow start side of each assay membrane. Therefore, the term “next to” can refer to two assay membranes being arranged in parallel to each other or with an angle in-between them, i.e. one assay membrane being tilted with respect to the other assay membrane.

Analogue to the function of the first assay membrane, the second assay membrane is configured to receive a second part of the at least one portion of the sample fluid from the sample release port and to indicate at least one second analyte in the second part of the at least one portion of the sample fluid and/or to indicate the at least one first analyte in the second part of the at least one portion of the sample fluid in a different sensitivity range/measurement range as the first assay membrane. In other words, the second assay membrane may test the first analyte in a more specific sensitivity/measurement range to further quantify the amount of the first analyte in the sample fluid or the second assay membrane may test a different analyte, i.e. a second analyte. This allows providing more information about the sample fluid, i.e. a higher number of test results in or on one single IVD test system. Therefore, one IVD test system (multiplexed assay) may be used where previously two or more IVD test system (single assay) were used to gain the same amount of information.

The assay membranes may therefore be cut and/or shaped in a size, specifically a width that fits the dimensions of the shared recessed assay membrane area. The assay membranes may be separated by a gap, i.e. a gap of air for example. Alternatively, the assay membranes may contact each other without having a gap in-between. The assay membranes may be provided on a shared substrate (such as a sheet) such that their orientation with respect to each other may be fixed and/or a gap between them is predefined on the substrate. The assay membranes may specifically comprise and/or be constituted of separate pieces of cellulose, nitrocellulose, glass fiber, nylon, and/or cellulose-based material. The function of the assay membranes are based on capillary forces, which drag the liquids from the flow start side towards the flow end side. Therefore, liquids are passively transported via the respective assay membrane. Different assay membrane material may cause different flow rates due to different capillary forces. All assay membranes may be made of the same material and therefore, the flow rate of all assay membranes may be identical. Alternatively, one or all assay membranes may be made of a material, which is different to another to achieve a specific flow rate for this assay membrane, which is different from the others.

The test carrier may already be equipped with the assay membranes such that the assay membranes are already positioned in and/or on the test carrier. Alternatively, the assay membranes may be provided separately to be positioned in and/or on the test carrier manually by a user for example or by a supplier.

The test carrier may specifically correspond to a centrifugal test carrier, in which the microfluid sample channel system is configured to guide the at least one portion of the sample fluid in at least one section/portion of the microfluid sample channel system, i.e. a portion between the sample application port and the sample release port, under the influence of a centrifugal force and/or under control of a centrifugal force.

Overall, the IVD test system, which may correspond to a microfluidic cartridge, specifically a centrifugal cartridge, allows for control of each of the single assay steps, thus yielding excellent and lab-like assay performance like high sensitivity, precision and accuracy.

The microfluid sample channel system may, without limitation, have a total path/trace length of about 5mm to 50mm, specifically of about 10mm to 25mm between the sample application port and the sample release port. The microfluid sample channel system may, without limitation, be configured to receive a volume of about 0,5 pL to 800pL, specifically of about 1 pL to 500pL. The microfluid sample channel system may, without limitation, comprise a channel portion with a width and/or diameter of about 0,05mm to 15mm, specifically of about 0, 1mm to 5mm.

One or more assay membranes may, without limitation, be configured to receive a fluid volume of about 0,5pL to 800pL, specifically of about IpL to 500pL.

The recessed areas and/or elements which may form the microfluid sample channel system or elements thereof, the microfluid washing buffer channel system or elements thereof and/or the test zone or elements thereof may comprise one or more steps, one or more different depths, one or more slopes and/or rounded and/or stepped surfaces to guide the flow of a fluid and/or liquid.

In general, it is an advantage of providing one cartridge for two or three or even more analytes over providing one cartridge for one single analyte, as the cartridge material used per assay may be reduced to approximately a half, a third or even less. This makes the invention specifically eco-friendly, as waste can be tremendously reduced. The IVD test system may further comprise a third assay membrane positioned in the shared recessed assay membrane area next to the first assay membrane and/or the second assay membrane and configured to receive a third part of the at least one portion of the sample fluid from the sample release port and to indicate at least one third analyte in the sample fluid, specifically in the third part of the at least one portion of the sample fluid and/or to indicate the at least one first analyte in the sample fluid, specifically in the third part of the at least one portion of the sample fluid in a different sensitivity/measurement range as the first assay membrane and/or the second assay membrane.

The third assay membrane may be positioned in parallel to the first assay membrane and/or the second assay membrane. Alternatively, the third assay membrane may be positioned having a tilted orientation with respect to the first assay membrane and/or the second assay membrane. A flow start side of the first and/or the second assay membranes and a flow start side of the third assay membrane may be positioned close to the sample release port, such that the sample that is released via the sample release port firstly arrives at the flow start side. If more than two assay membranes are provided, they may be oriented in a star-like arrangement, pointing towards a central spot, specifically a central spot, in which the sample release port is positioned and where the sample fluid is released.

The third assay membrane may have a corresponding function to the first and/or the second membrane, namely detecting an individual analyte or the same analyte as the first and/or the second membrane but in a different sensitivity/measurement range. The IVD test system may further comprise additional assay membranes with the corresponding functions, namely four, five, six, seven, eight, nine, ten or even more.

The third assay membrane may be separate from the first and/or the second membrane and with a gap and/or may share a common substrate.

The first assay membrane may rely on a first capture mechanism configured to capture the first analyte after being labeled with a first capture reagent and/or the second assay membrane may rely on a second capture mechanism different from the first capture mechanism to capture the second analyte after being labeled with a second capture reagent different from the first capture reagent, specifically when using a sandwich immunoassay (system) as test format. The first capture reagent and/or the second capture reagent may comprise one of a biotinylated antibody and/or a Digoxigenin (dig)-labeled antibody. These antibodies may be configured to bind with the first and/or the second analyte. The first capture mechanism and/or the second capture mechanism may comprise one of a streptavidin to capture the first or the second analyte via the biotinylated antibody, an anti-dig to capture the first or the second analyte via the dig-labeled antibody, a DNA-based system and/or an analyte specific antibody to directly capture the first or the second analyte.

It is an advantage to choose different capture mechanisms and corresponding different reagents to label different analytes such that these labeled analytes are specific to the capture mechanism or the capture mechanism is specific to the label of an analyte and the labeled analyte can be bound and/or captured in spatially separated areas and/or lines. In that way, it can be differentiated between analytes and therefore a multiplexed assay according to one embodiment may be realized. Such systems are typically based on sandwich immunoassays.

The first assay membrane and the second assay membrane may each have a general flow axis defined by a flow start side and a flow end side opposite the flow start side, through which the respective general flow axis may pass and wherein the general flow axis of the first assay membrane and the general flow axis of the second assay membrane may be aligned in parallel to each other such that the respective flow start side of each assay membrane is positioned closest to the sample release port of the test zone.

The general flow axis of the first assay membrane and/or the second assay membrane may correspond to a respective longitudinal axis of the assay membrane. The general flow axis may correspond to the direction, along which a fluid substantially flows and/or is passively transported when reaching the predefined flow start side. As a consequence thereof, the fluid flows from the flow start side of the respective assay membrane towards and/or to the flow end side of the assay membrane. It is to be understood that the fluid may also flow in another direction but the general flow axis and/or direction is (pre)defined by the flow start side and the flow end side, through which the general flow axis passes. The predefined flow start side may be the side which is closest to the line that should be passed by the fluid at first, such as the test (result) line.

The IVD test system may further comprise at least one waste pad positioned to contact the first assay membrane and/or the second assay membrane at the respective flow end side.

The waste pad may be configured and/or positioned for receiving a fluid waste portion of the sample fluid and/or a washing buffer solution and/or a washing solvent from the first assay membrane and/or from the second assay membrane at the respective flow end side. The fluid waste portion corresponds to the portion of the sample fluid that passes via at least one of the assay membranes and reaches the waste pad, which still has the volume capacity to receive the fluid waste portion.

The waste pad may collect the fluid or at least a portion of the fluid that has passed through or via the assay membrane(s). Therefore, the waste pad functions as a liquid reservoir and it determines the velocity of the flow rate via the assay membrane once the liquid reaches the waste pad. Similarly as the assay membranes, the waste pad may function based on capillary forces such that the liquid is dragged by the capillary forces of the waste pad. The first assay membrane and the second assay membrane and potentially additional assay membranes may share one waste pad, which is simple to assemble and efficient to use. Alternatively, at least one of the assay membranes may be connected and/or contacted to an individual waste pad that is configured to merely collect the fluid from that particular assay membrane and that is not shared among assay membranes. In one case, all provided assay membranes are contacted to their individual waste pads and the waste pads as well as the assay membranes are fluidically isolated, i.e. no liquid is flowing from one waste pad to the other or from one assay membrane to another. In this case, the flow rate may be set to individual values and/or is controlled not only by the assay membrane itself but also by the waste pad. Once the liquid arrives at the waste pad that contacts the assay membrane on the flow end side, it also affects the flow rate through the assay membrane.

The waste pad may be positioned on the test carrier at least partially in the test zone or beyond the test zone, but being in contact and/or being in fluidic contact with the assay membrane(s). The test carrier may comprise a specific waste pad area that is configured to receive one or more waste pads. This waste pad area may be recessed substantially in the shape of one or more waste pads. The waste pad area may have one or more barriers between the waste pads such that liquid flow between the barriers is stopped. The waste pad area may be at least partially comprised by the test zone, specifically the shared recessed assay membrane area or may be connected to the shared recessed assay membrane area and/or neighboring the shared recessed assay membrane area.

The test carrier may already be equipped with the waste pad such that the waste pad is already positioned in and/or on the test carrier. Alternatively, the waste pad may be provided separately to be positioned in and/or on the test carrier by a user for example or by a supplier.

One or more waste pads may, without limitation, have a length of approximately 0,5mm to 100mm, specifically of about 1mm to 70mm and more specifically of about 5 mm to 50 mm. One or more waste pads may, without limitation, have a width of approximately 0,01mm to 50mm, specifically of about 0,5mm to 30mm and more specifically of about 1mm to 20mm. One or more waste pads may, without limitation, have a thickness of approximately 0,05mm to 20mm, specifically of about 0,08mm to 15mm and more specifically of about 0,1mm to 10mm. One or more waste pads may, without limitation, comprise at least one of the following materials: cotton, cellulose, or synthetic fibers like polypropylene. One or more waste pads may, without limitation, be configured to receive a fluid volume of about 0,5pL to 700pL, specifically of about IpL to 500pL.

The IVD test system may further comprise a conjugate pad that may be configured to provide conjugates and separate components from the sample fluid and/or another pad with another function.

The test carrier may comprise a blood plasma separation element, which is integrated with the microfluid sample channel system and which is configured to extract a blood plasma from the sample fluid when the sample fluid comprises whole blood. The microfluid sample channel system may be configured to guide at least one portion of the sample fluid from the sample application port to the blood plasma separation element and at least a portion of the blood plasma from the blood plasma separation element to the sample release port.

The blood plasma separation element may correspond to an element of the microfluid sample channel, which differs in shape from a tube, a channel, and/or a pipe and carries a function of separating the plasma from a whole blood sample. If the test carrier corresponds to a centrifugal test carrier, the blood plasma separation element may be configured to separate the plasma from a whole blood sample under the influence and/or under the control of a centrifugal force. Alternatively or additionally, the blood plasma separation element may correspond to or comprise an element, which is configured to separate the blood plasma from the whole blood when passing the element, such as a plasma separation pad and/or a conjugation pad with a separation function, that comprises a membrane, which holds back components of the whole blood other than plasma. It may be required, specifically for an immunoassay, that a plasma is separated from the whole blood to further process (e.g. label) and analyze the sample.

The blood plasma separation element is integrated with the microfluid sample channel system, which may mean that blood plasma separation element is an element of the microfluid sample channel system and/or that the blood plasma separation element is fluidically connected or connectable with portions of the microfluid sample channel system. In other words, the blood plasma separation element may be connected and/or connectable with a part of the microfluid sample channel system and/or may correspond to a part, a portion and/or a component of the microfluid sample channel system in the form of a chamber, a hollow geometrical structure and/or a channel. The portion of the microfluid sample channel system may passively transport the sample fluid to the blood plasma separation element by capillary forces while the blood plasma separation element may perform its function based on other forces such as centrifugal forces, without limitation. However, it may also be possible that the function of the blood plasma separation element may be performed on the basis of capillary forces, for example if a plasma separation pad and/or a conjugation pad with separation function is involved.

The blood plasma separation element may, without limitation, have a volume of about, 0,5pL to 700pL, specifically of about IpL to 500pL and more specifically of about 5 pl to 300pl. The blood plasma separation element may be configured to extract a volume of the blood plasma from the sample fluid of about 0,5 pL to 400pL, specifically of about 0,8pL to 300pL and more specifically of about IpL to 200pL, without limitation.

The test carrier may further comprise at least one reagent chamber, which is integrated with the microfluid sample channel system for housing at least one reagent that is configured to bind with at least one of the analytes. The at least one reagent chamber may comprise: at least one capture reagent chamber for housing the first capture reagent and/or the second capture reagent. The at least one reagent chamber may already house and/or contain at least one reagent, specifically the first capture reagent and/or the second capture reagent. The microfluid sample channel system may be configured to guide at least one portion of the sample fluid from the sample application port to the at least one reagent chamber and at least a portion of the sample fluid inside the at least one reagent chamber to the release port, for example by means of capillary forces.

At least a portion of the sample fluid may be contacted, mixed and/or incubated with the at least one reagent, such that at least one analyte of interest can be labeled with a capture reagent designed to specifically couple to a binding site of the analyte. The at least one capture reagent chamber may specifically be configured for housing and/or housing more than one capture reagent. For example, the capture reagent chamber may house two capture reagents to respectively label two analytes of interest in the sample fluid. The capture reagent chamber may house even more than two capture reagents, such as three, four, five, six, seven, nine, ten or more capture reagents. The capture reagents may be provided in dried form and may only be solubilized by the sample fluid and/or a portion (a part and/or a component) thereof when being guided through the capture reagent chamber.

If the test carrier is a centrifugal test carrier, the sample fluid may be guided into the at least one capture reagent chamber under control of the centrifugal force, however if the passive transport of the fluid to the at least one capture reagent chamber is based on capillary forces, a centrifugal force may not be required, for example when the sample fluid is transported from the blood plasma separation element to the at least one capture reagent chamber. In more detail, at least a portion of the sample fluid may be guided under control of the centrifugal force via the microfluid sample channel system and potentially via other elements such as the blood plasma separation element to the capture reagent chamber. Further, after at least a portion of the sample fluid may be rested for a while in the capture reagent chamber for incubation, at least a portion of the sample fluid inside the capture reagent chamber may be guided via another portion and/or other elements of the microfluid channel system to the sample release port by means of capillary forces and/or centrifugal forces. The term “integrated with” may also in view of the capture reagent chamber refer to a component of and/or a component connected with at least a portion/part the microfluid channel system, specifically the capture reagent chamber may not have a function of passively transporting a sample fluid by means of capillary forces whereas other elements and/or portions of the microfluid channel system may have a function of passively transporting a sample fluid.

One or more reagent chambers may, without limitation, have a volume of about 0,5pL to 700pL, specifically of about IpL to 500pL. One or more reagent chambers may, without limitation, be configured to receive and/or house reagents with a mass of about Ipg to lOOmg, specifically of about 5pg to 50mg.

The at least one reagent chamber may comprise at least one detection reagent chamber for housing at least one first detection reagent that is configured to bind with the first analyte and/or for housing one second capture reagent that is configured to bind with the second analyte.

At least a portion of the sample fluid may be contacted, mixed and/or incubated with the at least one detection reagent, such that at least one analyte of interest can be labeled with a detection reagent designed to specifically couple to a (further) binding site of the analyte. The at least one detection reagent chamber may specifically be configured for housing and/or housing more than one detection reagent. For example, the detection reagent chamber may house two detection reagents to respectively label two analytes of interest in the sample fluid. The detection reagent chamber may house even more than two detection reagents, such as three, four, five, six, seven, nine, ten or more capture reagents. The detection reagents may be provided in dried form and may only be solubilized by the sample fluid and/or a portion (a part and/or a component) thereof when being guided through the detection reagent chamber.

If the test carrier is a centrifugal test carrier, the sample fluid may be guided into the at least one detection reagent chamber under control of the centrifugal force, however if the passive transport of the fluid to the at least one detection reagent chamber is based on capillary forces, a centrifugal force may not be required, for example when the sample fluid is transported from the capture reagent chamber to the at least one detection reagent chamber. In more detail, at least a portion of the sample fluid may be guided under control of the centrifugal force and/or a capillary force via the microfluid sample channel system and potentially via other elements such as the blood plasma separation element and/or the capture reagent chamber to the detection reagent chamber. Further, after at least a portion of the sample may be rested for a while in the detection reagent chamber for incubation, at least a portion of the sample fluid inside the detection reagent chamber may be guided via another portion and/or other elements of the microfluid channel system to the sample release port by means of capillary forces and/or centrifugal forces. The term “integrated with” may also in view of the detection reagent chamber refer to a component of and/or a component connected with at least a portion/part the microfluid channel system, specifically the detection reagent chamber may not have a function of passively transporting a sample fluid by means of capillary forces whereas other elements and/or portions of the microfluid channel system may have a function of passively transporting a sample fluid by means of capillary forces.

The test carrier may already be equipped with the reagents (capture and/or detection reagent) such that the reagents are already present in and/or on the test carrier. Alternatively, the reagents may be provided to be positioned in and/or on the test carrier by a user for example or by a supplier.

It is an advantage to provide reagent chambers dedicated for the mixing and incubation of the sample fluid with the reagents as the incubation term may be controlled individually in each chamber, for example by a low rotational rate of the centrifuge and/or by stopping the rotation of the centrifuge and/or by resting a portion of the sample fluid inside the reagent chamber. Alternatively or in addition, reagents may be provided in or on a pad such as a conjugation pad, through which the at least a portion of the sample fluid may pass - in that case, the incubation term may be controlled by the flow rate through the pad. A conjugation pad may be provided between portions of the microfluid channel system and/or in the test zone. Further, the reagents may be provided as a dry powder or in a solution. A dry form such as a powder may be stable for a long term.

If only two assay membranes are provided in the test assembly, the first assay membrane and the second assay membrane may each have a width without limitation of approximately 1,5 mm to 2mm, which may be measured in a perpendicular direction to the respective general flow axis, for example parallel to one or more of the lines such as the test line; or if three assay membranes are provided in the test assembly, the first assay membrane, the second assay membrane and the third assay membrane may each have a width without limitation of approximately 1mm to 1,3mm, which may be measured in a perpendicular direction to the respective general flow axis. In a case, in which a single membrane is provided for a testing of a single analyte, the width might range without limitation between approximately 3mm and 4mm. The width of an assay membrane is typically measured perpendicularly to the general flow direction of the assay membrane. The more assay membranes are provided, the smaller the width must be chosen to fit all assay membranes next to each other in the test zone. One or more assay membranes may have a length of approximately 5mm to 25mm, specifically of approximately 10mm to 20mm, and more specifically of approximately 15mm to 18mm. The length of an assay membrane is typically measured along the general flow direction and or perpendicular to a line such as a test line. One or more assay membranes may, without limitation, have a thickness of approximately 0, 1mm to 10mm. One or more assay membranes may comprise at least one of the following materials: cellulose, nitrocellulose, glass fiber, nylon, and/or cellulose-based material.

The test zone may comprise a washing buffer release port and the test carrier may further comprise without limitation: a microfluid washing buffer channel system for guiding and/or passively transporting a washing buffer for example based on capillary and/or centrifugal forces; a washing buffer reservoir-receiving portion for housing and/or receiving a washing buffer reservoir. The test carrier may comprise the washing buffer reservoir being fluidically connected or may be configured to be fluidically connected with the washing buffer release port via the microfluid washing buffer channel system for providing the assay membranes at the respective flow start side with a predetermined amount of washing buffer. A washing buffer reservoir may correspond to a washing buffer blister configured to be fluidically connected with the washing buffer release port via the microfluid washing buffer channel system for providing the assay membranes at the respective flow start side with a predetermined amount of washing buffer, for example under influence/control of the centrifugal force. The fluidic connection may be established by puncturing the washing buffer blister with a sharp element and/or a needle. This may be performed in an automated manner.

The blister may, without limitation, comprise at least one of the following materials: aluminium, PVC (polyvinyl chloride), PET (polyethylene terephthalate), and PVDC (poly vinylidene chloride).

A washing buffer reservoir-receiving portion may correspond to a recess in the test carrier for housing and/or receiving the washing buffer reservoir.

A washing buffer system may allow providing the assay membrane with a washing buffer to remove unspecifically adsorbed and/or weakly bound molecules and/or substances from the assay membrane to achieve a correct test result that provides a high degree of sensitivity and selectivity at the same time.

The test carrier may already be equipped with the washing buffer reservoir such that the washing buffer reservoir and specifically the washing buffer is already present in and/or on the test carrier. Alternatively, the washing buffer reservoir may be provided to be positioned in and/or on the test carrier by a user for example or by a supplier.

The washing buffer reservoir may, without limitation, have a volume of about lOpL to 5mL, specifically of about lOOpL to 3mL. The microfluid washing buffer channel system may, without limitation, have a total path/trace length of about 1mm to 20cm, specifically of about 5mm to 7cm between the opening of the washing buffer reservoir or the starting point of the microfluid washing buffer channel system and the washing buffer release port. The microfluid washing buffer channel system may be configured, without limitation, to receive a volume of about IpL to 5mL, specifically of about 50pL to 2mL. The microfluid washing buffer channel system may comprise, without limitation, a channel portion with a width and/or diameter of about 0,5mm to 75mm. The washing buffer reservoir-receiving portion may, without limitation, have a round, an oval, a polygonal and/or a tapered shape. The washing buffer reservoir-receiving portion may have a diameter or a length and/or a width, without limitation, between about 0,1pm and 2cm. The washing buffer reservoirreceiving portion may have a ring-shaped holding frame. The washing buffer reservoir may, without limitation, have a round, an oval, a polygonal and/or a tapered shape. The washing buffer reservoir may, without limitation, have diameter or a length and/or a width in its cross section between about 0, 1 pm and 2cm, specifically between about 5 pm and 1 cm.

The first analyte and/or the second analyte may comprise without limitation at least one of a cardiac parameter, specifically a cardiac analyte, such as cTropT, NTproBNP, D-Dimer, PCT, BM2, BM3, creatinine, potassium, sodium chloride. Alternatively or in addition, other analytes and/or parameters than the above listed may be probed by the IVD test system. The listed analytes may provide information about the cardiac health of a patient. Specifically, a cardiac infarction/comary thrombosis and/or other cardiac diseases may be identified by one or more of these parameters. The IVD test system allows multiplexing and/or better quantifying one or more of these parameters to provide a medical doctor and/or health care worker to better and more efficiently identify a disease and/or an acute health condition threatening a patient, such as a cardiac disease. As the IVD test system can provide several test results in a short time, the possibility to safe a patient’s life increases tremendously.

The multiplexed diagnostic assay may correspond to an immunochromatographic assay and the at least one first detection reagent and/or the at least one second detection reagent may comprise a fluorescent molecule. Immunochromatographic assays typically provide reliable, sensitive and/or selective test results. An assay membrane may therefore correspond to an immunochromatographic assay membrane

The test carrier may correspond to a centrifugal microfluidic test carrier and at least a section and/or portion of the microfluidic channel system comprising the microfluid sample channel system and the microfluid washing buffer system may be configured to at least partially guide the at least one portion of the sample fluid from the sample application port to the sample release port under influence of a centrifugal force and/or at least a portion of the washing buffer fluid from the blister to the washing buffer release port. By choosing specific rotation frequencies and/or by stopping a rotation, the flow may be controlled by the according centrifugal forces when rotated and/or by the capillary forces when stopping a rotation. Alternatively or in addition, in at least a section and/or portion of the microfluid sample channel system, a capillary force may drive the transport of the sample fluid.

For guiding a portion of a sample fluid through the entire micro fluid sample channel system, a program for predetermined frequencies of rotation may be run/performed. The program may comprise an acceleration of the ration to a certain value for moving the sample fluid, a slowing down for reducing the movement of the sample fluid, a stopping for letting the sample fluid rest in a position (for example for incubation) and/or a repeated acceleration to further move the sample fluid towards the sample release port. The centrifugal microfluidic test carrier is therefore configured to guide the at least one portion of the sample fluid at least partially from the sample application port to the sample release port under influence and/or under control of a centrifugal force.

According to a second aspect of the invention an IVD test apparatus comprises: a centrifuge with a cartridge support plate that is configured to be rotated around a centrifugal axis by the motor of the centrifuge; a controller that is configured to control the motor of the centrifuge and the rotation of the cartridge support plate; and at least one cartridge, which comprises and/or corresponds to the IVD test system comprising the centrifugal microfluidic test carrier, wherein the cartridge is configured to be fixed on the cartridge support plate to be rotated together with the rotating cartridge support plate around the centrifugal axis.

The IVD test apparatus provides all the advantages and technical effects, which are already described for the IVD test system or any according embodiment thereof, which is comprised by the IVD test apparatus. The IVD test system may be provided to the IVD test apparatus in the form of a cartridge. The cartridge may either correspond to the IVD test system or may comprise the IVD test system according to any one of the described embodiments.

The cartridge support plate may be configured to receive two or more IVD test systems in a position off the centrifugal axis which passes the cartridge support plate perpendicularly when it is positioned correctly inside IVD test apparatus, preferably such that the two or more IVD test systems can be positioned symmetrically around the centrifugal axis.

According to a second aspect of the invention a method of performing a multiplexed diagnostic assay comprises the steps of providing the IVD test apparatus as previously described; positioning and/or fixing the at least one cartridge on the cartridge support plate; initiating the transport of the at least one portion of the sample fluid through the microfluid sample channel system to the first assay membrane and the second assay membrane, such that the at least one portion may be transported across the first assay membrane and the second assay membrane; detecting whether the first assay membrane indicates the presence of the first analyte - 1 - and/or whether the second assay membrane indicates the presence of the second analyte.

The method of performing a multiplexed diagnostic assay provides all the advantages and technical effects, which are already described for the IVD test system or any embodiment thereof.

Initiating the transport of the at least one portion of the sample fluid through the microfluid sample channel system may comprise the addition of the sample fluid into or onto the sample application port. This may be followed by a passive transport by means of capillary forces generated by the walls of the microfluid sample channel system. The transport of a portion of the sample fluid may additionally at least in a portion of the microfluid sample channel system be supported by centrifugal forces when rotating the cartridge support plate with the cartridge in the centrifuge around the rotation axis at at least one first predetermined rotation frequency that is configured to guide and/or transport the at least one portion of the sample fluid in at least a section between the sample application port and the sample release port through the microfluid sample channel system to efficiently and/or quickly provide the first assay membrane and the second assay membrane and potentially the third assay membrane with the at least one portion of the sample fluid such that the multiplexed diagnostic assay may be performed at a high efficiency.

Rotating the cartridge support plate with the cartridge in the centrifuge around the rotation axis at at least one first predetermined rotation frequency that is configured to guide, move and/or transport the at least one portion of the sample fluid from the sample application port through the microfluid sample channel system to the sample release port may comprise following a program of accelerating, decelerating and stopping the rotation. For example, the program may comprise at least one of the following method steps:

A step may be comprised by the method that includes starting the rotation by accelerating the cartridge support plate with the cartridge in the centrifuge around the rotation axis at at least one first predetermined rotation frequency oi that is configured to guide the at least one portion of the sample fluid from the sample application port through a first portion of the microfluid sample channel system to the blood plasma separation element. The first predetermined rotation frequency oi may be between around 0 and 100Hz, specifically between around 55Hz and 85Hz, more specifically at around 70Hz. If the transport only relies on the capillary forces, the first predetermined rotation frequency (Bi may equal zero. A step may be comprised by the method that includes rotating the cartridge support plate at at least one second predetermined rotation frequency 02 that is configured to separate/ extract the blood plasma from the whole blood. The second predetermined rotation frequency 0)2 may be between around 50Hz and 115Hz, specifically at around 70Hz to 100Hz, in some embodiments, the second predetermined rotation frequency 02 may exceed 100Hz or even 115Hz.

A step may be comprised by the method that includes rotating the cartridge support plate at at least one third predetermined rotation frequency 03 that is configured to guide, move and/or transport at least one portion of the plasma from the blood plasma separation element via a second portion of the microfluid sample channel system to the capture reagent chamber. The third predetermined rotation frequency 03 may be between around 0 and 100Hz, specifically between around 55Hz and 85Hz, more specifically at around 70Hz. If the transport only relies on the capillary forces, the third predetermined rotation frequency 03 may equal zero.

A step may be comprised by the method that includes stopping the rotation for a predetermined first period - if not already stopped - configured to allow solubilization, mixing and/or incubation of the plasma with the capture reagent inside the capture reagent chamber such that at least one capture reagent may label a predetermined analyte via specifically binding to a first binding site of the predetermined analyte.

A step may be comprised by the method that includes rotating the cartridge support plate at at least one fourth predetermined rotation frequency 04 that is configured to guide, move and/or transport at least one portion of the plasma with the labeled analyte from the capture reagent chamber via a third portion of the microfluid sample channel system to the detection reagent chamber. The fourth predetermined rotation frequency 0)4 may be between around 0 and 100Hz, specifically between around 55Hz and 85Hz, more specifically at around 70Hz. If the transport only relies on the capillary forces, the fourth predetermined rotation frequency 04 may equal zero.

A step may be comprised by the method that includes stopping the rotation for a predetermined second period - if not already stopped - configured to allow solubilization, mixing and/or incubation of the plasma (that has an analyte, which is labeled with the capture reagent) with the capture reagent inside the detection reagent chamber such that at least one detection reagent may label the predetermined analyte via specifically binding to a second binding site of the predetermined analyte. A step may be comprised by the method that includes rotating the cartridge support plate at at least one fifth predetermined rotation frequency 05 that is configured to guide, move and/or transport at least one portion of the plasma with the twice- labeled/sandwi ch-labeled analyte from the detection reagent chamber via a fourth portion of the microfluid sample channel system to the sample release port to provide the first assay membrane and the second assay membrane with at least a portion of the plasma having the sandwich-labeled analyte. The fifth predetermined rotation frequency 05 may be between around 0 and 100Hz, specifically between around 55Hz and 85Hz, more specifically at around 70Hz. If the transport only relies on the capillary forces, the fifth predetermined rotation frequency 05 may equal zero.

A step may be comprised by the method that includes stopping the rotation for a predetermined third period - if not already stopped - configured to allow the multiplexed assay to be performed by letting at least a portion of the plasma having the sandwich-labeled analyte flow along the respective general flow axis of the first and the second assay membranes from the flow start side towards the flow end side and the at least one waste pad.

A step may be comprised by the method that includes puncturing a washing buffer reservoir to establish a fluid connection between the washing buffer reservoir and the microfluid washing buffer channel system.

A step may be comprised by the method that includes rotating the cartridge support plate at at least one sixth predetermined rotation frequency ®6 that is configured to guide, move and/or transport at least one portion of the washing buffer from the washing buffer reservoir via a first portion of the microfluid washing buffer channel system to a washing buffer aliquot element and/or chamber. The sixth predetermined rotation frequency ®6 may be between around 0 and 100Hz, specifically between around 55Hz and 85Hz, more specifically at around 70Hz. If the transport only relies on the capillary forces, the sixth predetermined rotation frequency ®6 may equal zero.

A step may be comprised by the method that includes rotating the cartridge support plate at at least one seventh predetermined rotation frequency 07 that is configured to aliquot/partition at least one predetermined portion of the washing buffer from the total amount of washing buffer in the washing buffer aliquot element and/or chamber. The seventh predetermined rotation frequency 07 may be between around 30Hz and 70Hz, specifically at around 40Hz to 60Hz, such as 50Hz, in some embodiments, the seventh predetermined rotation frequency 07 may exceed 50Hz or even 70Hz. A step may be comprised by the method that includes rotating the cartridge support plate at at least one eighth predetermined rotation frequency os that is configured to to guide, move and/or transport at least one aliquot of the washing buffer from the washing buffer aliquot element and/or chamber via a second portion of the microfluid washing buffer channel system to a washing buffer release port at the sample zone to provide the first assay membrane and the second assay membrane with at least a portion of the at least one aliquot of the washing buffer. The eighth predetermined rotation frequency os may be between around 0 and 100Hz, specifically between around 55Hz and 85Hz, more specifically at around 70Hz. If the transport only relies on the capillary forces, the eighth predetermined rotation frequency co 8 may equal zero.

A step may be comprised by the method that includes stopping the rotation for a predetermined fourth period - if not already stopped - configured to allow a washing step to be performed by letting at least a portion of the at least one aliquot of the washing buffer flow along the respective general flow axis of the first and the second assay membranes from the flow start side towards the flow end side and the at least one waste pad. After finishing this program, the test result may be read from the assay membranes by eye and/or by camera and/or another detection device.

The washing buffer aliquot element may be configured to receive, without limitation, between about 0,05 pL and 150pL, specifically between about IpL and lOOpL. The washing buffer aliquot element may be configured to partition the buffer in volumes, without limitation, of about 0,05 pL and 150pL, specifically between about IpL and lOOpL into to two, three, four or five partitions.

It is self-explanatory that the features relating to labeling and/or detecting the predetermined analyte are realized regardless of whether the actual sample fluid comprises the predetermined analyte of interest. The multiplexed assay is for determining whether or not one or two or even more analytes are present and therefore, also a sample that does not contain the specific one or two or even more analytes may be tested.

Detailed Description of the Invention

In the following, some example embodiments will be described in detail, wherein the invention should not be understood to be limited to the example embodiments described. The following examples and figures are provided to aid the understanding of the present invention, the true scope, of which is set forth in the appended claims. Single features being described in a particular embodiment may be arbitrarily combined, given that they are not excluding each other. In addition, different features, which are provided together in the example embodiments are not to be considered restrictive to the invention.

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 exaggerated relative to other elements whereas other elements may have been left out or represented in a reduced number in order to enhance clarity and improve understanding of the aspects of the present disclosure. The same reference numerals are used among different embodiments and examples for the same or similar elements or elements that have similar or the same effects.

Description of the Figures

Fig. la is a schematic drawing of preparing a sandwich-labeled analyte according to the known art;

Fig. lb is a schematic drawing of detecting a sandwich-labeled analyte on an immunochromatographic assay membrane according to the known art;

Fig- 2 is a schematic drawing in perspective top view of an IVD test system comprising an immunochromatographic assay membrane in a centrifugal microfluidic test carrier according to an example;

Fig. 3a is a schematic perspective top view of the IVD test system comprising the immunochromatographic assay membrane in the centrifugal microfluidic test carrier for detecting an analyte on one assay membrane according to the example of Fig. 2;

Fig. 3b is a schematic perspective top view of an IVD test system comprising two immunochromatographic assay membranes in a centrifugal microfluidic test carrier for multiplexed detecting of analytes according to an embodiment;

Fig- 4 is an exploded perspective top view of an IVD test system comprising three immunochromatographic assay membranes in a centrifugal microfluidic test carrier for multiplexed detecting of analytes according to another embodiment;

Fig. 5a is a schematic perspective top view of the IVD test system according to an embodiment of Fig. 3b;

Fig. 5b is a schematic drawing of a first capture mechanism of a first assay membrane provided as the immunochromatographic assay of Fig. 5a; Fig. 5c is a schematic drawing of a second capture mechanism of a second assay membrane provided as the immunochromatographic assay of Fig. 5a;

Fig. 6a is a schematic drawing in front view of an IVD test apparatus comprising an IVD test system according to an embodiment;

Fig. 6b is a schematic drawing in top view of a cartridge support plate with three cartridges of the IVD test apparatus of Fig. 6a according to an embodiment;

Fig- 7 is a schematic flow diagram of a method of performing a multiplexed diagnostic assay according to an embodiment; and

Fig. 8a-Fig. 8h are schematic drawings of the cutlines A-A’, a-a’ , B-B’ and b-b’ indicated in Fig. 3a and Fig. 3b according to different embodiments.

Fig. la is a schematic drawing describing the method of preparing a sandwich- labeled analyte 31c (analyte labeled with two reagents at two different binding sites) according to the known art and Fig. lb is a schematic drawing describing the method of detecting the sandwich-labeled analyte 31c on an immunochromatographic assay membrane 6 according to the known art. The methods related to Fig. la and Fig. lb are described further above, in the background section.

In general, when using immunochromatography in immunoassays for multiplexing, i.e. for the simultaneous determination of several analytes from the same sample in one measurement run, conventionally, an assay membrane with more than one test line is typically used. Each test line is coated with analyte-specific antibodies for specifically capturing the analyte of interest. The conventional test however comprises assay interferences: Each of the test lines with the corresponding immobilized reagents in the panel format are a source for non-specific interactions with the assay reagents and analytes for the assay reagents of the other analytes, which have to flow along the other test lines. Non-specific interactions of the analytespecific detection antibodies with the other reagents flowing along the assay membrane and with the capture zone reagents are well known to the experts. Therefore, accuracy and signal -to-noise ratios, which may arise from dependencies in the signal recoveries from donor to donor can be improved by the present invention.

Furthermore, the present invention can improve or even optimize the distance between the test lines to reduce and/or prevent cross-talk between the test lines during the signal detection. It is thereby not required to provide longer membranes, which might be needed and requires a new design of the test carrier, offering more space for the membrane. The present invention therefore has the advantage that, test panels for single parameter tests and test panels for multiplexing can be used with the identical platform, hardware, equipment, and/or test carrier. No different cartridge designs are therefore required. No additional designs for the test carrier are required to be involved in the production pipeline, which avoids complexity in material management and production.

It may also not be required to design a test carrier with separated channels (incl. assay membranes) for each of the multiple analytes behind the common blood application zone. Therefore, the size and complexity of the test carrier is not required to vary and/or experience an increase in complexity, which might result in increasing production costs. In addition, it may be avoided that the sample amount required for running the multiplex assay increases.

Therefore, the present invention has the advantage that a platform for single and multiplexed immunochromatography may be provided. The platform may offer flexibility to perform single-testing or multiplex-testing on the same POC platform without requiring major changes in design of the test carrier and instrument as well as the needed sample volume.

The term parameter specifically comprises an analyte. In general, the term parameter may also comprise a property of a sample fluid, a hematocrit (Het) value, the state of coagulation or the like.

Fig- 2 is a schematic drawing of an IVD test system 1c comprising one single immunochromatographic assay membrane 6 in or on a centrifugal microfluidic test carrier 2 according to an example. All elements except for the one single membrane 6 may be adopted in embodiments of the invention, namely in the IVD test systems la and lb of Fig. 3b, Fig. 4 and Fig. 5a. In other words, the elements of the centrifugal microfluidic test carrier 2 as well as the waste pad 9 may be used identical to such elements and components of embodiments of the invention. Again, in other words, the IVD test system 1c, which is shown in Fig. 2 may be transformed into an IVD test system la, lb according to one of the embodiments of the invention by replacing the single assay membrane 6 by two or three or even more assay membranes 6, 7, 8, which are narrower than the single assay membrane shown in Fig- 2 Therefore, all features described for the example of Fig. 2 and not being directly related to the assay membrane 6 may be adopted to realize embodiments of the invention. The IVD test system la of Fig. 3b is for multiplexing and provides two assay membranes 6, 7. The IVD test system lb of Fig. 4 is for multiplexing and provides three assay membranes 6, 7, 8. The assay membrane 6 may be positioned, specifically loosely positioned, clamped, stuck, glued and/or fixed on the centrifugal microfluidic test carrier 2. The centrifugal microfluidic test carrier 2 is shown without one or two cover plates 2a, 2b and without a washing buffer reservoir 13 (shown in Fig. 4 for example). In Fig. 4, which refers to an embodiment of the invention for multiplexing based on three assay membranes 6, 7, 8, a center/core piece 2c is shown together with a first cover plate 2a and a second cover plate 2b wherein the first cover plate 2a is configured and/or sized and/or fit to cover a first side SI of the center/core piece 2c and the second cover plate 2b is configured and/or sized and/or fit to cover a second side S2 of the center/core piece 2c. The center/core piece 2c is therefore sandwiched by the first and the second cover plate 2a, 2b. It is conceivable that the center/core piece 2c is only covered by one single cover plate 2a or 2b and therefore, the center/core piece 2c may be considered an upper or a lower test carrier piece. In Fig. 2, only the center piece and/or the core piece 2c of the test carrier 2, which may correspond to an injection- molded element, is shown for simplicity. The microfluidic test carrier 2 (see Fig. 4) may therefore comprise the center/core piece 2c and one or both of the first cover plate 2a and the second cover plate 2b.

The microfluidic test carrier 2 may be considered together with the cover plate(s) 2a, 2b, the assay membrane 6, the waste pad 9 and the washing buffer reservoir 13 (not shown here) a functional cartridge 24 that can be used in an IVD test apparatus 20 as schematically displayed in Fig. 6a.

The functional elements of the IVD test system 1c, specifically the micro fluidic test carrier 2 are described in the following being explicitly applicable to embodiments of the invention. A sample application port 3 is provided on at least one element of the microfluidic test carrier 2. The sample application port 3 may be considered a starting point and/or entrance into the microfluid sample channel system 5 for the sample fluid 30 (not show here), which may, for example, be whole blood in the present case. The microfluidic test carrier 2 may be specifically suited to test analytes, which may be present in whole blood samples. However, it may be conceivable that another type of sample fluid is supposed to be tested and a test carrier may be suited to test anayltes, which may be present in such sample fluids like urine or saliva or other samples. A cover plate 2a, 2b might have a recess, a cutout and/or a hole at the position of the sample application port 3 to provide an access and/or an entry to a microfluid sample channel system 5. A first portion of the microfluid sample channel system 5 connects the sample application port 3 with a blood plasma separation element 10. In fact, the sample application port 3 and the blood plasma separation element 10 may be considered sections, portions and/or elements of the microfluid sample channel system 5 and the first portion of the microfluid sample channel system 5 connecting the sample application port 3 with a blood plasma separation element 10 may be considered a first connecting portion of the micro fluid sample channel system 5.

A second portion of the microfluid sample channel system 5 connects the blood plasma separation element 10 with a first reagent chamber I la. A third portion of the micro fluid sample channel system 5 connects the first reagent chamber I la with a second reagent chamber 11b. In fact, the reagent chambers I la, 11b may be considered sections, portions and/or elements of the microfluid sample channel system 5 and the said second and third portion of the microfluid sample channel system 5 may be considered a second and third connecting portion of the microfluid sample channel system 5. The first reagent chamber I la may house or may be configured to house at least one capture reagent 33 (capture antibody) and the second reagent chamber 11b may house or may be configured to house at least one detection reagent 32 (detection antibody). Alternatively, the first reagent chamber I la may house or may be configured to house at least one detection reagent 32 (detection antibody) and the second reagent chamber 1 lb may house or may be configured to house at least one capture reagent 33 (capture antibody). Alternatively, the first and the second reagent chambers I la, 11b may only house or may be configured to house one or more, for example two, three, four, five, six or more detection reagents 32 (detection antibody) or one or more, for example two, three, four, five, six or more capture reagents 32 (capture antibody). It may even be conceivable, that one or both reagent chamber(s) house(s) a capture and a detection reagent 32, 33 at the same time. In the case of a multiplexed test, one reagent chamber may house or may be configured to house several reagents 32, 33, which each bind to the respective binding sites of several analytes 31.

A fourth portion of the microfluid sample channel system 5 connects the second reagent chamber 1 lb with a sample release port 4b in the test zone 4 where the (processed) sample fluid 30 may be released to be provided to a flow start side 6b of the assay membrane 6, which is positioned in the shared recessed assay membrane area 4a. The assay membrane 6 is contacted at a flow end side 6c to a waste pad 9 that is positioned in a waste pad area 4d that neighbors the shared recessed assay membrane area 4a in the test zone 4. The assay membrane 6 may be positioned, specifically loosely positioned, clamped, glued and/or fixed to the waste pad 9 and may overlap the waste pad 9. Alternatively, the assay membrane 6 contacts the waste pad 9 without overlapping. The sample fluid 30 may then be passively transported along the general flow axis 6a of the assay membrane 6 to the waste pad 9. In other words, the sample fluid may flow along the general flow axis 6a.

A processed sample fluid 30 may be considered a sample fluid 30 that has been treated by at least one of the following steps, without limitation: aliquoting/partitioning, separating from other elements and/or parts of the applied sample, labelling an analyte in the sample fluid with a reagent, specifically a detection reagent and/or a capture reagent, diluting the sample fluid, purifying the sample fluid, purifying and/or enriching one or more analytes, solubilizing analytes and/or portions and/or fragments of the sample fluid 30.

On the other side of the test carrier 2, which is the side neighboring the side where the microfluid sample channel system 5 is located on the test carrier 2, a washing buffer reservoir-receiving portion 14 or a receiving portion/recess for receiving a washing buffer reservoir/blister 13 (not shown, see Fig. 4) is provided. The washing buffer reservoir-receiving portion 14 may correspond to a recess, a cut-out and/or a hole. A microfluid washing buffer channel system 12 may transport and/or guide the washing buffer from the blister 13 in an opened state towards the washing buffer release port 4c in the test zone 4. The blister 13 may be punctured by a needle or a sharp element to establish the opened state and a fluidic connection between the inside of the blister 13 and the microfluid washing buffer channel system 12. A first portion of the microfluid washing buffer channel system 12 connects the blister 13 with the washing buffer aliquot chamber 15, wherein the first portion of the microfluid washing buffer channel system 12 may be considered a first connection portion of the washing buffer aliquot chamber 15 and the washing buffer aliquot chamber 15 may be considered an element of the microfluid washing buffer channel system 12. The washing buffer aliquot chamber 15 has the function of partitioning/aliquoting the washing buffer into portions/aliquots, i.e. partitioned volumes of washing buffer, upon rotating the microfluidic test carrier 2. The partitioned washing buffer is further transported through the microfluid washing buffer channel system 12 towards the washing buffer release port 4c in the test zone 4. The entire volume of washing buffer of the blister 13 may be provided to the washing buffer release port 4c in several portions, for example in two, three, four, five, six or even more portions/aliquots.

The microfluidic test carriers 2 shown in Fig. 2, 3a, 3b, 4 and 5a respectively have a first side SI and a second S2 opposite the first side SI. The Fig. 2, 3a, 3b and 5a correspond to a top view onto the respective second side S2 of IVD test systems la, 1c. The Fig. 4 corresponds to a perspective top view onto the first side SI of the IVD test system lb.

As can be seen in Fig. 2, 3a, 3b and 5a, the mentioned functional elements of the respective micro fluidic test carrier 2 of the IVD test systems la, lb, 1c in most cases correspond to recessed elements and/or traces of the respective test carrier 2, specifically the test carrier core piece 2c. In these embodiments, the recessed trace is a trace in the surface of the second side S2 of the microfluidic test carrier 2 and a second cover plate 2b (not shown, see Fig. 4) may be attached and/or fixed to the test carrier core piece 2c to cover the second side S2 of the respective IVD test system la, lb, 1c. The second cover plate 2b may therefore at least partially seal and/or cover and/or enclose the microfluid sample channel system 5. A first cover plate SI may cover the opposite side, i.e. the first side of the microfluidic test carrier 2. The microfluid sample channel system 5 may therefore correspond to or comprise the recessed trace in the test carrier core piece 2c on the second side and the microfluid sample channel system 5 may substantially be defined an/or enclosed by the recessed surface of the second side S2 of the test carrier core piece 2c and the second cover plate 2b. There may or may not be an access to the microfluid sample channel system 5 from the first side SI potentially via a hole, cut-out and/or recess in the first cover plate la and/or the carrier support center piece 2c (e.g. sample application port 3). Alternatively or in addition, an access to the microfluid sample channel system 5 may be provided from the second side S2 potentially via a hole, cut-out and/or recess in the second cover plate 2b (e.g. sample application port 3). Alternatively or in addition, the microfluid sample channel system 5 may correspond to or comprise a recessed trace in one or both of the cover plates 2a, 2b.

The sample application port 3 may correspond to a hole in one of the cover plates 2a, 2b, i.e. the sample application port 3 may correspond to a hole in the first cover plate 2a or in the second cover plate 2b. The test carrier core piece 2c may have a starting point of the microfluid sample channel system 5 at the position that corresponds to the position of the sample application port 3. In other words, when the respective cover plate 2a, 2b is correctly fixed to the test carrier core piece 2c, the sample application port 3 is positioned right over or under the starting point of the micro fluid sample channel system 5.

The test zone 4 may correspond to a recess in the test carrier core piece 2c. The test zone 4 may specifically correspond to a recess in the test carrier core piece 2c with different depths. For example, the recess of the test zone 4 in the region of the sample release port 4b and in the region of the washing buffer release port 4c may be somewhat deeper than the recess of the test zone 4 in the shared recessed assay membrane area 4a and/or in the waste pad area 4d. Further, the recess in the shared recessed assay membrane area 4a may have a different depth than the recess in the waste pad area 4d. For example, the recess in the shared recessed assay membrane area 4a may have a different depth than the recess in the waste pad area 4d as the waste pad 9 and the assay membrane(s) 6, 7 may have different thicknesses and/or may overlap in some regions. The recess in the test zone 4 may specifically be dimensioned to receive a waste pad 9 and one or more assay membranes 6, 7. The depth of the recesses may respectively match the thickness of the assay membrane(s) 6, 7, 8 and/or the waste pad 9 and/or an overlap of the assay membrane(s) 6, 7, 8 with the waste pad 9.

Fig. 3a is a schematic perspective top view of the IVD test system la according to the example of Fig. 2. Fig. 3b is a schematic perspective top view of an IVD test system la for multiplexing comprising two immunochromatographic assay membranes 6, 7 in the centrifugal microfluidic test carrier 2 of Fig. 2. Fig. 3a and Fig. 3b are shown next to each other to demonstrate that the identical centrifugal microfluidic test carrier 2 may be used for detecting a single analyte using one assay membrane 6 (Fig. 3a) and for multiplexing using two assay membranes 6, 7 (Fig. 3b). In other words, the centrifugal microfluidic test carriers 2 for use with one single assay membrane 6 (Fig. 3a) and for multiplexing using two assay membranes 6, 7 (Fig. 3b) are identical. The IVD test system la of Fig. 3b is configured for multiplexed detecting of analytes 31a, 31b on two assay membranes 6, 7 according to an embodiment. The same centrifugal microfluidic test carrier 2 may even be used for multiplexing using three, four, five, six or more assay membranes if they are chosen to have a smaller width than one or two assay membranes.

The first assay membrane 6 of the IVD test system la for multiplexing has a general flow axis 6a, a flow start side 6b and a flow end side 6c. The first assay membrane 6 is contacted with the waste pad 9 at its flow end side 6c. The second assay membrane 7 of the IVD test system la for multiplexing has a general flow axis 7a, a flow start side 7b and a flow end side 7c. The second assay membrane 7 is contacted with the waste pad 9 at its flow end side 7c. The first assay membrane 6 and the second assay membrane 7 are aligned next to each other, specifically parallel to each other with respect to the general flow axis 6a and 7a such that the flow start side 6b of the first assay membrane 6 and the flow start side 7b of the second assay membrane 7 are positioned next to each other and the flow end side 6c of the first assay membrane 6 and the flow end side 7c of the second assay membrane 7 are positioned next to each other. Both assay membranes 6, 7 share a single waste pad 9. The assay membranes 6, 7 are separated by a gap 16. The gap 16 may be an air gap and/or a gap that is not filled with a membrane material while the two assay membranes 6, 7 may be positioned and/or fixed on a substrate. However, it is not required in all embodiments that the two assay membranes 6, 7 are on a substrate, specifically a shared substrate. The two assay membranes 6, 7 may for example be fixed to and/or supported by one or more waste pads 9.

Different cuts through the test carrier core piece 2c of test carriers according to different embodiments shown in Fig. 3a and Fig. 3b are shown in Fig. 8a-h.

In Fig. 3a, a schematic cutline a-a’ along the length axis of the assay membrane 6 is indicated. Further, a cutline A-A’ across, i.e. perpendicular to the cutline a-a’ and the length axis of the assay membrane 6 is indicated.

Fig. 8a is a schematic drawing that shows the cut along the cutline A-A’ according to an embodiment. The assay membrane 6 is supported by the bottom/supporting surface 39 of the recessed assay membrane area 4a. The recessed assay membrane area 4a is confined by recess walls 38 of the shared recessed assay membrane area 4a indicated on both sides. In other words, the assay membrane 6 is received by and/or embedded into the recessed assay membrane area 4a. The assay membrane 6 may be only positioned in the recessed assay membrane area 4a and/or clamped into the recessed assay membrane area 4a and/or adhesively attached onto the bottom/supporting surface 39 of the recessed assay membrane area 4a.

Fig. 8b, Fig. 8c and Fig. 8d are schematic drawings that show the cut along the cutline a-a’ according to different embodiments. Fig. 8b shows the assay membrane 6 and the waste pad 9 being positioned in the test zone 4 and being flush with each other. The bottom/supporting surface 39 of the test zone 4 specifically the recessed assay membrane area 4a is a plane flat surface/flat plane with no step(s). In another embodiment, the assay membrane 6 and the waste pad 9 may overlap with each other and/or may be fluidically connected with each other via another pad (not shown). Compared to Fig. 8b, Fig. 8c and Fig. 8d show steps in the bottom/supporting surface 39 of the test zone 4 where the level of the bottom/supporting surface 39 supporting the waste pad 9 is lower than the bottom/supporting surface 39 supporting the assay membrane 6 in Fig. 8c and where the level of the bottom/supporting surface 39 supporting the assay membrane 6 is lower than the bottom/supporting surface 39 supporting the waste pad 9 in Fig. 8d. In both cases, in which steps are provided between the area where the assay membrane 6 is mainly supported and the area where the waste pad 9 is mainly supported, preferably the waste pad 9 and the assay membrane 6 are overlaid with each other to provide a reliable fluidic connection between them. The step may have a depth of 0,l-3mm, specifically 0,5-2mm.

Fig. 8e and Fig. 8f are schematic drawings that show the cut along the cutline B-B’ according to different embodiments. Fig. 8e is a schematic drawing that shows the cut along the cutline B-B’ according to an embodiment. The assay membranes 6, 7 are contacted with each other, having a distance of 0mm between them. The assay membranes 6, 7 are supported by the bottom/ supporting surface 39 of the recessed assay membrane area 4a. The recessed assay membrane area 4a is confined by recess walls 38 of the shared recessed assay membrane area 4a indicated on both sides. In other words, the assay membranes 6, 7 are received by and/or embedded into the recessed assay membrane area 4a. The assay membranes 6, 7 may be only positioned in the recessed assay membrane area 4a and/or clamped into the recessed assay membrane area 4a and/or adhesively attached onto the bottom/supporting surface 39 of the recessed assay membrane area 4a. Fig. 8f is a schematic drawing that shows the cut along the cutline B-B’ according to an embodiment. Compared to Fig. 8e, the assay membranes 6, 7 in this embodiment are not directly contacted with each other and/or have a gap between them with a distance of d>0mm between them. The bottom/supporting surface 39 of the recessed assay membrane area 4a is a plane flat surface/flat plane with no step(s) such that there is no barrier between the first assay membrane 6 and the second assay membrane 7. In general, in all shown embodiments the bottom/supporting surface 39 of the recessed assay membrane area 4a may be a plane flat surface/flat plane with no step(s) such that there is no barrier between the two assay membranes 6, 7.

Fig. 8g and Fig. 8h are schematic drawings that show the cut along the cutline b-b’ according to different embodiments. The embodiment of Fig. 8g corresponds to the embodiment of Fig. 8b, except for having two assay membranes 6, 7 instead of only one single assay membrane 6. The embodiment of Fig. 8h corresponds to the embodiment of Fig. 8d, except for having two assay membranes 6, 7 instead of only one single assay membrane 6. Preferable, the shared recessed assay membrane area 4a, specifically shown in Fig. 8e-8h are plane surfaces with no wall and/or protrusion between two assay membranes 6, 7. If however, a protrusion should be provided between two assay membranes 6, 7 or in general between at least two of multiple assay membranes (for example to better align and/or to support alignment of an assay membrane 6, 7 in the test zone), the protrusion may not have the same height as the assay membrane, specifically have a lower height and/or may not have the same length as the assay membrane 6, 7 specifically be shorter than the assay membrane 6, 7. Specifically, the recessed assay membrane area 4a should not correspond to a substantially bifurcated pathway for a fluid before arriving at the assay membranes 6, 7 but should correspond to a commo n/shared reservoir from which fluids may be received by the assay membranes 6, 7 (specifically substantially at the same time).

The width w of the (shared) recessed assay membrane area 4a may range between approx. 0,1cm and 3 cm, specifically between approx. 0,5 cm and 2cm and more specifically between approx. 0,8cm and 1,5 cm. The edge-to-edge distance d between two neighboring assay membranes may range between approx. 0 and 1cm, specifically between approx. 0,1 and 5mm and more specifically between approx. 0,3 and 1mm. The width w of the (shared) recessed assay membrane area 4a may fit the width of one assay membrane 6 or - if multiple assay membranes are provided - the combined widths of the provided assay membranes 6, 7. Specifically, the width w of the (shared) recessed assay membrane area 4a may correspond to a value between approx. 1 to 2, specifically approx. 1,1 to 1,7 and more specifically approx. 1,2 to 1,5 times the width of one assay membrane 6, or - if multiple assay membranes are provided - times the combined widths of the provided assay membranes 6, 7.

The length 1 of the (shared) recessed assay membrane area 4a including the area that receives the waste pad 9 may range between approx. 1cm and 7cm, specifically between approx. 2cm and 5cm and more specifically between approx. 2,5cm and 3 cm. The length 1 of the (shared) recessed assay membrane area 4a including the area that receives the waste pad 9 may fit the length of the assay membrane(s) 6, and the length of the waste pad 9. Specifically, The length 1 of the (shared) recessed assay membrane area 4a including the area that receives the waste pad 9 may correspond to a value between approx. 1 to 1,5, specifically approx. 1 to 1,3 and more specifically approx. 1 to 1,2 times the lengths of one of the provided assay membrane(s) 6, 7 combined with the waste pad 9. The length h of the (shared) recessed assay membrane area 4a excluding the length h of the area that receives the waste pad 9 may range between approx. 0,5cm and 4cm, specifically between approx. 1cm and 3cm and more specifically between approx. 1,5cm and 2 cm. The length h of the area that receives the waste pad 9 may range between approx. 0,2cm and 3cm, specifically between approx. 0,5cm and 2cm and more specifically between approx. 0,8cm and l,2cm.Fig. 4 is an exploded perspective top view of an IVD test system lb comprising three immunochromatographic assay membranes in a centrifugal microfluidic test carrier 2 for multiplexed detecting of analytes according to another embodiment. The test carrier core piece 2c of Fig. 4 is shown from the opposite side (top view from first side S2) than Fig. 2, Fig. 3a and Fig. 3b (top view from second side S2). The test carrier 2 may have all the features and advantages described in conjunction with Fig. 2, Fig. 3a and Fig. 3b and vice versa. The IVD test system lb of Fig. 4 comprises a test carrier 2 having a test carrier core piece 2c, which may be produced by injection molding, having a first cover plate 2a for at least partially covering the first side SI of the test carrier core piece 2c and having a second cover plate 2b for at least partially covering the second side S2 of the test carrier core piece 2c. The test carrier core piece 2c is sandwiched between the first cover plate 2a and the second cover plate 2b. This configuration with first and second cover plates 2a, 2b may also apply to the embodiments shown in Fig. 2, Fig. 3a and Fig. 3b. Alternatively, it may be conceivable that the test carrier core piece 2c is only covered on the first side SI by the first cover plate 2a and a second cover plate 2b is not required. The first cover plate 2a may act as a top cover for placing a label thereon with information and/or specifications regarding the test type, the sample type, the sample amount, the brand, the charge number, the model number, design elements, bar codes, patient and/or sample information and/or identification notes etc. The second cover plate 2b may act as a sealing lid. The first cover plate 2a comprises cutouts and/or holes to provide access to the blister 13, the microfluid sample channel system 5 and/or the assay membranes 6, 7, 8.

The IVD test system lb of Fig. 4 further comprises a first assay membrane 6, which may be configured to detect at least one first analyte, a second assay membrane 7 which may be configured to detect at least one second analyte and a third assay, membrane 8, which may be configured to detect at least one third analyte. Each assay membrane 6, 7, 8 may be configured to detect an individual analyte. In other words, the first analyte, the second anaylte and the third anaylte may be different analytes. Alternatively, at least one of the assay membranes may be configured to detect a same anaylte as another assay membrane but in in a different sensitivity range.

The IVD test system lb of Fig. 4 further comprises a shared waste pad 9 and a blister/ washing buffer reservoir 13 for storing the washing buffer. The IVD test system lb as shown in Fig. 4 may be considered a cartridge, which may be used in an IVD test apparatus 20 as for example shown in Fig. 6a.

The test carrier 2 may be dimensioned on the longest side between approximately 3cm to 12cm, specifically between approximately 4cm to 10cm and more preferably between approximately 5cm to 7cm. The test carrier 2 may be dimensioned on the shorter side, which is measured perpendicularly to the longest side and possibly parallel to the general flow axis of the assay membranes 6, 7, 8 when inserted into/onto the cartridge between approximately 2,5cm to 10cm, specifically between approximately 3 cm to 7cm and more preferably between approximately 4cm to 6cm. The test carrier 2 (with both cover plates 2a, 2b) may have a thickness of approximately 0, 1cm to 1cm, preferably of approximately 0,3 cm to 0,5 cm.

If only two assay membranes 6, 7 are provided in the test assembly la as shown for example in Fig. 3b, the first assay membrane 6 and the second assay membrane 7 may each have a width 6d, 7d of approximately 1,3mm to 2,3mm, specifically of approximately 1,5 mm to 2mm measured in a perpendicular direction to the respective general flow axis 6a, 7a.

If three assay membranes 6, 7, 8 are provided in the test assembly lb as shown for example in Fig. 4, the first assay membrane 6, the second assay membrane 7 and the third assay membrane 8 may each have a width 6d, 7d, 8d of approximately 0,8mm to 1,5mm, specifically of approximately 1mm to 1,3mm measured in a perpendicular direction to the respective general flow axis 6a, 7a, 8a.

Fig. 5a is a schematic perspective top view of the IVD test system la according to an embodiment of Fig. 3b. Fig. 5b is a schematic drawing of a first capture mechanism of a first assay membrane 6 provided as the immunochromatographic assay of Fig. 5a. Fig. 5c is a schematic drawing of a second capture mechanism of a second assay membrane 7 provided as the immunochromatographic assay of Fig. 5a. Together with Fig. 5a-c, the multiplexed assay is described in more detail, as follows.

The first analyte 31a that may be detected on the first assay membrane 6 in this embodiment may correspond to cardiac Troponin T (TnT). The second analyte 31b that may be detected on the second assay membrane 7 in this embodiment may correspond to NTproBNP. The first reagent chamber I la houses a first capture reagent 33a to bind to a binding site of TNT and a first capture mechanism 34a provided on the first assay membrane 6 and a second capture reagent 33b to bind to a binding site of the NTproBNP and a second capture mechanism 34b provided on the second assay membrane 7. The first capture reagent 33a may in the present embodiment correspond to a dry capture Ab against the first anaylte 31a, e.g. biotinylated, such as MAB-M-11.7-F(ab’)2-Bi and the first capture mechanism 34a may correspond to streptavidin. The second capture reagent 33b may in the present embodiment correspond to a dry capture Ab against the second analyte 31b, e.g. digoxigeninated, such as MAB-S-1.21.3-F(ab’)2-Dig and the second capture mechanism 34b may correspond to anti-dig. The second reagent chamber 1 lb houses a first detection reagent 32a to bind to another binding site of TNT and a second detection reagent 32b to bind to another binding site of the NTproBNP. The first detection reagent 32a may in the present embodiment correspond to a dry detection Ab against the first analyte 31a, e.g. a fluorescent, such as MAB-M-17.57.511-IgG- JG9-Lx. The second detection reagent 32b may in the present embodiment correspond to a dry detection Ab against the second analyte 31b, e.g. a fluorescent, such as MAB-M-18.4.34-IgG-JG9-Lx.

In Fig. 5b, the first assay membrane 6 for detecting the first analyte 31a is shown and in Fig. 5c, the second assay membrane 7 for detecting the second analyte 31b is shown. Both assay membranes 6, 7 are similar to the example shown in Fig. la and Fig. lb illustrating the concept of a known POC test. A whole blood sample may represent a sample fluid 30 in the present case, being received from the patient. The sample fluid 30 contains in the present case the first analyte 31a and the second analyte 31b to demonstrate the principle. Of course, also sample fluids 30 may be tested, which do not contain one or more analytes 31 of interest. In that case, the test result for the specific analyte 31 of interest is negative.

By centrifugation, plasma 30a is extracted from the whole blood sample 30 in and/or by the blood plasma separation element 11. In the present case, the plasma 30a contains the first analyte 31a and the second analyte 31b. Either, the centrifuge is started to rotate at a frequency that is suitable to move the plasma 30a from the blood plasma separation element 11 to the capture reagent chamber I la or the transportation process solely rely on capillary forces of the microfluid sample channel system 5. The plasma 30a is mixed and incubated with the dried first and the second capture reagents 33a, 33b for a certain predetermined amount of time, in which the centrifuge does not rotate or is slowed down or stopped. The plasma 30a solubilizes the capture reagents 33a, 33b, which then respectively couple to the binding sites of the first and the second analytes 31a, 31b and therefore label the analyte 31a, 31b for the binding process, which will take place later. Either, the centrifuge is started again to rotate at a frequency that is suitable to move the plasma 30a from the capture reagent chamber I la to the detection reagent chamber 1 lb or the transportation process solely relies on capillary forces of the microfluid sample channel system 5. The plasma 30a solubilizes the detection reagents 32a, 32b, which then respectively couple to the other binding sites of the first and the second analytes 31a, 31b and therefore label the analyte 31a, 31b for detection, which will be performed later.

The assay membranes 6, 7 each have a general flow axis 6a, 7a, a flow start side 6b, 7b and a flow end side 6c, 7c. The assay membranes 6, 7 may in some cases be contacted with one or more waste pads 9 at the flow end sides 6c, 7c. Fig. 5b and Fig. 5c respectively show the assay membranes 6, 7 after the serum 30a was applied and after the labeled analytes 31a, 31b, 31c are bound on and/or by the assay membranes 6, 7.

The plasma 30a with the labeled analytes 31a, 31b, 31c is provided to the assay membranes 6, 7 at the respective flow start sides 6b, 7b via the sample release port 4b. At least a portion, specifically a part and/or a component of the plasma 30a is passively transported by capillary forces substantially along the general flow axis 6a, 7a of each assay membrane 6, 7 from the flow start sides 6b, 7b to the flow end sides 6c, 7c. The assay membranes 6, 7 each comprise three lines 35, 36, 37, a control line 35, a calibration line 36 and a capture and test result line 37. The control line 35 (assay control) of the first membrane 6 is pre-coated with the first analyte 31a of interest and the control line 35 of the second membrane 7 is pre-coated with the second analyte 3 lb of interest. The calibration line 36 (instrument calibration) of the first membrane 6 is pre-coated with the fluorescent label comprised in the first detection reagent 32a (detection antibody) and the calibration line 36 (instrument calibration) of the second membrane 7 is pre-coated with the fluorescent label comprised in the second detection reagent 32b. The capture and test result line 37 of the first membrane 6 is coated with the first capture mechanism 34a (streptavidin). The capture and test result line 37 of the second membrane 7 is coated with the second capture mechanism 34b (anti-dig).

The serum 30a is applied to the assay membranes 6, 7 on the respective flow start sides 6b, 7b and therefore firstly passes the capture and test result lines 37 where the analytes 31a, 31b respectively sandwiched between the two reagents 31a, 31b, 32a, 32b are specifically bound/captured via the capture antibodies 33a, 33b that bind to the first and second capture mechanisms 34a, 34b if the labelled analytes 31a, 31b are present in the serum 30a. The residual portion of the serum 30a passes the calibration lines 36 where no further binding process takes place and then it passes the control lines 35 where residual unbound detection reagent 32a, 32b comprising the detection antibody is at least partially specifically bound via the binding site of the analytes 3 la, 3 lb provided on the pre-coated control lines 35.

The control line 35 provides a control of whether or not the serum 31 was mixed with the reagents 32a, 32b by capturing the unbound detection reagents 32a, 32b. The control line 35 is specifically significant if the test result is negative, since in that case, the result might be negative due to the absence of the analytes 31 a, 3 lb but it may also be negative (specifically false-negative) due to the absence of the fluorescent labels 32a, 32b. The calibration lines 36 serve for calibration purposes and provide a pre-coated area with the fluorescent labels used in combination with the detection antibodies 32a, 32b to label the analytes 31a, 31b for detection.

Finally, the assay membranes 6, 7 are washed with a buffer solution provided via the washing buffer release port 4c to remove unbound reagents 3 la, 3 lb, 32a, 32b and a CCD image may be recorded to read out the test result(s). A test result of the assay membranes 6, 7 schematically shown in Fig. 5b and Fig. 5c would be positive for TnT and NTproBNP.

Fig. 6a is a schematic drawing of an IVD test apparatus 20 comprising an IVD test system la, lb according to an embodiment. The IVD test apparatus 20 comprises a centrifuge 21 with a cartridge support plate 23 that is configured to be rotated around a centrifugal axis 25 by a motor of the centrifuge 21. The IVD test apparatus 20 further comprises a controller 22 that is configured to control the motor of the centrifuge 21 and the rotation of the cartridge support plate 23. The IVD test apparatus 20 comprises at least one cartridge 24, which comprises the IVD test system la and/or corresponds to the IVD test system la according to an embodiment as described herein, wherein the cartridge 24 is configured to be fixed on the cartridge support plate 23 to be rotated together with the rotating cartridge support plate 23 around the centrifugal axis 25. Preferably, several cartridges 24, such as two, three, four, five or even more cartridges 24 may be fixed on the cartridge support plate 23. If each cartridge 24 provides a different multiplexed test for detecting two different analytes, six analytes may be detected if three cartridges 24 are provided. Alternatively, the multiplexed tests may be identical and the cartridges 24 may be loaded with samples from different patients.

Fig. 6b is a schematic drawing in top view of a cartridge support plate 23 with three cartridges 24 of the IVD test apparatus 20 of Fig. 6a according to an embodiment. As can be seen from above, the cartridges 24 are equally distributed around the centrifugal axis 25.

Fig- 7 is a schematic drawing of a method 100 of performing a multiplexed diagnostic assay according to an embodiment. The microfluidic cartridge thereby allows for control of each of the single assay steps, thus yielding excellent and lablike assay performance like high sensitivity, precision and accuracy.

The method 100 of performing the multiplexed diagnostic assay may be performed using an IVD test system la for performing a multiplexed diagnostic assay as described according to one of the embodiments. The method 100 may comprise the steps of providing 101 an IVD test apparatus 20 as described together with Fig. 7. The IVD test apparatus 20 may comprise: a centrifuge 21 with a cartridge support plate that is configured to be rotated around a centrifugal axis 25 by a motor of the centrifuge 21; a controller 22 that may be configured to control the motor of the centrifuge 21 and the rotation of the cartridge support plate 23; at least one cartridge 24, which comprises the IVD test system la and/or corresponds to the IVD test system la according to an embodiment as described herein, wherein the cartridge 24 is configured to be fixed on the cartridge support plate 23 to be rotated together with the rotating cartridge support plate 23 around the centrifugal axis 25.

The method 100 may further comprise positioning 102 and/or fixing the at least one cartridge 24 on the cartridge support plate 23; rotating 103 the cartridge support plate 23 with the cartridge 24 in the centrifuge 21 around the rotation axis 25 at at least one first predetermined rotation frequency that is configured to guide the at least one portion 30, 31 of the sample fluid 30 at least partially from the sample application port 3 through the microfluid sample channel system 5 to the sample release port 4b to provide 105 the first assay membrane 6 and the second assay membrane 7 with the at least one portion 30, 31 of the sample fluid 30 such that the multiplexed diagnostic assay 104 may be performed; detecting 105 whether the first assay membrane indicates the presence of the first analyte and/or whether the second assay membrane indicates the presence of the second analyte. The rotation program (order and frequencies) may be optimized to the cartridge design such that each function is precisely performed/carried out based on the design of the elements of the micro fluid sample channel system 5.

The method may in some embodiments comprise at least one of the following steps: Specifically, the above step “rotating 103 the cartridge support plate 23 with the cartridge 24 in the centrifuge 21 around the rotation axis 25 at at least one first predetermined rotation frequency that is configured to guide the at least one portion 30, 31 of the sample fluid 30 from the sample application port 3 through the microfluid sample channel system 5 to the sample release port 4b to provide 105 the first assay membrane 6 and the second assay membrane 7 with the at least one portion 30, 31 of the sample fluid 30 such that the multiplexed diagnostic assay 104 may be performed” may comprise or involve further steps. For example, the rotating 103 may correspond to a performance of a rotating program, in which the cartridge support plate 23 with the cartridge 24 is rotated in the centrifuge 21 around the rotation axis 25 at at least one first predetermined rotation frequency, which allows to move at least one portion of the sample fluid in at least a section or portion from the sample application port to the blood plasma separation element 10. The performance of the rotating program may comprise or involve a rotation at the at least one first predetermined rotation frequency or another predetermined rotation frequency, which is configured to perform the blood plasma separation. The performance of the rotating program may comprise or involve a rotation at the at least one first predetermined rotation frequency or another predetermined rotation frequency, which is configured to move at least a portion of the plasma from the blood plasma separation element 10 to the first reagent chamber I la. This optional step may accelerate the transport that is driven by capillary forces. The performance of the rotating program may comprise or involve a stopping of the rotation and allowing a binding reaction between the first analyte 31a and the first capture reagent 33a and between the second analyte 31b and the second capture reagent 33b to take place if the first analyte 31a and the second analyte 31b are present in the sample fluid 30.

The performance of the rotating program may comprise or involve a rotation at the at least one first predetermined rotation frequency or another predetermined rotation frequency, which is configured to move at least a portion of the plasma from the first reagent chamber I la to the second reagent chamber 11b. This optional step may accelerate the transport that is driven by capillary forces. The performance of the rotating program may comprise or involve a stopping of the rotation and allowing a binding reaction between the first analyte 31a and the first detection reagent 32a and between the second analyte 31b and the second detection reagent 32b to take place if the first analyte 31a and the second analyte 3 lb are present in the sample fluid 30.

The performance of the rotating program may comprise or involve rotating the cartridge support plate 23 with the cartridge 24 in the centrifuge 21 at at least one first predetermined rotation frequency or another predetermined rotation frequency that is configured to provide the first assay membrane 6 and the second assay membrane 7 with the at least one portion 30, 31 of the sample fluid 30 such that the first part of at least one portion 30, 31 of the sample fluid 30 flows along the general flow axis 6a of the first assay membrane 6 and thereby allowing a capture process of the first analyte 31a on the first assay membrane 6 if the first analyte 31a is present in the sample fluid 30 and such that the second part of at least one portion 30, 31 of the sample fluid 30 flows along the general flow axis 7a of the second assay membrane 7 and thereby allowing a capture process of the second analyte 3 lb on the second assay membrane 7 if the second analyte 3 lb is present in the sample fluid 30.

The performance of the rotating program may comprise or involve opening the washing buffer reservoir 13. The performance of the rotating program may comprise or involve rotating the cartridge support plate 23 with the cartridge 24 in the centrifuge 21 at at least one first predetermined rotation frequency or another predetermined rotation frequency that is configured to guide at least a portion of the washing buffer from the washing buffer reservoir 13 to the washing buffer release port 4c via the micro fluid washing buffer channel system 12 to provide the first assay membrane 6 and the second assay membrane 7 with the at least one portion of the washing buffer. The last step may comprise or involve an aliquotation step, in which the rotation frequency is configured to partition the washing buffer solution in several portions by means of the washing buffer aliquot chamber. Alternatively or in addition the washing buffer solution may be transported/guided through at least a portion/section of the microfluid washing buffer channel system 12 solely by capillary forces without the requirement of a rotation.

In the following, general statements directed to the principle, the effects, the advantage and embodiments of the invention are made.

The IVD test systems la, lb are shown to comprise a waste pad 9, but in other embodiments they might not provide a waste pad 9. The IVD test systems la, lb are shown to comprise two reagent chambers I la, 11b, however; at least one of the reagent chambers I la, 11b may be divided in sub-chambers for housing a first reagent in one sub-chamber and a second reagent in another sub-chamber. The IVD test system la, lb may also comprise, in other embodiments, only one reagent chamber 11. The IVD test system la, lb may also comprise more than two reagent chambers I la, 11b, for example three, four, five, six or more. The IVD test systems la, lb are shown to comprise recessed and open microfluid channels 5 and channel elements 10, 11, 12, 15 as long as no cover plate is applied, however, in other embodiments, the microfluid channels 5 and channel elements 10, 11, 12, 15 may also correspond to substantially and/or at least partially closed channel structures, which do not require a cover plate to gate the microfluidics through the channel. The IVD test systems la, lb are shown to comprise a recessed test zone 4, however, in other embodiments, the test zone 4 may not be recessed.

The IVD test systems la, lb are shown to comprise a washing buffer reservoirreceiving portion 14, which corresponds to a cutout in the test carrier 2c. In other embodiments, the washing buffer reservoir-receiving portion 14 may correspond to a recess and/or an area to provide space for the washing buffer reservoir 13. In other embodiments, the washing buffer reservoir 13 may be integrated in the test carrier 2, 2c. The test carrier 2, 2c may for example comprise a chamber for housing the washing buffer. In other embodiments the washing buffer reservoir 13 may be fixedly connected to the test carrier 2, 2c. The washing buffer reservoir 13 may be glued, thermally fused and/or welded with/to the test carrier 2, 2c. The IVD test systems la, lb are shown to comprise two or three assay membranes 6, 7, 8 being positioned in parallel to each other with respect to the respective general flow axis 6a, 7a, 8a. In other embodiments, more than three assay membranes may be provided. In other embodiments, the assay membranes may be positioned next to each other without being aligned in parallel. For example, the assay membranes 6, 7, 8 may be positioned with respect to each other in a star-like and/or a fan-like assembly/orientation being characterized by an angle other than 0°, 180° and/or 360° that is enclosed by the length axes and/or the general flow axes of two assay membranes.

The microfluidic test carrier may be described for performing the multiplexed determination of two, three or even more analytes in parallel by using only one microfluidic cartridge design with a tool-box like approach. The sample volume may range approximately between 10-100pL, specifically between approximately 15- 70pL and more specifically between approximately 30-35pL of a sample fluid such as whole blood. Depending on the number of analytes to be multiplexed, two, three or more assay membranes are placed in the microfluidic test carrier 2. For example, the membrane width 6d, 7d, 8d in a single-analyte-test may be approximately 3-4mm for one assay membrane, in a dual-analyte-test may be approximately l,5-2mm for one assay membrane out of two and in a three-analyte-test may be approximately 1- 1,3mm for one assay membrane out of three. The length for all membranes may be identical, e.g. approximately 17mm. When assuming sandwich immunoassay as test format, the capture zone for the first analyte may contain streptavidin for capturing biotinylated antibodies, for the second analyte the capture zone may contain anti-dig for capturing dig-labeled antibodies, and for the third analyte the capture zone may contain analyte-specific antibodies for the direct capture of the corresponding analyte.

In the following, some general definitions are provided.

A serum is the clear liquid part of the blood hat can be separated from clotted blood. Plasma corresponds to the clear liquid part of blood which contains the blood cells. Serum differs from plasma, the liquid portion of normal unclotted blood containing the red and white cells and platelets. It is the clot that makes the difference between serum and plasma. The term "whole blood" as used herein contains all components of blood, for examples white and red blood cells, platelets, and plasma. “Detecting an analyte” may refer to the mere qualitative detection of presence of at least one analyte of interest. “Detecting an analyte” may specifically comprise the meaning of quantitative determination of the level of the analyte, as mostly used herein. The term "determining" the level of the analyte of interest, as used herein refers to the quantification or qualification of the analyte of interest, e.g. determining or measuring the level of the analyte of interest in the sample.

In this context “presence of at least one analyte of interest” encompass the qualification of the analyte of interest by its absolute value and/or its relative signal value to an internal standard and/or reference and/or other analyte and/or a limit of analyte concentration which is matched with its conentration.

In this context "level" or "level value" encompasses the absolute amount, the relative amount or concentration as well as any value or parameter which correlates thereto or can be derived therefrom.

In the context of the present disclosure, the term “analyte”, “analyte molecule”, or “analyte(s) of interest” are used interchangeably referring the chemical species to be analyzed via mass spectrometry. Chemical species suitable to be analyzed via mass spectrometry, i.e. analytes, can be any kind of molecule present in a living organism, include but are not limited to nucleic acid (e.g. DNA, mRNA, miRNA, rRNA etc.), amino acids, peptides, proteins (e.g. cell surface receptor, cytosolic protein etc.), metabolite or hormones (e.g. testosterone, estrogen, estradiol, etc.), fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids (e.g. Vitamin D), molecules characteristic of a certain modification of another molecule (e.g. sugar moieties or phosphoryl residues on proteins, methyl-residues on genomic DNA) or a substance that has been internalized by the organism (e.g. therapeutic drugs, drugs of abuse, toxins, etc.) or a metabolite of such a substance. Such analyte may serve as a biomarker. In the context of present invention, the term “biomarker” refers to a substance within a biological system that is used as an indicator of a biological state of said system.

List of reference numerals

1 In Vitro diagnostic (IVD) test system for multiplexing la In Vitro diagnostic (IVD) test system with one single assay membrane

2 test carrier

2a first cover plate

2b second cover plate

2c test carrier core piece 3 sample application port test zone a shared recessed assay membrane area b sample release port c washing buffer release port d waste pad area

5 microfluid sample channel system

6 first assay membrane

6a general flow axis

6b flow start side

6c flow end side

6d width of first assay membrane

7 second assay membrane

7a general flow axis

7b flow start side

7c flow end side

7d width of second assay membrane

8 third assay membrane

8a general flow axis

8b flow start side

8c flow end side

8d width of third assay membrane

9 waste pad

10 blood plasma separation element

11 reagent chamber

I la capture reagent chamber

11b detection reagent chamber

12 microfluid washing buffer channel system

13 washing buffer reservoir/blister

14 washing buffer reservoir-receiving portion in test carrier

15 washing buffer aliquot chamber

16 gap 0 IVD test apparatus 1 centrifuge 2 controller 3 cartridge support plate 4 cartridge 5 rotation axis 0 sample fluid, specifically whole blood sample 0a plasma separated from the whole blood sample 1 analyte in the plasma 1a first analyte in the plasma lb second analyte in the plasma 1c sandwiched analyte (first, second or third) labeled with a detection antibody and/or a capture antibody detection reagent/antibody ’ fluorescent label a first detection reagent/antibody b second detection reagent/antibody 3 capture reagent/antibody 3a first capture reagent/antibody 3b second capture reagent/antibody capture system/ mechanism a first capture system/ mechanism b second capture system/ mechanism 5 control line 6 calibration line 7 capture and test result line 8 recess walls of the shared recessed assay membrane area 9 Bottom/supporting surface of the shared recessed assay membrane area

Claims

Patent Claims

1. In Vitro diagnostic (IVD) test system (la, lb) for performing a multiplexed diagnostic assay, wherein the IVD test system (la, lb) comprises: a test carrier (2) comprising: a sample application port (3) configured to receive a sample fluid (30); a test zone (4) comprising a shared recessed assay membrane area (4a) and a sample release port (4b) for releasing at least one portion (30, 30a) of the sample fluid (30) to the shared recessed assay membrane area (4a); and a microfluid sample channel system (5) configured to guide the at least one portion (30, 30a) of the sample fluid (30) from the sample application port (3) to the sample release port (4b); the IVD test system (la, lb) further comprising: a first assay membrane (6) positioned in the shared recessed assay membrane area (4a) and configured to receive a first part of the at least one portion (30, 30a) of the sample fluid (30) from the sample release port (4b) and to indicate at least one first analyte (31a) in the sample fluid (30); and a second assay membrane (7) positioned in the shared recessed assay membrane area (4a) next to the first assay membrane (6) and configured to receive a second part of the at least one portion (30, 30a) of the sample fluid (30) from the sample release port (4b) and to indicate at least one second analyte (3 lb) in the sample fluid (30) and/or to indicate the at least one first analyte (31a) in the sample fluid (30) in a different sensitivity range as the first assay membrane (6).

2. The IVD test system (lb) of claim 1, further comprising a third assay membrane (8) positioned in the recessed assay membrane area (4a) next to the first assay membrane (6) and/or the second assay membrane (7) and configured to receive a third part of the at least one portion (30, 30a) of the sample fluid (30) from the sample release port (4b) and to indicate a third analyte in the sample fluid (30).

3. The IVD test system (la, lb) of claim 1 or 2, wherein the first assay membrane (6) relies on a first capture mechanism (34a) to capture the first analyte (31a) after being labeled with a first capture reagent (33 a); the second assay membrane (7) relies on a second capture mechanism (34b) different from the first capture mechanism (34a) to capture the second analyte (31a) after being labeled with a second capture reagent (33b) different from the first capture reagent (33a), optionally wherein the first capture reagent (33 a) and/or the second capture reagent (33b) comprises one of a biotinylated antibody or a Digoxigenin (dig)- labeled antibody, and optionally wherein the first capture mechanism (34a) and/or the second capture mechanism (34b) comprises one of a streptavidin to capture the first or the second analyte (3 la, 3 lb) via the biotinylated antibody, an anti-dig to capture the first or the second analyte via the dig-labeled antibody, a DNA- based system and/or an analyte specific antibody to directly capture the first or the second analyte (31a, 31b).

4. The IVD test system (la, lb) of any one of the preceding claims, wherein the first assay membrane (6) and the second assay membrane (7) each have a general flow axis (6a, 7a) defined by a flow start side (6b, 7b) and a flow end side opposite (6c, 7c) the flow start side (6b, 7b), through which the respective general flow axis (6a, 7a) passes and wherein the general flow axis (6a) of the first assay membrane (6) and the general flow axis (7a) of the second assay membrane (7) are aligned in parallel such that the respective flow start side (6b, 7b) is positioned closest to the sample release port (4b) of the test zone (4).

5. The IVD test system (la, lb) of claim 4, further comprising at least one waste pad (9) positioned to contact the first assay membrane (6) and/or the second assay membrane (7) at the respective flow end side (6c, 7c).

6. The IVD test system (la, lb) of any one of the preceding claims, wherein the test carrier comprises a blood plasma separation element (10), which is integrated with the microfluid sample channel system (5) and which is configured to extract a blood plasma (30a) from the sample fluid (30) when the sample fluid comprises whole blood, optionally wherein the microfluid sample channel system (5) is configured to guide at least one portion of the sample fluid (30) from the sample application port (3) to the blood plasma separation element (10) and at least a portion of the blood plasma (30a) from the blood plasma separation element (10) to the release port (4b).

7. The IVD test system (la, lb) of any one of claims 3 to 6, wherein the test carrier (2) further comprises at least one reagent chamber (11), which is integrated with the microfluid sample channel system (5) for housing at least one reagent (32, 33) that is configured to bind with at least one of the analytes (31, 31a, 3 lb), specifically wherein the at least one reagent chamber (11) comprises at least one capture reagent chamber (I la) housing the first capture reagent (33a) and/or the second capture reagent (33b).

8. The IVD test system (la, lb) of claim 7, wherein the at least one reagent chamber (11) comprises at least one detection reagent chamber (1 lb) for housing at least one first detection reagent (32a) that is configured to bind with the first analyte (31a) and/or for housing one second capture reagent (32b) that is configured to bind with the second analyte (31b).

9. The IVD test system (la, lb) of any one of the preceding claims, wherein, if only two assay membranes (6, 7) are provided in the test assembly, the first assay membrane (6) and the second assay membrane (7) each have a width 6d, 7d of approximately 1,5 mm to 2mm measured in a perpendicular direction to the respective general flow axis (6a, 7a); or wherein, if three assay membranes (6, 7, 8) are provided in the test assembly, the first assay membrane (6), the second assay membrane (7) and the third assay membrane (8) each have a width 6d, 7d, 8d of approximately 1mm to 1,3mm measured in a perpendicular direction to the respective general flow axis (6a, 7a).

10. The IVD test system (la, lb) of one of the preceding claims, wherein the test zone (4) comprises a washing buffer release port (4c) and wherein the test carrier (2) further comprises a microfluid washing buffer channel system (12) for guiding a washing buffer; a washing buffer reservoir-receiving portion (14) for housing and/or receiving a washing buffer reservoir (13); optionally further comprising the washing buffer reservoir (13) configured to be fluidically connected with the washing buffer release port (4c) via the microfluid washing buffer channel system (12) for providing the assay membranes at the respective flow start side with a predetermined amount of washing buffer.

11. The IVD test system (la, lb) of any one of the preceding claims, wherein the first analyte (31a) and/or the second analyte (3 lb) comprises at least one of a cardiac analyte, specifically cTropT, NTproBNP, D-Dimer, PCT, BM2, BM3, creatinine, potassium, sodium chloride.

12. The IVD test system (la, lb) of any one of claims 8 to 11, wherein the multiplexed diagnostic assay corresponds to an Immunochromatographic assay and the at least one first detection reagent (32a) and/or the at least one second detection reagent (32b) comprises a fluorescent molecule.

13. The IVD test system (la, lb) of any one of the preceding claims, wherein the test carrier (2) corresponds to a centrifugal microfluidic test carrier and wherein the microfluidic channel system (5) is configured to guide the at least one portion of the sample fluid (30) from the sample application port (3) to the sample release port (4b) under influence of a centrifugal force.

14. An IVD test apparatus (20) comprising a centrifuge (21) with a cartridge support plate that is configured to be rotated around a centrifugal axis (25) by the motor of the centrifuge (21); a controller (22) that is configured to control the motor of the centrifuge (21) and the rotation of the cartridge support plate (23); at least one cartridge (24), which comprises the IVD test system (la, lb) and/or corresponds to the IVD test system (la, lb) of claim 13, wherein the cartridge (24) is configured to be fixed on the cartridge support plate (23) to be rotated together with the rotating cartridge support plate (23) around the centrifugal axis (25).

15. A method (100) of performing a multiplexed diagnostic assay comprising the steps of providing (101) the IVD test apparatus (20) of claim 14; positioning (102) and/or fixing the at least one cartridge (24) on the cartridge support plate (23); initiating (103, 104) the transport of the sample fluid (30) through the microfluid sample channel system to the first assay membrane (6) and the second assay membrane (7); detecting (105) whether the first assay membrane indicates the presence of the first analyte and/or whether the second assay membrane indicates the presence of the second analyte.